WO2024183750A1 - A novel immune cell and use thereof for treating diseases - Google Patents

A novel immune cell and use thereof for treating diseases Download PDF

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Publication number
WO2024183750A1
WO2024183750A1 PCT/CN2024/080277 CN2024080277W WO2024183750A1 WO 2024183750 A1 WO2024183750 A1 WO 2024183750A1 CN 2024080277 W CN2024080277 W CN 2024080277W WO 2024183750 A1 WO2024183750 A1 WO 2024183750A1
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cell
fold
cancer
tumor
full length
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PCT/CN2024/080277
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French (fr)
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Jing Xu
Luhan Yang
Yaqi Lv
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Qihan Hong Kong Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to immunology, chimeric antigen receptors, and chimeric autoantigen receptors, specifically relates to a novel engineered immune cell (e.g., engineered natural killer (NK) cells) or a population thereof.
  • the immune cells can be engineered to exhibit improved characteristics as compared to control cell (e.g., a non-engineered immune cell) .
  • the present disclosure provides a method of preparing the cell as well as a method for treating autoimmune diseases by using the cell or a composition comprising the cell.
  • 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
  • Induced pluripotent stem cells were originally developed by Japanese scientist Shinya Yamanaka in 2006 by transfering a combination of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) into adult somatic cells with a viral vector and obtaining a pluripotent stem cell similar to embryonic stem cells after a reprogramming process.
  • Human induced pluripotent stem cells have the following advantages: no ethical issue is involved, sources of cells are easily acquired from adult cells (such as skin, blood, and the like) , the cells possess strong differentiation ability, are able to differentiate into different functional cells, can be infinitely expanded, are cost-effective, and have high cell consistency.
  • the present disclosure provides a novel immune cell or a population thereof, a method of preparing the cell, use of the cell or a composition comprising the cell for the manufacture of a medicament for treating diseases.
  • Some aspects of the present disclosure provides engineered immune cells (e.g., engineered natural killer (NK) cells) and methods and/or uses for treatment of diseases such as cancer by using the engineered immune cells or a composition or a kit comprising the engineered immune cells.
  • engineered immune cells e.g., engineered natural killer (NK) cells
  • NK natural killer
  • the present disclosure provides the following technical solutions to solve the technical problems existing in the prior art:
  • the cell is (a) an induced pluripotent cell (iPSC) , a clonal iPSC, or an iPS cell line cell, or embryonic stem cell (ESC) ; (b) a derivative cell obtained from differentiating the cell of (a) ; or (c) an immune cell derived from an animal body,
  • iPSC induced pluripotent cell
  • ESC embryonic stem cell
  • the cell comprises a component which comprising an ectodomain, a transmembrane domain, and an intracellular domain;
  • the ectodomain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, and a full length or at least a portion of CD64 or its variant; and
  • transmembrane domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at least a portion of the native or modified transmembrane domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, CD25, CD122, CD132, CD127, CD218, CD360 and ICAM-1 polypeptide,
  • the intracellular domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at least a portion of the native or modified intracellular domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, ICAM-1, CD25, CD122, CD132, CD127, CD218, CD360, and CD3 ⁇ , and wherein the cell has enhanced or acquired ADCC (antibody-dependent cell-mediated cytotoxicity) or ADCP (antibody-dependent cell-mediated phagocytosis) in comparison to a control cell or population
  • the component further comprises a full length or at least a portion of the native or modified costimulatory domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, and NKp80 polypeptide.
  • the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant; or
  • the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant; or when the ectodomain is a full length or at least a portion of CD64 or its variant, the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant.
  • the transmembrane domain and the intracellular domain are selected from the group consisting of CD32a, CD64, and CD89; or
  • the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89; or
  • the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89.
  • a chimeric antigen receptor (CAR) or a T cell receptor (TCR) or the nucleotide coding sequence thereof;
  • hypo-immunity regulator a hypo-immunity regulator, or the nucleotide coding sequence thereof
  • CAR or the TCR specifically recognizes a tumor antigen selected from the group consisting of Adhesion G protein-coupled receptor E2 (ADGRE2) , Armadillo repeat-containing X-linked protein 3 (ARMCX3) , Carbonic Anhydrase IX (CA1X) , CCRI, CCR4, Carcinoembryonic Antigen (CEA) , CD3 ⁇ , CD5, CD7, CD8, CD9, CD10, CD19, CD20, CD22, CD25, CD26, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD52, CD56, CD70, CD74, CD82, CD99, CD123, CD133, CD138, CD200, CD269 (BCMA) , CD S, CLEC12A, Collectin Liver 1 (CLL1) , an antigen of a cytomegalovirus (CMV) infected cell (e.g., CMV) infected cell (e.g
  • pathogen antigen is derived from HIV, HBV, EBV, HPV, Lasse Virus, Influenza Virus, or Coronavirus.
  • transmembrane domain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 11-13
  • the costimulatory domain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 14-16
  • the signaling domain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to SEQ ID NO: 17.
  • the CAR comprises or consists of (i) an scFv having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 18 and 20-21 or (ii) an scFv encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NO: 22.
  • the CAR comprises or consists of (i) a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 25-29; or (ii) an amino acid encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NO: 30.
  • the persistence component comprises or consists of (i) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting SEQ ID NOs: 31-37; (ii) an amino acid sequence encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 38-39.
  • hypo-immunity regulator comprises a reduced expression of one or more genes selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and a NKG2DL (e.g., ULBP1) , and/or an enhanced expression of one or more genes selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, CD59, CD3, CD4, CD80, 41BBL, and CD131, preferably selected from the group consisting of PD-L2, TGF-beta, CD46, CD55, and CD59, preferably the hypo-immunity regulator comprises or consists of an amino acid sequence
  • the immune activity component comprises a reduced expression of one or more genes selected from the group consisting of TGFb receptor, TIGIT, PD1, PDL1, SIGLEC9, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, CD96, LAG3, and 2B4, and/or an enhanced expression of one or more genes selected from the group consisting of NCR, NKp30, NKp44, NKp46, NKG2D, NKp80, DNAM1, other NK activating receptors, chemokines or chemikine receptors.
  • the safety switch component 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 tumor microenvironment component comprises a reduced or an enhanced expression of one or more genes selected from the group consisting of KLRD1, CD96, CD244, CCR4, CCR9, CXCR6, CCR2, CXCR2, CX3CR1, KLRC2, TGFBR2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, MIR21, MIR181B1, MIR181A1 MIR144, and MIR150.
  • iPSC induced pluripotent cell
  • ESC embryonic stem cell
  • step b) introducing the component as defined in any one of items 1-6 into the cell provided by step a) ;
  • step c) optionally introducing the at least one of the feature as defined in any one of items 7-22 into the cell provided by step b) .
  • composition or a kit comprising the cell or population thereof of any one of items 1-22.
  • the tumor is selected from the group consisting of: Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer
  • autoimmune disease is selected from the group consisting of rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE) , myasthenia gravis (MG) , 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.
  • MGUS multiple sclerosis
  • Figure 1 shows a diagram of chimeric Fc receptor designs
  • Figure 2 shows mRNA level of chimeric receptor genes in transduced NK-92 cells evaluated by qPCR
  • Figure 3A-3C show surface expression of chimeric receptors in transduced NK-92 cells evaluated by FACS
  • Figure 4 shows ADCC of trastuzumab mediated by FcR-engineered NK92 cells against SKOV3 at the effector to target ratio of 1: 1 following incubation for 5 hours;
  • Figures 5A-5C show FcR5 (CD16/32) is superior to hnCD16 and CD64/16 to mediate ADCC;
  • Figure 6 shows that FcR-engineered NK92 cells were stimulated as indicated for 4 hours, and percentage (A) and MFI (B) of each FcR was determined by flow cytometry;
  • Figure 7A-7B show surface expression of chimeric receptors in engineered iPSC clones evaluated by FACS;
  • Figure 8 shows that the expression of TRA-181, a human pluripotent stem cell marker, was evaluated by FACS;
  • Figure 9 shows that iPSC expressing chimeric Fc receptors were differentiated into CD56+ NK cells
  • FIGS. 10A-10B show that FcR-expressing iNK cells were stimulated as indicated for 4 hours, and percentage (A) and MFI (B) of each FcR was determined by flow cytometry;
  • Figure 11 shows that ADCC mediated by FcR-engineered iPSC-NK cells with different dose of Rituximab against Raji cells at the effector to target ratio of 1: 1 following incubation for 5 hours;
  • Figure 12 shows serial killing assay to examine the ADCC of engineered iPSC-NK cells with rituximab
  • FIG. 13A is a schematic of antibody-based CAR structure design of the present invention.
  • ScFv stands for single chain variable fragment
  • TM stands for transmembrane domain
  • CSD stands for co-stimulatory domain
  • SD stands for signaling domain.
  • Figures 13B-13C shows targeted cytotoxicity of NK cells comprising one of the different chimeric receptor polypeptides, against CD19-presenting target cells.
  • Figure 13D is a schematic of the binding mechanism of a T cell receptor (TCR) -based CAR of the present invention.
  • TCR-like ScFv specifically recognizes peptide-major histocompatibility complex (pMHC) on the antigen presenting cells (APCs) .
  • Figure 13E is a schematic of the binding mechanism of a pMHC-based CAR of the present invention.
  • PMHC specifically recognizes TCR on the self-reactive T cells.
  • Figures 14B-14C illustrate engineered NK cells comprising heterologous human IL-15 as a persistency component
  • Figure 15 illustrates the surface expression of IL-15 in iNK cells differentiated from hIL15-IL15Ra fused-1 iPSC clones detected by FACS with APC (allophycocyanin) conjugated anti-IL-15 antibody.
  • PW15, PW18, and PW23 are clones expressing membrane-bound IL-15;
  • Figure 16 illustrates an in-vitro growth curve of eNK cells differentiated from mbIL-15-expressing iPSC clones (KB-15) cultured with or without IL-2;
  • FIGS. 17A-17E illustrate expression of CD56+, NKG2A+, NKp30+, NKp44+, and NKp46+ among WT iNK and iNK differentiated from different mbIL-15-iPSC clones.
  • FIG. 17A the percentage of CD56+ cells in the total differentiated cells.
  • FIG. 17B the percentage of NKG2A+ in the CD56+population.
  • FIG. 17C the percentage of NKp30+ in the CD56+ population.
  • FIG. 17D the percentage of NKp44+ in the CD56+ population.
  • FIG. 17E the percentage of NKp46+ cells in the CD56+population;
  • FIG. 18A-18C illustrate properties of NK cells differentiated from iPSC clones expressing secreted IL-15.
  • FIG. 18A the percentage of CD56+ cells in the total differentiated cells among WT iNK cells and iNK differentiated from different sIL15-iPSC clones.
  • FIG. 18B the concentration of IL-15 in culture medium from WT iNK cells and iNK cells differentiated from iPSC clones expressing secreted IL-15 (KA08) .
  • FIG. 18C an in-vitro growth curve of WT eNK cells and eNK cells differentiated from secretory IL-15-expressing iPSC clones (OQ-20) , in absence of an exogenous cytokine;
  • FIGS 19A-19C illustrate a screen of engineered NK cells for persistency.
  • FIG. 19A method design for in-vitro screening of the NK persistency related genes comparing culturing with low or high cytokine.
  • FIG. 19B the percentage of indel in FCER1G deficient editing when cultured with low or high cytokine.
  • FIG. 19C the percentage of indel in PTPN2 deficient editing when cultured with low or high cytokine;
  • FIGs 20A-20G illustrate a screen of engineered NK cells for persistency.
  • FIG. 20A the method design for in-vivo screening of the NK persistency related genes comparing in vitro culture and in-vivo growth.
  • FIG. 20A the method design for in-vivo screening of the NK persistency related genes comparing in vitro culture and in-vivo growth.
  • FIG. 20B the percentage of indel in STAT3 deficient editing in mouse liver versus being cultured with high cytokine.
  • FIG. 20C the percentage of indel in STAT3 deficient editing in mouse spleen versus being cultured with high cytokine.
  • FIG. 20D the percentage of indel in STAT3 deficient editing in mouse bone marrow (BM) versus being cultured with high cytokine.
  • FIG. 20E the percentage of indel in PTPN2 deficient editing in mouse liver versus being cultured with high cytokine.
  • FIG. 20F the percentage of indel in PTPN2 deficient editing in mouse spleen versus being cultured with high cytokine.
  • FIG. 20G the percentage of indel in PTPN2 deficient editing in mouse bone marrow (BM) versus being cultured with high cytokine;
  • Figure 21 illustrates a testing scheme for introducing a hypoimmunity regulator via editing and differentiating iPSC
  • FIG. 22A-22Q illustrate confirmed establishment of edit-1 clones to edit-9 clones.
  • FIG. 22A illustrates FACS analysis of edit-1 clones (hiPSC electroporated with pre-mixed ribonucleoprotein [RNP] targeting B2M) .
  • FIG. 22B illustrates Sanger sequencing of edit-1 clones.
  • FIG. 22C illustrates Sanger sequencing of edit-2 clones (hiPSC electroporated with RNP targeting CIITA) .
  • FIG. 22D illustrates FACS analysis of edit-3 clones (hiPSC electroporated with two RNPs targeting B2M and CIITA) .
  • FIG. 22E illustrates Sanger sequencing of edit-3 clones.
  • FIG. 22A illustrates FACS analysis of edit-1 clones (hiPSC electroporated with pre-mixed ribonucleoprotein [RNP] targeting B2M) .
  • FIG. 22B illustrates Sanger sequencing of edit-1 clones.
  • FIG. 22F illustrates FACS analysis of edit-4 clones (hiPSC electroporated with a construct overexpressing PD-L1, PD-L2, TGF- ⁇ , HLA-E, HLA-G, CD47, IL-10, CCL-21, CD46, CD55, CD59 and two RNPs, targeting B2M and CIITA) .
  • FIG. 22G illustrates Sanger sequencing of edit-4 clones.
  • FIG. 22H illustrates FACS analysis of edit-5 clones (hiPSC electroporated with a construct overexpressing of PD-L1, HLA-E, CD47, IL-10, CCL-21 and two RNPs, targeting B2M and CIITA) .
  • FIG. 22I illustrates Sanger sequencing of edit-5 clones.
  • FIG. 22J illustrates FACS analysis of edit-6 clones (hiPSC electroporated with a construct overexpressing PD-L1, HLA-E, CD47, CD46, CD55, CD59 and two RNPs, targeting B2M and CIITA) .
  • FIG. 22K illustrates Sanger sequencing of edit-6 clones.
  • FIG. 22L illustrates FACS analysis of edit-7 clones (hiPSC electroporated with a construct overexpressing PD-L1, HLA-E, CD47, CCL-21, CD55 and two RNPs, targeting B2M and CIITA) .
  • FIG. 22M illustrates Sanger sequencing of edit-7 clones.
  • FIG. 22J illustrates FACS analysis of edit-6 clones (hiPSC electroporated with a construct overexpressing PD-L1, HLA-E, CD47, CD46, CD55, CD59 and two RNPs, targeting B2M and CIITA) .
  • FIG. 22K illustrates Sanger sequencing of
  • FIG. 22N illustrates FACS analysis of edit-8 clones (hiPSC electroporated with a construct overexpressing of CD47 and two RNPs, targeting B2M and CIITA) .
  • FIG. 22O illustrates Sanger sequencing of edit-8 clones.
  • FIG. 22P illustrates FACS analysis of edit-9 clones (hiPSC electroporated with a construct overexpressing PD-L1, PD-L2, TGF- ⁇ , HLA-E, HLA-G, CD47, IL-10, CCL-21, CD46, CD55, CD59, two RNPs, targeting B2M and CIITA) ;
  • FIG. 22Q illustrates Sanger sequencing of edit-9 clones;
  • Figure 23 is a summary of the gene edits from edit-1 through edit-9;
  • FIGS. 24A-24S illustrate functional properties of edit-1 clones to edit-9 clones.
  • FIG. 24A cell lysis when different edited iPSC clones co-incubating with human complement.
  • FIG. 24B cell lysis when different edited iPSC clones co-incubating with cord blood-derived natural killer (CBNK) .
  • FIG. 24C cell counts of CD56+ cells among different iNK differentiated from corresponding edited iPSC. The iPSC clone number was shown in the parenthesis.
  • FIG. 24D CD56+ percentage among different iNK differentiated from corresponding edited iPSC. The iPSC clone number was shown in the parenthesis.
  • FIG. 24A cell lysis when different edited iPSC clones co-incubating with human complement.
  • FIG. 24B cell lysis when different edited iPSC clones co-incubating with cord blood-derived natural killer (CBNK) .
  • FIG. 24C cell counts
  • FIG. 24E NKG2A+ percentage among different iNK differentiated from corresponding edited iPSC. The iPSC clone number was shown in the parenthesis.
  • FIG. 24F NK cell-mediated lysis of K562 cells when co-cultured with corresponding eNKs at a single time point.
  • FIG. 24G NK cell-mediated lysis of K562 cells when co-cultured with corresponding eNKs over 6 days.
  • FIG. 24H the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with peripheral blood mononuclear cell (PBMC) from donor 1.
  • PBMC peripheral blood mononuclear cell
  • FIG. 24I the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 2.
  • FIG. 24J the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 3.
  • FIG. 24K the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 4.
  • FIG. 24L the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 5.
  • FIG. 24M the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 6.
  • FIG. 24N the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 1.
  • FIG. 24O the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 2.
  • FIG. 24P the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 3.
  • FIG. 24Q the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 4.
  • Figures 25A-25C illustrate enhanced NK cell activity conferred by aCD19-CAR
  • Figures 26A-26B illustrate enhanced IL-15 signaling conferred by IL-15-RF
  • FIGS. 27A-27C illustrate anti-tumor activity of QN-019a (anti-CD19 CAR NK) cells in Nalm6 NOG tumor mouse model.
  • Figure 28A-B illustrates that QN-019a NK cells effectively inhibited tumor growth in the mice and QN-019a NK cell+Rituximab combined treatment showed lower tumor fluorescence values than the QN-019a NK cell only group.
  • achimeric transmembrane receptor includes a plurality of chimeric transmembrane receptors.
  • the term “about” or “approximately” generally mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1%of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • 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.
  • an algal cell e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C.
  • seaweeds e.g., kelp
  • a fungal cell e.g., a yeast cell, a cell from a mushroom
  • an animal cell e.g., fruit fly, cnidarian, echinoderm, nematode, etc.
  • a cell from a vertebrate animal e.g., fish, amphibian, reptile, bird, mammal
  • a cell from a mammal e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.
  • a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell) .
  • reprogramming generally refers to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state.
  • a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state.
  • a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state.
  • differentiated generally refers to a process by which an unspecialized ( “uncommitted” ) or less specialized cell acquires the features of a specialized cell such as, e.g., an immune cell.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized ( “committed” ) position within the lineage of a cell.
  • the term “committed” generally refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • pluripotent generally refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) .
  • embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm.
  • Pluripotency can be a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell) , which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell) .
  • iPSCs induced pluripotent stem cells
  • differentiated cells e.g., differentiated adult, neonatal, or fetal cells
  • iPSCs reprogrammed stem cells
  • the iPSCs produced do not refer to cells as they are found in nature.
  • iPSCs can be engineered to differentiation directly into committed cells (e.g., natural killer (NK) cells.
  • NK natural killer
  • iPSCs can be engineered to differentiate first into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) ) , which can be further induced to differentiate into committed cells (e.g., NK cells) .
  • tissue-specific stem cells e.g., hematopoietic stem cells (HSCs)
  • HSCs hematopoietic stem cells
  • ESCs generally refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm.
  • ESCs can be engineered to differentiation directly into committed cells (e.g., NK cells) .
  • ESCs can be engineered to differentiate first into tissue-specific stem cells (e.g., HSCs) , which can be further induced to differentiate into committed cells (e.g., NK cells) .
  • isolated stem cells generally refers to any type of stem cells disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells (MSCs) , etc. ) that are isolated from a multicellular organism.
  • HSCs can be isolated from a mammal’s body, such as a human body.
  • an embryonic stem cells can be isolated from an embryo.
  • isolated generally refers to a cell or a population of cells, which has been separated from its original environment.
  • a new environment of the isolated cells is substantially free of at least one component as found in the environment in which the “un-isolated” reference cells exist.
  • An isolated cell can be a cell that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue or biopsy sample.
  • the term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, isolated form a cell culture or cell suspension. Therefore, an isolated cell is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.
  • hematopoietic stem and progenitor cells generally refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., into NK cells) and include, multipotent hematopoietic stem cells (hematoblasts) , myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors.
  • hematoblasts multipotent hematopoietic stem cells
  • HSCs Hematopoietic stem and progenitor cells
  • myeloid monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells
  • lymphoid lineages T cells, B cells, NK cells
  • HSCs can be CD34+ hematopoietic cells capable of giving rise to both mature myeloid and lymphoid cell types including T cells, NK cells and B cells.
  • immune cell generally refers to a differentiated hematopoietic cell.
  • Non-limiting examples of an immune cell can include an NK cell, a T cell, a monocyte, an innate lymphocyte, a tumor-infiltrating lymphocyte, a macrophage, a granulocyte, etc.
  • NK cell or “Natural Killer cell” generally refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3) .
  • NK cells that are phenotypically CD3-and CD56+, expressing at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both in same or different degrees) , and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceR ⁇ , and EAT-2.
  • isolated subpopulations of CD56+ NK cells can exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/or DNAM-1.
  • nucleotide generally refers to a base-sugar-phosphate combination.
  • a nucleotide can comprise a synthetic nucleotide.
  • a nucleotide can comprise a synthetic nucleotide analog.
  • Nucleotides can be monomeric units of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) ) .
  • nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP) , uridine triphosphate (UTP) , cytosine triphosphate (CTP) , guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
  • Such derivatives can include, for example, [ ⁇ S] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them.
  • nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs dideoxyribonucleoside triphosphates
  • Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots.
  • Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM) , 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE) , rhodamine, 6-carboxyrhodamine (R6G) , N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) , 6-carboxy-X-rhodamine (ROX) , 4- (4′dimethylaminophenylazo) benzoic acid (DABCYL) , Cascade Blue, Oregon Green, Texas Red, Cyanine and 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) .
  • FAM 5-carboxyfluorescein
  • JE 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein
  • fluorescently labeled nucleotides can include [R6G] dUTP, [TAMRA] dUTP, [R110] dCTP, [R6G] dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110] ddCTP, [TAMRA] ddGTP, [ROX] ddTTP, [dR6G] ddATP, [dR110] ddCTP, [dTAMRA] ddGTP, and [dROX] ddTTP available from Perkin Elmer, Foster City, Calif.
  • Nucleotides can also be labeled or marked by chemical modification.
  • a chemically-modified single nucleotide can be biotin-dNTP.
  • biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP) , biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP) , and biotin-dUTP (e.g. biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP) .
  • polynucleotide oligonucleotide, ” or “nucleic acid, ” as used interchangeably herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form.
  • a polynucleotide can be exogenous or endogenous to a cell.
  • a polynucleotide can exist in a cell-free environment.
  • a polynucleotide can be a gene or fragment thereof.
  • a polynucleotide can be DNA.
  • a polynucleotide can be RNA.
  • a polynucleotide can have any three dimensional structure, and can perform any function, known or unknown.
  • a polynucleotide can comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase) . If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. 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.
  • thiol containing nucleotides thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine.
  • Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA (tRNA) , ribosomal RNA (rRNA) , short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA) , nucleic acid probes, and primers.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • the term “gene” generally refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript.
  • 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: 93) , wherein “x” denotes any amino acid.
  • an ankyrin repeat motif can comprise the following sequence: DxxGxTPLHLAxxxGxxx
  • 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 host 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 would not be rejected by the host’s immune system (e.g., antibody-mediated complement cytotoxicity, or antibody-dependent cellular cytotoxicity (ADCC) ) and/or (ii) would be rejected at a slower rate by the host’s immune system as compared with a control cell without the enhanced expression or reduced expression of the hypo-immunity regulator.
  • the host’s immune system e.g., antibody-mediated complement cytotoxicity, or antibody-dependent cellular cytotoxicity (ADCC)
  • ADCC antibody-dependent cellular cytotoxicity
  • 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. ”
  • enhanced hypo-immunity e.g., enhanced resistance against ADCC
  • a population of engineered immune cells e.g., a population of engineered NK cells
  • an antibody e.g., SSEA-4 antibody
  • in vivo e.g., upon administration to a subject’s bloodstream
  • engineering of an immune cell e.g., NK cells as disclosed herein can enhance the immune cell’s resistance against immune rejection (e.g., ADCC) by at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95%, at least or up to about 100%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%.
  • ADCC immune rejection
  • the enhanced resistance against immune rejection (e.g., ADCC) can be ascertained in vitro in a medium comprising at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, or at least or up to about 80%, human complement.
  • a medium comprising at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, or at least or up to about 80%, human complement.
  • 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.
  • the immune cell of the present disclosure comprising ADCC component
  • the present disclosure provides a cell or a population thereof, wherein a) the cell is (a) an induced pluripotent cell (iPSC) , a clonal iPSC, or an iPS cell line cell, or embryonic stem cell (ESC) ; (b) a derivative cell obtained from differentiating the cell of (a) ; or (c) an immune cell derived from an animal body, b) the cell comprises a component which comprising an ectodomain, a transmembrane domain, and an intracellular domain.
  • iPSC induced pluripotent cell
  • ESC embryonic stem cell
  • the ectodomain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, and a full length or at least a portion of CD64 or its variant.
  • the transmembrane domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at least a portion of the native or modified transmembrane domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, CD25, CD122, CD132, CD127, CD218, CD360, and ICAM-1 polypeptide.
  • the intracellular domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at least a portion of the native or modified intracellular domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, ICAM-1, CD25, CD122, CD132, CD127, CD218, CD360, and CD3 ⁇ .
  • the cell has enhanced or acquired ADCC (antibody-dependent cell-mediated cytotoxicity) or ADCP (antibody-dependent cell-mediated phagocytosis) in comparison to a control cell or population thereof without the component.
  • the component further comprises a full length or at least a portion of the native or modified costimulatory domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, and NKp80 polypeptide.
  • the cell is an iPSC, a clonal iPSC, or an iPS cell-line cell.
  • the cell is an ESC, preferably a human ESC or a non-human animal ESC.
  • the cell is a derivative cell obtained from differentiating an iPSC, a clonal iPSC, or an iPS cell-line cell.
  • the cell is a derivative cell obtained from differentiating an ESC.
  • the cell is an immune cell derived from an animal body, preferably a human or a non-human animal.
  • the cell is a hemopoietic cell, an NK cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, or the derivative thereof.
  • the NK cell is an iPSC NK cell, a PBNK cell, a CBNK cell, or an NK92 cell.
  • the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant.
  • the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant.
  • the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant.
  • the transmembrane domain and the intracellular domain are selected from the group consisting of CD32a, CD64, and CD89.
  • the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89.
  • the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89.
  • the component comprises or consists of a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of SEQ ID NOs: 1-7.
  • 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 regulate 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.
  • the engineered immune cell comprises an ADCC component
  • the ADCC component is selected from the group consisting of: a) a full length or at least a portion of a high affinity non-cleavable CD16 (hnCD16) ; b) a full length or at least a portion of CD16a; c) a full length or at least a portion of CD32a; d) a full length or at least a portion of CD89; e) a full length or at least a portion of CD64; f) at least a portion of CD16a fused with at least a portion of CD32a or CD89 or CD64; g) at least a portion of CD32a fused with at least a portion of CD89 or CD64; and h) at least a portion of CD64 fused with at least a portion of CD89.
  • the ADCC component comprises or consists of a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of SEQ ID NOs: 8-10.
  • a heterologous gene encoding a heterologous CD16 variant that (i) exhibits higher binding affinity to its target (e.g., monomeric IgG) and (ii) is non-cleavable can be introduced to the cell (i.e., hnCD16) .
  • the heterologous CD16 variant can be a modified CD16 comprising, for example, F176V and S197P, as disclosed herein.
  • the heterologous CD variant can be a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) an ectodomain of a different cell surface protein, such as a glycoprotein (e.g., CD64) , that exhibits enhanced binding to the target (e.g., monomeric IgG) as compared to an unmodified CD16.
  • a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) an ectodomain of a different cell surface protein, such as a glycoprotein (e.g., CD64) , that exhibits enhanced binding to the target (e.g., monomeric IgG) as compared to an unmodified CD16.
  • a heterologous gene as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein.
  • the heterologous gene may not and need not be integrated into the genome of the engineered cell.
  • the enhanced CD16 signaling of the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., SHP-1) via Western blotting or (ii) expression of a downstream gene (e.g., CD25, IFN-gamma, TNF, etc. ) via Western blotting or PCR techniques.
  • a downstream signaling protein e.g., SHP-1
  • a downstream gene e.g., CD25, IFN-gamma, TNF, etc.
  • the CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be greater than CD16 signaling of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold,
  • enhanced CD16 signaling of the engineered immune cell e.g., the engineered NK cell comprising hnCD16
  • the engineered immune cell can be characterized by an increase in phosphorylation of a downstream signaling protein by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up or
  • enhanced CD16 signaling of the engineered immune cell e.g., the engineered NK cell comprising hnCD16
  • the engineered immune cell can be characterized by an increased expression of a downstream gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold,
  • the CD16 signaling of the engineered immune cell e.g., the engineered NK cell comprising hnCD16
  • the CD16 signaling of a control 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 up
  • 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 as compared to a control cell, as disclosed herein.
  • the cell furhter comprises at least one feature selected from the group consisting of: (i) a chimeric antigen receptor (CAR) or a T cell receptor (TCR) , or the nucleotide coding sequence thereof; (ii) a persistence component, or the nucleotide coding sequence thereof; (iii) a hypo-immunity regulator, or the nucleotide coding sequence thereof; (iv) an immune activity component, or the nucleotide coding sequence thereof; (v) a safety switch component, or the nucleotide coding sequence thereof; (vi) a gene modification to enhance or reduce the expression of one or more endogenous or heterologous genes; and/or (vii) a tumor microenvironment component or the nucleotide coding sequence thereof.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • the cell further comprises at least two features selected from the group consisting of: (i) - (vii) . In some cases, the cell further comprises at least three features selected from the group consisting of: (i) -(vii) . In some cases, the cell further comprises at least four features selected from the group consisting of: (i) - (vii) . In some cases, the cell further comprises at least five features selected from the group consisting of: (i) - (vii) . In some cases, the cell further comprises all six features selected from the group consisting of: (i) - (vii) . In some cases, the cell further comprises (i) and (ii) .
  • the cell additionally comprises at least one, at least two, at least three, at least four, or all five features selected from the group consisting of (iii) - (vii) .
  • the cell further comprises a gene modification to enhance or reduce the expression of one or more endogenous or heterologous genes.
  • the cell further comprises a gene modification to enhance the expression of one or more endogenous or heterologous genes, preferably an endogenous gene.
  • the cell further comprises a gene modification to reduce the expression of one or more endogenous or heterologous genes, preferably an endogenous gene.
  • the cell further comprises a gene modification to disrupt the expression of one or more endogenous or heterologous genes, preferably an endogenous gene.
  • the gene modification comprises a reduced or disrupted expression of FcR ⁇ .
  • 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) or a T cell receptor (e.g., at least 1, 2, 3, 4, 5, or more different types of T cell receptors) .
  • the engineered immune cell can be engineered to express a chimeric polypeptide receptor or a T cell receptor transiently or permanently.
  • a recombinant chimeric polypeptide receptor or a recombinant T cell 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 e.g., DNA or RNA
  • encoding the chimeric polypeptide receptor or the T cell receptor can be delivered to the engineered immune cell.
  • 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 Fc
  • 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.
  • the engineered immune cell comprises a CAR or a TCR, the CAR or the TCR specifically recognize a tumor antigen selected from the group consisting of Adhesion G protein-coupled receptor E2 (ADGRE2) , Armadillo repeat-containing X-linked protein 3 (ARMCX3) , Carbonic Anhydrase IX (CA1X) , CCRI, CCR4, Carcinoembryonic Antigen (CEA) , CD3 ⁇ , CD5, CD7, CD8, CD9, CD10, CD19, CD20, CD22, CD25, CD26, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD52, CD56, CD70, CD74, CD82, CD99, CD123, CD133, CD138, CD200, CD269 (BCMA) , CD S, CLEC12A, Collectin Liver 1 (CLL1) , an antigen of a cytomegalovirus (CMV) inf
  • the CAR or the TCR specifically recognize a pathogen antigen derived from a virus, bacteria, fungi, parasite and protozoa capable of causing diseases. In some cases, the CAR or the TCR specifically recognize a pathogen antigen derived from HIV, HBV, EBV, HPV, Lasse Virus, Influenza Virus, or Coronavirus. In some cases, the engineered immune cell comprises a CAR, the CAR specifically recognize a tumor antigen of CD19 or CD33.
  • the engineered immune cell comprises a CAR, wherein the CAR comprises at least one of the following domains: a) a transmembrane domain, such as a full length or at least a portion of the native or modified transmembrane domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, and ICAM-1 polypeptide; b) a costimulatory domain, such as a full length or at least a portion of the native or modified costimulatory domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, and NKp80 polypeptide; and c) a signaling domain, such as a full length or at least a portion of the native or modified signaling domain selected from CD3 ⁇
  • the engineered immune cell comprises a CAR, wherein the CAR comprises at least one of a transmembrane domain of CD8, a costimulatory domain of 2B4, and a signaling domain of CD3 ⁇ .
  • the transmembrane domain of CAR has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 11-13
  • the costimulatory domain of CAR has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 14-16
  • the signaling domain of CD3 ⁇ has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NO: 17.
  • the CAR further comprises: (i) an anti-CD33 scFv having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 18 and 20-21, or (ii) an anti-CD33 scFv encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NO: 22.
  • the CAR further comprises a CD8 hinge domain having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 23-24.
  • the CAR comprises or consists of (i) a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 25-29, or (ii) a sequence encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NO: 30.
  • a hinge domain (e.g., the linker between the extracellular antigen binding domain and the transmembrane domain) of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.
  • a transmembrane domain of a CAR in the engineered immune cell can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.
  • the hinge domain and the transmembrane domain of a CAR as disclosed herein can be derived from the same protein (e.g., CD8) .
  • the hinge domain and the transmembrane domain of the CAR as disclosed herein can be derived from different proteins.
  • a signaling domain of a CAR can comprise at least or up to about 1 signaling domain, at least or up to about 2 signaling domains, at least or up to about 3 signaling domains, at least or up to about 4 signaling domains, at least or up to about 5 signaling domains, at least or up to about 6 signaling domains, at least or up to about 7 signaling domains, at least or up to about 8 signaling domains, at least or up to about 9 signaling domains, or at least or up to about 10 signaling domains.
  • a signaling domain (e.g., a signaling peptide of the intracellular signaling domain) of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of a polypeptide of CD3 ⁇ , 2B4, DAP10, DAP12, DNAM1, CD137 (41BB) , IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D, or any combination thereof.
  • the signaling domain CAR in the engineered immune cell can comprise a full length or at least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell comprises the chimeric polypeptide receptor (e.g., CAR) that comprises at least CD8 transmembrane domain and one or more of: (i) 2B4 signaling domain and (ii) DAP10 signaling domain.
  • the engineered cell e.g., the engineered NK cell
  • the chimeric polypeptide receptor e.g., TFP or CAR
  • the 2B4 signaling domain can be flanked by the CD8 transmembrane domain and the DAP10 signaling domain.
  • the DAP10 signaling domain can be flanked by the CD8 transmembrane domain and the 2B4 signaling domain.
  • the chimeric polypeptide receptor as disclosed herein can further comprise yet an additional signaling domain derived from CD3 ⁇ .
  • An antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor can be a cell surface marker, a secreted marker, or an intracellular marker.
  • Non-limiting examples of an antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor (e.g., TFP or CAR) or TCR 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, CD123, 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
  • antigen of the antigen binding moiety of the chimeric polypeptide receptor or TCR 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 (CD40
  • 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 or TCR 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, CD23, CD24, CD30, CD33, CD38, CD44v7/8, CDC27, CDK- 4, CEA, CLCA2, Cyp-B, DAM-10, D
  • antigen of the antigen binding moiety of the chimeric polypeptide receptor or TCR 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 (e.g., 1, 2, 3, 4, or more) members selected from the group comprising BCMA, CD20, CD22, CD30, CD33, CD38, CD70, CD123, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100.
  • Non-limiting examples of the NKG2D ligand comprises one or more (e.g., 1, 2, 3, 4, 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 CD23.
  • the engineered immune cell s endogenous gene encoding CD23 can be modified to effect reduced expression or activity of the endogenous CD23.
  • 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 CD123.
  • the engineered immune cell ’s endogenous gene encoding CD123 can be modified to effect reduced expression or activity of the endogenous CD123.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38.
  • the engineered immune cell ’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38.
  • the subject engineered immune cells comprising the chimeric polypeptide receptor against CD38 can be capable of targeting and effecting death (or degradation) of plasma cells.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38.
  • the engineered immune cell is an engineered NK cell that is derived from an isolated ESC or an induced stem cell (e.g., iPSC) .
  • the engineered immune cell’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38.
  • 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 CD7.
  • the engineered immune cell s endogenous gene encoding CD7 can be modified to effect reduced expression or activity of the endogenous CD7.
  • 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 CD19 and/or CD33.
  • the CAR comprises at least one of a transmembrane domain of CD8, a costimulatory domain of 2B4, and a signaling domain of CD3 ⁇ .
  • 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 T cell or 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 cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell.
  • the heterologous cytokine can comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) .
  • IL interleukin
  • the antigen is not CD19.
  • the antigen binding moiety may not and need not exhibit any specific binding to CD19, but rather a specific binding to an antigen (e.g., one or more antigens) that is not CD19.
  • the engineered immune cell (e.g., an engineered NK cell) as disclosed herein can comprise a heterologous receptor that is a respective receptor of the heterologous cytokine as disclosed herein (e.g., heterologous IL-15 receptor (IL-15R, such as IL-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
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell.
  • the heterologous cytokine can further comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) .
  • IL interleukin
  • the engineered immune cell may and need not comprise a heterologous receptor that is a respective receptor of the heterologous cytokine (e.g., a heterologous IL-15R) .
  • the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be of the same species as that of the engineered immune cell (e.g., the engineered NK cell) .
  • both the heterologous cytokine and the engineered immune cell can be of human origin.
  • the heterologous cytokine can be of a different species than that of the engineered immune cell.
  • a heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be introduced to the engineered immune cell (e.g., engineered NK cell) by contacting a heterologous polynucleotide encoding the heterologous cytokine to the engineered immune cell.
  • the heterologous polynucleotide can be integrated into the engineered immune cell’s chromosome (e.g., nuclear chromosome) .
  • the heterologous polynucleotide may not and need not be integrated into the chromosome of the engineered immune cell.
  • an 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, and IL-36.
  • 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.
  • FMDV foot-and-mouth disease virus
  • EAV equine rhinitis A virus
  • TaV Thosea asigna virus
  • PTV-I porcine tescho virus-1
  • cardioviruses such as Theilovirus (e.g., Theiler'
  • 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
  • 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) .
  • the heterologous cytokine e.g., heterologous IL-15 ⁇ or IL-15 ⁇ for heterologous IL-15
  • an expression cassette encoding a heterologous fusion polypeptide comprising (i) the heterologous cytokine that is coupled to (ii) a heterologous receptor can be introduced to the engineered immune cell.
  • the heterologous cytokine may not and need not be cleavable from the heterologous receptor.
  • Non-limiting examples of the heterologous receptor can include a respective receptor of the heterologous cytokine (e.g., heterologous IL-15 ⁇ or IL-15 ⁇ for heterologous IL-15) , or a different receptor such as a common gamma chain ( ⁇ C ) receptor or a modification thereof.
  • An expression cassette as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein. Alternatively, the expression cassette may not and need not be integrated into the genome of the engineered cell.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced signaling of an endogenous signaling pathway that involves the heterologous cytokine (e.g., the heterologous IL, such as the heterologous IL-15) and/or the heterologous receptor (e.g., the heterologous IL receptor, such as the heterologous IL-15R) as disclosed herein.
  • the enhanced signaling of the endogenous signaling pathway as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., JAK3, STAT3, STAT5, etc.
  • a downstream gene e.g., Mcl1, Cdk4/6, Mki67, Tnf, Gzmb, Gzmc, Ifng, etc. for IL-15/IL-15R
  • PCR polymerase chain reaction
  • enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor can be characterized by an increase in phosphorylation of a downstream signaling protein by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about
  • enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor can be characterized by an increased expression of a downstream gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1- fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a heterologous cytokine as disclosed herein, wherein the heterologous cytokine is bound to a membrane (e.g., plasma membrane) of the engineered immune cell.
  • the heterologous cytokine can comprise a heterologous IL as disclosed herein (e.g., a heterologous IL-15) .
  • the engineered immune cell can further comprise one, two, or all of: (a) a different heterologous cytokine (e.g., a heterologous cytokine as disclosed herein, other than the one that is bound to the membrane of the subject cell) , (b) reduced expression or activity of an endogenous immune regulator polypeptide, and (c) a safety switch.
  • a different heterologous cytokine e.g., a heterologous cytokine as disclosed herein, other than the one that is bound to the membrane of the subject cell
  • the endogenous immune regulator polypeptide is not B2M.
  • the endogenous immune regulator can be, for example, a polypeptide other than B2M.
  • the engineered immune cell (e.g., an engineered NK cell) can comprise the different heterologous cytokine and one or both of (b) the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) and (c) the safety switch.
  • the engineered immune cell comprise the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) and one or both of (a) the different heterologous cytokine and (c) the safety switch.
  • the engineered immune cell comprise the safety switch and one or both of (a) the different heterologous cytokine and (b) the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) .
  • the engineered immune cell comprise all of (a) , (b) , and (c) .
  • the 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 present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a CD16 variant as disclosed herein for enhanced CD16 signaling in the engineered NK cell.
  • the CD16 variant e.g., a heterologous CD16 variant
  • the engineered immune cell can further comprise reduced expression or activity of an endogenous immune regulator polypeptide as compared to a control cell, as disclosed herein.
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced CD16 signaling as compared to a control cell, as disclosed herein.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) .
  • the engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • the engineered NK cell can be treated with inhibitors (e.g., small molecule inhibitors) of the endogenous cytokine signaling.
  • the engineered NK cell can comprise reduced expression of endogenous IL-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 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
  • the endogenous receptor can be a respective receptor of any cytokine as disclosed herein (e.g., a respective receptor of any IL as disclosed herein) .
  • the endogenous receptor can be a respective receptor of IL (e.g., IL-17R for IL-7 signaling) .
  • IL-17R can include IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, and variants thereof.
  • the endogenous IL-17R comprises IL-17RA.
  • the reduced expression or activity of the endogenous cytokine e.g., an endogenous IL, such as IL-17
  • endogenous receptor thereof as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc. for IL-17) or (ii) expression of a downstream gene via Western blotting or PCT techniques.
  • a downstream signaling protein e.g., PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc.
  • a downstream gene of IL cytokine can include a chemokine (e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc. ) , a cytokine (e.g., IL-6, TNFa, G-CSF, GM-CSF, etc. ) , an acute phase response molecule (e.g., SAA, CRP, lipocalin 2/24p3, etc. ) , and/or an enzyme (e.g., a metalloproteinase, such as MMP1, MMP3, MMP9, MMP13) .
  • a chemokine e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc.
  • a cytokine e.g., IL-6, TNFa, G-CSF, GM-CSF, etc.
  • reduced expression or activity of the endogenous cytokine e.g., the endogenous IL, such as IL-17
  • engineered immune cell e.g., engineered NK cell
  • reduced expression or activity of the endogenous cytokine can be characterized by a decrease in phosphorylation of a downstream signaling protein of the endogenous cytokine by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-
  • reduced expression or activity of the endogenous cytokine e.g., the endogenous IL, such as IL-17
  • engineered immune cell e.g., engineered NK cell
  • reduced expression or activity of the endogenous cytokine can be characterized by a decrease in the expression of a downstream gene of the endogenous cytokine by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least
  • the engineered immune cell e.g., the engineered NK cell
  • the engineered immune cell can exhibit enhanced expression profile of a specific cell marker for a committed immune cell (e.g., a NK cell marker) as compared to a control cell that does not exhibit the reduced activity of the endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) as disclosed herein.
  • a specific cell marker for committed NK cells can include CD57 or killer immunoglobulin-like receptors (KIR) .
  • KIR can comprise KIR2D and/or KIR3D.
  • KIR2D can comprise KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and/or KIR2DS5.
  • KIR3D can comprise KIR3DL1, KIR3DL2, KIR3DL3, and/or KIR3DS1.
  • the enhanced expression profile of the specific cell marker for the committed immune cell (e.g., CD57 or KIR for NK cells) as disclosed herein can be ascertained by a number of methods, including, but are not limited to, Western blotting or PCR techniques.
  • the expression of the specific cell marker for a committed immune cell (e.g., CD57 or KIR or NK cells) in the engineered immune cell of the present disclosure can be greater than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise one or both of: (i) a heterologous transcription factor (e.g., a heterologous STAT) , (ii) reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling as disclosed herein) , and (iii) reduced expression or activity of endogenous enzyme (e.g., a ligase, such as CBL-B) .
  • the engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • the heterologous transcription factor can comprise at least 1, 2, 3, 4, 5, or more different types of heterologous transcription factor.
  • the heterologous transcription factor can comprise at most 5, 4, 3, or 2 different types of transcription factor.
  • 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 present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of a specific endogenous cell marker for a committed immune cell as disclosed herein (e.g., a NK cell marker, such as KIR) as compared to a control cell.
  • a specific endogenous cell marker is KIR.
  • the engineered immune cell can further comprise one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein, (b) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein, (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell, as disclosed herein, (d) an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell, and (e) reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein
  • a heterologous cytokine e.g., a heterologous IL, such as IL-15
  • the engineered immune cell can comprise the chimeric polypeptide receptor and one or more of (e.g., 1, 2, 3, or 4 of) : (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the engineered immune cell can comprise the heterologous cytokine and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (c) the CD16 variant for enhanced CD16 signaling, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the engineered immune cell can comprise the CD16 variant for enhanced CD16 signaling and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the CD16 variant for enhanced CD16 signaling and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the engineered immune cell can comprise the heterologous immune regulator and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
  • the heterologous immune regulator e.g., 1, 2, 3, or 4 of
  • the engineered immune cell can comprise the reduced expression or activity of an endogenous immune regulator polypeptide and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, and (d) the heterologous immune regulator.
  • an endogenous immune regulator polypeptide e.g., 1, 2, 3, or 4 of
  • the reduced expression or activity of the specific endogenous cell marker for the committed immune cell e.g., KIR for NK cells
  • KIR for NK cells
  • the reduced expression or activity of the specific endogenous cell marker for the committed immune cell can be ascertained by a number of methods, including, but are not limited to, Western blotting or PCR techniques.
  • 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 present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise a heterologous cytokine (e.g., a heterologous IL) , as disclosed herein that is not IL-15.
  • the heterologous cytokine comprises IL-21 or variants thereof.
  • the engineered immune cell can be derived from an induced stem cell (e.g., iPSC) .
  • a control cell can be a cell can be an immune cell, such as a NK cell, used for comparison purposes.
  • a control cell can be a cell that does not comprise a heterologous cytokine (e.g., IL-15) .
  • a control cell can be a cell that does not comprise a CD16 variant for enhanced CD16 signaling.
  • a control cell can be a cell that a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen.
  • a control cell can be a cell that comprises a heterologous IL-15R.
  • a control cell can be a cell that does not comprise a membrane bound heterologous cytokine (e.g., IL-15) .
  • a control cell can be a cell that does not exhibit reduced expression or activity of an endogenous immune regulator polypeptide.
  • a control cell can be a cell that does not exhibit reduced expression or activity of an endogenous cytokine (e.g., IL-17) or a receptor thereof (e.g., IL-17R) .
  • a control cell can be a cell that does not comprise a heterologous transcription factor (e.g., STAT) .
  • a heterologous transcription factor e.g., STAT
  • a control cell can be a cell that does not exhibit reduced expression or activity of a specific endogenous cell marker for a committed immune cell (e.g., a NK cell marker, such as KIR) .
  • a control cell can be a cell that does not comprise a heterologous immune regulator polypeptide.
  • a control cell can be a cell that does not exhibit reduced expression or activity of one or more (e.g., 1, 2, 3, or 4) of: endogenous CD94, endogenous CD96, endogenous TGF beta receptor, or endogenous SHIP2.
  • a control cell can be a cell that does not exhibit reduced expression or activity of one or more (e.g., 1, 2, 3, 4, or 5) of: endogenous CD80, endogenous CD86, endogenous ICOSL, endogenous CD40L, endogenous MICA or MICB, or endogenous NKG2DL.
  • a control cell can be a cell that does not exhibit reduced expression or activity of ICAM1.
  • a control cell can be a cell that does not comprise a heterologous PDL2 or heterologous TGF beta.
  • a control cell can be a cell that does not comprise one or more (e.g., 1, 2, 3, 4, or 5) of: heterologous CCL21, heterologous IL-10, heterologous CD46, heterologous CD55, or heterologous CD59.
  • a control cell can be a cell that does not comprise heterologous IL-21.
  • a control cell can be a cell that is not derived from a cell line.
  • a control cell can be a cell that is not derived from an isolated ESC.
  • a control cell can be a cell that is not derived from an iPSC.
  • the present disclosure provides a population of engineered immune cells comprising any one of the engineered immune cells as disclosed herein (e.g., a population of engineered NK cells comprising any one of the engineered NK cells as disclosed herein) .
  • An engineered immune cell (e.g., an engineered NK cell) of the population of immune cells (e.g., the population of NK cells) can comprise a heterologous polypeptide, wherein the heterologous polypeptide comprises a heterologous IL-15 (e.g., heterologous secretory IL-15, and/or membrane-bound IL-15, such as IL15-IL15 receptor fusion, e.g., IL15-IL15 receptor alpha fusion) .
  • IL-15 e.g., heterologous secretory IL-15, and/or membrane-bound IL-15, such as IL15-IL15 receptor fusion, e.g., IL15-IL15 receptor alpha fusion
  • the engineered immune cell comprises a persistence component, the persistence component is selected from the group consisting of: a) , wherein the persistence component is selected from the group consisting of: a) a full length or at least a portion of IL15, preferably in secretion form; b) a full length or at least a portion of IL15 fused with a full length or at least a portion of IL15Ra; c) a transgene for p-STAT5 enhancement; d) a modification to an endogenous gene for p-STAT5 enhancement, preferably a disruption of an endogenous gene; e) a reduced expression of one or more genes selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, CD70; and f) an enhanced expression of one or more genes selected from the group consisting of CD25, CD122, and N
  • the persistence component comprises or consists of (i) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting SEQ ID NOs: 31-37; or (ii) an amino acid sequence encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 38-39.
  • An activity level (e.g., persistence level) of the population of engineered immune cells in an environment that is substantially free of an exogenous interleukin can be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about or more greater than a control persistence level of a comparable population of immune cells (e.g., engineered to comprise a comparable heterologous IL-15 as disclosed herein) in a control environment comprising the exogenous interleukin.
  • an exogenous interleukin e.g., IL-2, IL-15, etc.
  • Such persistence level may ascertained after the population of immune cells are in the environment for at least or up to about 1 day, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 11 days, at least or up to about 12 days, at least or up to about 13 days, at least or up to about 14 days, at least or up to about 15 days, at least or up to about 16 days, at least or up to about 17 days, at least or up to about 18 days, at least or up to about 19 days, at least or up to about 20 days, at least or up to about 21 days, at least or up to about 22 days, at least or up to about 23 days, at least or up to about 24 days, at least or up to about 25 days, at least or up to about 26 days, at least
  • the amount of the exogenous interleukin (e.g., IL-2, IL-15, etc. ) in the environment can be at least or up to about 1 unit per milliliter (U/ml) , at least or up to about 5 U/mL, at least or up to about 10 U/mL, at least or up to about 15 U/mL, at least or up to about 20 U/mL, at least or up to about 30 U/mL, at least or up to about 40 U/mL, at least or up to about 50 U/mL, at least or up to about 60 U/mL, at least or up to about 80 U/mL, at least or up to about 100 U/mL, at least or up to about 150 U/mL, at least or up to about 200 U/mL, at least or up to about 300 U/mL, at least or up to about 400 U/mL, or at least or up to about 500 U/mL.
  • the environment can be in vitro, ex vivo, or in viv
  • the population of engineered NK cells as disclosed herein can exhibit at least or up to about 10%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 95%, or more persistence (or survival rate) after being in the environment that is substantially free of the exogenous interleukin (e.g., IL-2) for at least or up to about 1 day, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 11 days, at least or up to about 12 days, at least or up to about 13 days, at least or up
  • the population of engineered NK cells as disclosed herein can exhibit at least about 50%survival after at least about 5 days (50%, after 9 days, 25%after 15 days) in the environment that is substantially free of the exogenous interleukin (e.g., IL-2) .
  • the population of engineered NK cells as disclosed herein can exhibit at least about 50%survival after at least about 9 days in the environment that is substantially free of the exogenous interleukin (e.g., IL-2) .
  • the population of engineered NK cells as disclosed herein can exhibit at least about 25%survival after at least about 15 days in the environment that is substantially free of the exogenous interleukin (e.g., IL-2) .
  • the population of engineered NK cells as disclosed herein can exhibit enhanced persistence by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about
  • NK cells for at least or up to about 1 day, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 11 days, at least or up to about 12 days, at least or up to about 13 days, at least or up to about 14 days, at least or up to about 2 weeks, at least or up to about 3 weeks, at least or up to about 4 weeks, at least or up to about 6 weeks, or at least or up to about 8 weeks, as compared to that of a comparable population of NK cells lacking the heterologous polypeptide comprising the heterologous IL-15 (e.g., lacking a heterologous membrane-bound IL-15) .
  • the population of engineered NK cells as disclosed herein can exhibit enhanced persistence by at least about 7-fold after
  • having such enhanced persistence level by having the heterologous polypeptide comprising the enhanced IL-15 signaling can reduce the amount of exogenous proteins (e.g., IL-2) required for the production of the engineered immune cells, enhance the efficiency of producing the engineered immune cells, and/or reduce overall cost of the immune cell therapy.
  • Any enhanced level of persistence ascertained in vitro can be translated to an event in vivo (e.g., in the blood stream of a subject in need thereof) .
  • an exogenous interleukin can comprise an interleukin that is not secreted by the cell or the population of cells (e.g., the engineered immune cell (s) , such as the engineered NK cells as disclosed herein) , and can be artificially added to the environment.
  • an exogenous interleukin may comprise a recombinant interleukin protein that is added to a medium.
  • an exogenous interleukin may comprise a recombinant interleukin protein that is administered to a subject in need thereof.
  • the term “persistence” as used herein may generally refer to a presence of at least a portion of a population of cells (e.g., a population of engineered immune cells, such as a population of engineered NK cells as disclosed herein) remaining in an environment after introducing the population of cells to the environment (e.g., in an in vitro medium, in the serum after intravenous (IV) administration, etc. ) .
  • a persistence may be ascertained by a duration of time that at least a portion of the population of cells remain in the environment at a detectable level.
  • persistence of a population of cells may correlate to the half-life of the population of cells in the environment (e.g., medium, blood stream, etc. ) .
  • a population of cells of interest e.g., a population of engineered immune cells, such as a population of engineered NK cells
  • a population of cells having a greater persistence level (e.g., at least 5%greater) in an environment after a period of time (e.g., after at least about 5 days) than a control population of cells in a comparable environment after a comparable period of time may indicate that a greater proportion (e.g., at least 5%greater) of the size of the population of cells have survived as compared to the control population of cells.
  • the engineered immune cell comprises a gene modification to enhance or reduce the expression of one or more endogenous or heterologous genes, wherein the gene modification comprises a reduced or disrupted expression of endogenous CD33 and/or CD38.
  • the reduced or disrupted expression of endogenous CD33 and/or CD38 does not impact growth and/or function of the cell, preferably does not impact differentiation of the cell.
  • the reduced or disrupted expression of endogenous CD33 and/or CD38 is achieved by using RNAi or a gene editing system.
  • endogenous CD33 and/or CD38 is reduced by RNAi selected from the group consisting of siRNA-, shRNA-, micoRNA-, and circular RNA-mediated RNA interferences.
  • RNAi selected from the group consisting of siRNA-, shRNA-, micoRNA-, and circular RNA-mediated RNA interferences.
  • the expression of endogenous CD33 and/or CD38 is disrupted by the gene editing system selected from the group of CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these systems, preferably by CRISPR-Cas9 system.
  • the CRISPR-Cas9 system comprises an sgRNA having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of SEQ ID NOs: 40-45 for the disruption of CD33 and/or an sgRNA having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of SEQ ID NOs: 46-51 for the disruption of CD38.
  • the CRISPR-Cas9 system comprises an sgRNA of any one of SEQ ID NOs: 40-45 for the disruption of CD33 and/or an sgRNA of any one of SEQ ID NOs: 46-51 for the disruption of CD38.
  • 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.
  • any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit one or more heterologous genes (e.g., knocked-in) : CD3, CD4, CD80, 41BBL, and CD131.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprise one or more (e.g., 1, 2, 3, 4, 5, or more) hypo-immunity regulators.
  • the engineered immune cell exhibits reduced expression or activity of one or more (e.g., 1, 2, 3, 4, 5, or more) hypo-immunity regulators from: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous ICOSL, (iv) endogenous CD40L, (v) endogenous MICA or MICB, or (vi) endogenous NKG2DL.
  • the engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • the engineered immune cell comprises a hypo-immunity regulator
  • the hypo-immunity regulator comprises a reduced expression of one or more genes selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and a NKG2DL (e.g., ULBP1) , and/or an enhanced expression of one or more genes selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, CD59, CD3, CD4, CD80, 41BBL, and CD131, preferably selected from the group consisting of PD-L2, TGF-beta, CD46, CD55, and CD59.
  • the hypo-immunity regulator comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 57-58.
  • 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 present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprise a hypo-immunity regulator (e.g., ICAM1) .
  • the engineered immune cell further comprises one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein, (b) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein, and (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell.
  • a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein
  • a heterologous cytokine e.g., a heterologous IL, such as IL-15
  • CD16 variant for enhanced CD16 signaling as compared to a control cell.
  • the engineered immune cell (e.g., the engineered NK cell) comprises a chimeric polypeptide receptor as disclosed herein and one or both of: (b) the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein and (c) the CD16 variant for enhanced CD16 signaling.
  • the heterologous cytokine e.g., a heterologous IL, such as IL-15
  • the engineered immune cell (e.g., the engineered NK cell) comprise the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein and one or both of: (a) the chimeric polypeptide receptor as disclosed herein and (c) the CD16 variant for enhanced CD16 signaling.
  • the engineered immune cell (e.g., the engineered NK cell) comprises the CD16 variant for enhanced CD16 signaling and one or both of: (a) the chimeric polypeptide receptor as disclosed herein and (b) the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein.
  • 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 present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein.
  • the endogenous immune regulator polypeptide comprise a hypo-immunity regulator (e.g., ICAM1) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • the reduced expression or activity of the endogenous ICAM1 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell, as disclosed herein.
  • the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell.
  • 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 present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell.
  • the immune regulator polypeptide comprises a hypo-immunity regulator.
  • the hypo-immunity regulator can comprise one or more (e.g., 1, 2, 3, 4, or more) members from: (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.
  • the engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) .
  • the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
  • 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 present disclosure provides an engineered immune cell (e.g., an engineered NK cell) .
  • the engineered immune cell can exhibit reduced expression or activity of one or more (e.g., 1, 2, 3, 4, 5, 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 (e.g., 1, 2, 3, 4, 5, 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 further comprises an immune activity component
  • the immune activity component comprises a reduced expression of one or more genes selected from the group consisting of TGFb receptor, TIGIT, PD1, PDL1, SIGLEC9, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, CD96, LAG3, and 2B4, and/or an enhanced expression of one or more genes selected from the group consisting of NCR, NKp30, NKp44, NKp46, NKG2D, NKp80, DNAM1, other NK activating receptors, chemokines or chemikine receptors.
  • the engineered immune cell further comprises a safety switch component
  • the safety switch component 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 engineered immune cell further comprises a tumor microenvironment component
  • the tumor microenvironment component comprises a reduced or an enhanced expression of one or more genes selected from the group consisting of KLRD1, CD96, CD244, CCR4, CCR9, CXCR6, CCR2, CXCR2, CX3CR1, KLRC2, TGFBR2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, MIR21, MIR181B1, MIR181A1 MIR144, and MIR150.
  • 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 e.g., ESCs or iPSCs
  • pluripotency characteristics of the 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 (vi) formation of embryoid bodies consisting of cells from the three somatic lineages.
  • pluripotent stem cell markers including, but not limited to SSEA1 (mouse only)
  • 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 or the population thereof has enhanced or acquired ADCC and optionally at least one of the following characteristics: (ii) improved persistency and/or survival; (iii) reduced fratricide; (iv) improved tumor microenvironment; (v) reduced immunogenicity, and (vi) improved safety, in comparison to a control cell (e.g., non-engineered immune cell or immune cell lacking the relevant feature (s) or component (s) ) or population thereof.
  • a control cell e.g., non-engineered immune cell or immune cell lacking the relevant feature (s) or component (s)
  • the engineered immune cell has an improved persistency and/or survival in at least one of following circumstances: a) in combination with antibodies targeting CD38; and b) under high oxidative stress conditions.
  • the engineered immune cell has a reduced fratricide in at least one of following circumstances: a) reduced antigen expression in the cell when the cell comprise a CAR targeting the antigen; b) reduced relevant receptor expression when its ligand is expressed in the cell; and c) reduced ligand expression when its receptor is expressed in the cell.
  • the engineered immune cell has a reduced immunogenicity in at least one of following circumstances: a) in combination with a monoclonal antibody to reduce immune cells including T cell, B cell, NK cell, and/or microphage of a host of the cell; b) in combination with a therapeutic agent to reduce immune cells including T cell, B cell, NK cell, and/or microphage of a host of the cell; and c) in combination with a therapeutic agent to reduce the activity of a host immune cell.
  • the engineered immune cell has an improved tumor microenvironment in at least one of following circumstances: a) with therapeutic agents for microenvironment improvement; and b) in combination with chemokine antagonist or agonist to improve cell infiltration.
  • the present disclosure provides a method of preparing the engineered immune cell or population thereof, the method comprises the following steps: a) providing a cell which is (a) an induced pluripotent cell (iPSC) , a clonal iPSC, or an iPS cell line cell, or embryonic stem cell (ESC) ; (b) a derivative cell obtained from differentiating the cell of (a) ; or (c) an immune cell derived from an animal body; b) introducing the component as defined in any one of items 1-6 into the cell provided by step a) ; and c) optionally introducing at least one of the following features into the cell provided by step b) : (i) a chimeric antigen receptor (CAR) ; (ii) a persistence related component; (iii) a hypo-immunity regulator; (iv) an immune activity component; (v) a safety switch component; (vi) a gene modification to enhance or reduce the expression of one or more endogenous or
  • 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
  • the co-therapeutic agent comprises an anti-CD20 antibody.
  • the present disclosure provides a composition or a kit comprising the engineered immune cell or population thereof.
  • the composition or a kit further comprises one or more therapeutic agents.
  • the one or more therapeutic agents are selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA) , mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD) .
  • IMD immunomodulatory drug
  • the checkpoint inhibitor comprised in the composition or the kit comprises: (a) one or more antagonists checkpoint molecules comprising PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA/B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha) , TLR3, VISTA, NKG2A/HLA-E, or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab,
  • the therapeutic agents comprised in the composition or the kit comprise one or more of azacytidine, venetoclax, MG132, decitabine, dasatinib, cytarabine, pomalidomide, and the derivatives thereof.
  • the antibody comprised in the composition or the kit comprises: (a) anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD 123, anti-GD2, anti-PDL1, and/or anti-CD38 antibody; (b) one or more of rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, certuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and their humanized or Fc modified variants or fragments and their functional equivalents and biosimilars.
  • 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, ) , acetyl
  • 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 composition or the kit of the present invention comprises the cell or population thereof of any one of items 1-16.
  • the composition or the kit further comprises an antibody selected from the group consisting of: 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, A
  • 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, infection disease, autoimmune disease, 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) obtained from the subject 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, a target disease of a subject, or can be used for anti-aging.
  • 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.
  • 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 retin
  • AIED autoimmune inner ear disease
  • 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
  • the target disease is T cell leukemia (TCL) , such as T-cell acute lymphoblastic leukemia (T-ALL) .
  • TCL T cell leukemia
  • T-ALL T-cell acute lymphoblastic leukemia
  • a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to CD7 as disclosed herein
  • a heterologous cytokine e.g.,
  • 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 and/or CD123) as disclosed herein,
  • a heterologous cytokine e.g., IL-15
  • a 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.
  • the present disclosure provide a method for treating disease in a subject suitable for adoptive cell therapy by using the engineered immune cell or population thereof or the composition or the kit of the present disclosure, and/or a use of the engineered immune cell or population thereof or the composition or the kit of the present disclosure in the manufacture of a medicament for treating a disease in a subject suitable for adoptive cell therapy, wherein the disease is selected from the group consisting of an autoimmune disorder; a hematological malignancy; a solid tumor; cancer, or a virus infection.
  • the present disclosure provide a method for reducing or preventing allorejection by using the engineered immune cell or population thereof or the composition or the kit of the present disclosure, and/or a use of the engineered immune cell or population thereof or the composition or the kit of the present disclosure in the manufacture of a medicament for reducing or preventing allorejection.
  • the present disclosure provide a method for treating acute myeloid leukemia in a subject by using the engineered immune cell or population thereof in combination with an anti-CD38 monoclonal antibody and/or azacytidine, and/or a use of the engineered immune cell or population thereof in combination with an anti-CD38 monoclonal antibody and/or azacytidine in the manufacture of a medicament for treating acute myeloid leukemia.
  • the present disclosure provide a method for treating disease in a subject by using a composition or a kit comprising an immune cell derived from an animal body and one or more therapeutic agents, and/or a use of a composition or a kit comprising an immune cell derived from an animal body and one or more therapeutic agents in the manufacture of a medicament for treating a disease in a subject, wherein the disease is selected from the group consisting of an autoimmune disorder; a hematological malignancy, preferably acute myeloid leukemia; a solid tumor; cancer, or a virus infection.
  • the one or more therapeutic agents are selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA) , mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD) .
  • a peptide a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA) , mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD)
  • Example 1 Engineered NK cells
  • Table 1 illustrates examples of engineered NK cells with or without genetic modifications, along with possible functions, and therapeutic indications.
  • therapeutic indications can include acute myeloid leukemia (AML) , multiple myeloma (MM) , Myelodysplastic syndrome (MDS) , B cell leukemia, T cell leukemia, solid tumor, blood cancer, infection disease, autoimmune diseases, and/or aging.
  • Example 2 Component for enhancing ADCC
  • NK cells can be engineered to exhibit enhanced ADCC.
  • exemplary amino acid sequences are:
  • SEQ ID NO: 4 Ectodomain of CD32a with H131 variant +Transmembrane domain of CD32a+Intracellular domain of CD32a
  • SEQ ID NO: 5 Ectodomain of CD16 with F158V+Transmembrane domain of CD89+Intracellular domain of CD89
  • SEQ ID NO: 6 Ectodomain of CD64+Transmembrane domain of CD89+Intracellular domain of CD89
  • SEQ ID NO: 7 Ectodomain of CD32a with H131 variant +Transmembrane domain of CD89+Intracellular domain of CD89
  • Fc ⁇ R fusions are designed and constructed to enhance ADCC (see Table 2) .
  • FcR5 CD16/32
  • FcR6 CD64/32
  • FcR-engineered NK92 cells against SKOV3 at the effector to target ratio of 1: 1 following incubation for 5 hours (see Figure 4) .
  • FcR-engineered NK92 cells were stimulated as indicated for 4 hours, and percentage (A) and MFI (B) of each FcR was determined by flow cytometry (see Figure 6) .
  • TRA-181 a human pluripotent stem cell marker
  • iPSC expressing chimeric Fc receptors were differentiated into CD56+ NK cells (see Figure 9) .
  • FcR-expressing iNK cells were stimulated as indicated for 4 hours, and percentage (A) and MFI (B) of each FcR was determined by flow cytometry (see FIG. 18A-10B) .
  • Example 3 Engineered NK cells with enhanced survival and/or persistency
  • NK cells can be engineered to comprise at least (i) a heterologous transcription factor (e.g., STAT) and (ii) reduced expression or activity of an endogenous cytokine receptor (e.g., endogenous IL receptor, such as IL-17R) .
  • a heterologous transcription factor e.g., STAT
  • an endogenous cytokine receptor e.g., endogenous IL receptor, such as IL-17R
  • NK cells are generated from isolated ESCs or iPSCs.
  • the NK cells are engineered to express a heterologous STAT (e.g., STAT3 and/or STAT5B) .
  • a gene encoding the heterologous STAT is incorporated into the NK cell’s genome via either viral transduction or via action of a gene editing moiety as disclosed herein.
  • the NK cells are also engineered to exhibit reduced expression or activity of endogenous IL-17R (i.e., STAT3 + IL-17R - NK cells) .
  • NK cells with either one of (i) the heterologous STAT and (ii) reduced expression or activity of IL-17R, or non-engineered NK cells are used as a control.
  • the engineered STAT3 + IL-17R - NK cells can be cultured in vitro to assess viability and growth (or proliferative capacity) of the engineered STAT3 + IL-17R - NK cells in absence of an exogenous cytokine.
  • the NK cells are cultured in culture medium without the addition of exogenous cytokines for 3-6 weeks.
  • the engineered STAT3 + IL-17R - NK cells exhibit a significantly higher number of NK cells as compared to the control cells, indicating the enhanced survival and persistency of the engineered STAT3 + IL-17R - NK cells in vitro.
  • the engineered STAT3 + IL-17R - NK cells can be administered in NCG mice having a Raji xenograft model.
  • NCG mice are triple immunodeficient and lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function to host the xenograft model.
  • the engineered STAT3 + IL-17R - NK cells and the control cells are each administered into the respective Raji xenograft model mice via intravenous (IV) tail vein injection, at a dose of about 1 ⁇ 10 6 cells per animal.
  • mice injected with the engineered STAT3 + IL-17R - NK cells exhibit higher NK cell concentrations in the peripheral blood from about 7 days to about 28 days post-infusion, demonstrating the enhanced survival and persistency of the engineered STAT3 + IL-17R - NK cells in vivo.
  • Cells of interest can be engineered with (i) reduced expression of at least one endogenous gene (e.g., loss-of-function of one or more immune regulating polypeptides) and/or (ii) enhanced or introduced expression of at least one additional gene (e.g., at least one transgene encoding one or more additional immune regulating polypeptides) .
  • endogenous gene e.g., loss-of-function of one or more immune regulating polypeptides
  • additional gene e.g., at least one transgene encoding one or more additional immune regulating polypeptides
  • NK cells e.g., cord blood NK (CBNK) cells, NK cells derived from iPSCs, etc.
  • BCL3 transcription coactivator BCL3 transcription coactivator
  • CBLB Cbl proto-oncogene B
  • CDK8 loss-of-function of cyclin dependent kinase 8
  • FCER1G loss-of-function of Fc fragment of IgE receptor Ig
  • FCER1G loss-of-function of interleukin 17A
  • IL17F loss-of-function of interleukin 17F
  • IPP5D/SHIP1 loss-of-function of suppressor of cytokine signaling 1 (SOCS1) ; loss-of-function of suppressor of cytokine signaling 2 (SOCS2) ; loss-of-function of suppressor of cytokine signaling 3 (SOCS3) ; loss-of-function of signal transducer and activator of transcription 3 (STAT3) ; loss-of-function of BCL3 transcription coactivator (BCL3) ; loss-of-function of Cbl proto-oncogene B (CBLB) ; loss-of-function of
  • the loss-of function gene editing can be fulfilled by one or more gene editing moieties as disclosed herein, such as CRIPSR/Cas9 system.
  • NK cells e.g., CBNK cells
  • the medium e.g., Lymphocyte Serum-Free Medium KBM 581(Corning) , 10%Human male AB serum, 1%MEM Non-Essential Amino Acids Solution (100X, Gibco) , 1%L-Glutamine (200 mM) (Gibco) , 0.02%Vitamin C, 200U/mL IL-2) .
  • cells e.g., about 1x10 7 cells
  • RNP guide RNA/Cas9 protein complex
  • the guide RNA and Cas9 are transfected, e.g., with the ratio of 2: 1 (guide RNA 75 picomole (pmol) , Cas9 protein 150 pmol) .
  • the transfected cells are recovered (e.g., using the medium with doubled Human male AB serum (20%) ) before downstream assays.
  • the editing efficiency is analyzed by fragment analysis or NGS.
  • NK cells can be utilized as a model population of cells to examine persistence enhancing gene edits.
  • a population of NK cells can be engineered comprise (i) reduced expression of at least one endogenous immune regulating polypeptide comprising one or more members selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, and CD70, and/or (ii) the enhanced or introduced expression of NKG2C.
  • the persistence level of the population of engineered NK cells in such mixture can be characterized by (i) an enrichment level of the population of engineered NK cells within the mixture in a sub-optimal environment that is greater than (ii) an enrichment level of the population of engineered NK cells within the mixture in an optimal environment.
  • the sub-optimal environment can comprise a lower amount (or concentration) of exogenous cytokine (e.g., exogenous IL, such as IL-2) .
  • the sub-optimal environment can comprise an amount of the exogenous cytokine that is at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more lower than that in the optimal environment.
  • the sub-optimal environment can be an in vitro medium, and the optimal environment can be an in vitro medium.
  • the sub-optimal environment can be an in vivo environment (e.g., blood stream of a subject) , and the optimal environment can be an in vitro medium.
  • the enrichment level of the population of engineered NK cells within such mixture can be ascertained by identifying an amount (or proportion) of cells exhibiting (i) the reduced expression of at least one endogenous immune regulating polypeptide comprising one or more members selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, and CD70, and/or (ii) the enhanced or introduced expression of NKG2C.
  • NK cells with reduced expression or activity level of a gene of interest may survive or persist longer in a sub-optimal environment (e.g., low cytokine) .
  • a sub-optimal environment e.g., low cytokine
  • NKs cells may exhibit high survival rate or persistence, regardless of whether the NK cells comprise the reduced expression or activity level of such gene of interest (e.g., FCER1G) .
  • a higher enrichment e.g., INDEL% in a mixture with different types of cells in the sub-optimal (or more challenging) environment can suggest that such loss-of-function gene may induce enhanced survival or persistence to the cell.
  • NK cells For example, to find the gene related to NK persistency, one or more of such population of engineered NK cells (e.g., eighteen kinds of gene-edited NK cells, such as gene-edited CBNK cells) were mixed in a mixture as disclosed herein and cultured using high cytokine (200 U/mL IL-2) or low cytokine (10 U/mL IL-2) . Editing percentage (%INDEL) of each gene was analyzed by NGS after 8 days culturing. Two mixtures were prepared using two individual electroporation of each gene, three replicates were set for each culturing condition. A flowchart showing the method design is depicted in FIG. 11A.
  • %INDEL Editing percentage
  • FCER1G deficient editing showed increased percentage when cultured with low cytokines, which indicated that FCER1G deficient NK cells had better persistency in low cytokine conditions, while PTPN2 had no significant difference in the same assay, as illustrated in FIG. 11B and FIG. 11C, respectively.
  • an immune regulator polypeptide that is not PTPN2 e.g., FCER1G
  • a cell e.g., stem cell, immune cell, such as NK cell, etc.
  • NK cells Similar to the in vitro screening for cell persistence, one or more of such population of engineered NK cells (e.g., eighteen kinds of gene-edited NK cells, such as gene-edited CBNK cells) were mixed in a mixture as disclosed herein and cultured using high cytokine (200U/mL IL-2) (more optimal environment) or injected to hIL-15-NCG mice (sub-optimal environment) , each mixed NK cells were injected to 5 mice with an amount (e.g., an amount of 1X10 7 NK cells /mouse) . Over time (e.g., after 8 days) , in vitro cultured NK cells or mouse tissue are harvested to extract the genome.
  • high cytokine 200U/mL IL-2
  • hIL-15-NCG mice sub-optimal environment
  • Editing percentage (%INDEL) of each gene is analyzed by NGS. Two mixtures were prepared using two individual electroporation of each gene, three replicates were set for in vitro culturing, five mice were used for each mixed NK cells injection. A flowchart showing the method design is depicted in FIG. 12A.
  • STAT3 deficient editing shown increased percentage in mouse tissue compared to cultured using high cytokine, which indicated that STAT3 deficient NK cells have better survival and persistency in vivo, while PTPN2 had no significant difference in the same assay.
  • an immune regulator polypeptide that is not PTPN2 e.g., STAT3
  • a cell e.g., stem cell, immune cell, such as NK cell, etc.
  • cells of interest can be engineered to exhibit enhanced cytokine signaling (e.g., enhanced IL-15 signaling by enhanced or introduced IL-15, such as heterologous secretory IL-15, heterologous membrane-bound IL-15, heterologous IL-15 cytokine-IL15 receptor fusion, etc. ) .
  • the cells of interest can be stem cells, such as isolated stem cells (e.g., embryonic stem cells) or induced stem cells (e.g., iPSCs) .
  • the cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. Alternatively, such immune cells can be immune cell lines (e.g., NK cell lines) .
  • Engineered NK cells e.g., NK cells
  • NK-92 cells were engineered with (i) hIL-15 knock in or (ii) hIL-15-hIL15R fusion polypeptide knock in.
  • Two variants of the hIL-15-hIL15R fusion polypeptide were tested.
  • the first variant i.e., hIL15-IL15Ra fused-1 or “fus1”
  • the first variant was designed with a linker between hIL-15 and hIL15R, which linker comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “GGGGS” (SEQ ID NO. 53) , e.g., “GGGGSGGGGSGGGGSGGGGSGGGGGGSGGGGS” (SEQ ID NO. 54) .
  • the second variant (i.e., hIL15-IL15Ra fused-2 or “fus2” ) was designed with a linker between hIL-15 and hIL15R, which linker comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “GGGGS” (SEQ ID NO. 53) and one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “EGKSSGSGSESKST” (SEQ ID NO. 55) , e.g., “EGKSSGSGSESKSTEGKSSGSGSESKSTGGGGS” (SEQ ID NO. 56) .
  • NK-92 cells with either of the hIL-15-hIL15R fusion polypeptide variant knocked-in were positive for hIL-15.
  • the engineered NK-92 cells expressing either variant of the hIL-15-hIL15R fusion polypeptide for enhanced IL-15 signaling exhibited longer persistency as compared to control NK-92 cells engineered express secretory form of IL-15.
  • Western blotting analysis revealed increased phosphorylation of IL-15-stimulated STAT5 in the NK-92 cells expressing either hIL15-IL15Ra fused-1 (fus1) or hIL15-IL15Ra fused-2 (fus2) , as compared to the secretory IL-15 (IL15) .
  • hIL15-IL15Ra fused-1 sequence (SEQ ID NO. 31) :
  • hIL15-IL15Ra fused-2 sequence (SEQ ID NO. 32) :
  • Membrane-bound IL-15 expression in iNK cells differentiated from hIL15-IL15Ra fused-1 iPSC clones The expression of IL15 was confirmed in several iNK cells differentiated from hIL15-IL15Ra fused-1 iPSC clones, PW15, PW18, and PW23. As shown in FIGURE 15, Fluorescence-activated Cell Sorting (FACS) was performed to quantify the surface expression of IL-15 in clones expressing membrane-bound IL-15 in comparison with controls, wild-type (wt) iNK cells and isotype. Thus, the clones were validated for expected overexpression level of membrane-bound IL-15.
  • FACS Fluorescence-activated Cell Sorting
  • KB-15 cells were eNK cells (e.g., NK cells differentiated from iPSCs and subsequently expanded) differentiated from iPSC clones expressing hIL15-IL15Ra, aCD19 CAR, and a CD16 variant for enhanced CD16 signaling.
  • eNK cells e.g., NK cells differentiated from iPSCs and subsequently expanded
  • iPSC clones expressing hIL15-IL15Ra, aCD19 CAR, and a CD16 variant for enhanced CD16 signaling.
  • the in-vitro growth of 2x10 7 KB-15 cultured with or without IL-2 (100U/mL) was monitored for 30 days. The cultured cells was collected and counted every 3 to 4 days, and the medium was renewed with corresponding medium meanwhile.
  • KB-15 cells were able to grow in the absence of exogenous cytokines as vigorously as the ones in the presence of exogenous cytokines.
  • the engineered NK cells comprising enhanced IL-15 signaling e.g., KB-15 NK cells comprising hIL15-IL15Ra
  • the engineered NK cells cultured in a medium substantially free of exogenous IL-2 exhibited enhanced persistence (e.g., on day 5, day 9, day 12, day 16, day 23, day 26, etc. ) than the engineered NK cells cultured in a medium comprising exogenous IL-2.
  • membrane-bound IL-15 can have a capability to induce downstream IL signaling (e.g., STAT pathway) to a higher degree than secreted IL (e.g., IL-2, IL-15) , but the secreted IL at sufficient amount can act as a competitor for the same membrane receptor to reduce the chance of binding between the membrane-bound IL-15 and the respective receptor (e.g., IL-15R) and the signaling thereof.
  • IL signaling e.g., STAT pathway
  • secreted IL e.g., IL-2, IL-15
  • IL15-IL15R fusion membrane-bound IL-15
  • the engineered NK cells comprising enhanced IL-15 signaling is administered in vivo, where an amount of cytokines such as IL-2 in the blood stream or in a tissue of interest may be low (or sub-optimal) , the engineered NK cells can exhibit optimal persistence or survival via self-induced activation of IL-15.
  • IPSC expressing membrane-bound IL-15 differentiated into iNK
  • the engineered iPSC were subjected for iNK differentiation. Specifically, 5x10 5 culture containing cells was collected and used for staining with NKG2A-PE, NKp30-PE, NKp44-PE, NKp46-PE and CD56-APC antibody. As shown in FIGs. 9A-9E respectively, CD56 + cells could be detected in the total differentiated cells, and NKG2A + , NKp30 + , NKp44 + , and NKp46 + cells could be detected in the CD56 + population among three exemplified clones, PW15, PW18 and PW23, which expressed membrane-bound IL-15. Therefore, iPSC expressing membrane-bound IL-15were proven to be able to differentiate into iNK cells.
  • iPSC expressing secretory IL-15 differentiated into iNK
  • the engineered iPSC were subjected for iNK differentiation. Three clones expressing secretory IL-15, PX27, PX33, and PX39, were tested. Specifically, 1x10 5 culture containing cells was collected and used for staining with CD56-APC antibody. FIG. 10A with the percentage of CD56 + cells in the total differentiated cells illustrates that iPSC expressing secretory IL-15 could differentiate into iNK.
  • iNK cells differentiated from iPSC clones expressing secreted IL-15, aCD19 CAR, and a variant for enhanced CD16 signaling (KA08) were cultured for 2 days without renewing the medium, and the supernatant was collected and diluted 10-fold to measure human IL-15 the Human IL-15 ELISA kit.
  • FIG. 10B showing the concentration of IL-15 in iNK culture medium proves that the iNK cells were validated for secreting human IL-15 into culture medium.
  • FIG. 10C shows the in-vitro growth curve of 5x10 6 eNK cells differentiated from iPSC clones expressing secretory IL-15, aCD19 CAR, and a variant for enhanced CD16 signaling (OQ-20) .
  • the cultured cells were collected and counted every 4 days, and the medium in absence of exogenous cytokines was renewed with corresponding medium meanwhile. The growth of the cells within 16 days was recorded and plotted as curves. Therefore, it has been proven that eNK cells expressing secretory IL-15 facilitated in-vitro growth without exogenous cytokines.
  • cells of interest can be engineered to comprise a heterologous chimeric polypeptide (e.g., a chimeric antigen receptor) comprising an antigen binding moiety against a specific antibody of a target cell (e.g., a cancer or tumor cell) , to exhibit enhanced cytotoxicity against such target cell.
  • a heterologous chimeric polypeptide e.g., a chimeric antigen receptor
  • the cells of interest can be immune cells (e.g., NK cells) .
  • immune cells can be derived from the stem cells as disclosed herein.
  • NK cells derived from stem cells one or more genetic modifications as disclosed herein can be introduced at (A) the stem cell state (e.g., iPSC state) , (B) the hematopoietic stem cell state, and/or (C) the NK cell state.
  • the stem cell state e.g., iPSC state
  • the hematopoietic stem cell state e.g., IL-12
  • C NK cell state
  • immune cells can be immune cell lines (e.g., NK cell lines) .
  • NK cells expressing a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to CD33 were generated.
  • Targeted cytotoxicity of NK92 cells with CD33-CAR integration on KG1 cells, a tumor cell line with high expression of CD33, with an E/T (Effector/Target) ratio of 1: 1 was tested.
  • WT-NK92 cell were used as unmodified control.
  • the targeted cytotoxicity of CD33-CAR on KG1 cells were greatly improved compared to control.
  • NK92 cells were engineered to express anti-CD33 CAR, then cultured in the presence of CD33+ KG1 cells to assess targeting of the Raji cells by the engineered anti-CD33 NK cells. Wild type (WT) NK92 cells were used as control.
  • the anti-CD33 CAR NK cells exhibited enhanced cytotoxicity against the KG1 cells (as ascertained by a reduced number of alive Raji cells) as compared to the control.
  • NK cells e.g., NK-92 cells
  • WT Wild type
  • NK-92 cells were used as control.
  • the anti-CD19 CAR NK cells exhibited enhanced cytotoxicity against the Raji cells (as ascertained by a reduced number of alive Raji cells) as compared to the control.
  • the anti-CD19 CAR NK cells exhibited enhanced expression of endogenous CD107a (indicative of cytotoxic granule release) as compared to the control.
  • the anti-CD19 CAR NK cells exhibited enhanced cytokine production (e.g., IFN-gamma and/or TNF-alpha production) as compared to the control.
  • NK cells expressing a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to BCMA was generated.
  • E/T (Effector/Target) ratios used were 1: 1; 1: 5 and 1: 10. WT-NK92 cell were used as unmodified control.
  • FIG. 5A illustrates different chimeric polypeptide receptor (e.g., CAR) constructs.
  • FIG. 5A Top schematically illustrates CD19 CAR (2B4) structure design. TM short for Transmembrane domain; SCFV short for single chain variable fragment.
  • FIG. 5A Middle schematically illustrates CD19 CAR (4-1-BB) structure design. TM short for Transmembrane domain; SCFV short for single chain variable fragment.
  • FIG. 5A bottom schematically illustrates CD19 CAR (CD28) structure design. TM short for Transmembrane domain; SCFV short for single chain variable fragment.
  • CD19 CAR CD28
  • FIGs. 5B and 5C shows targeted cytotoxicity against target cells by NK cells expressing one of the chimeric polypeptide receptor design shown in FIG. 5A.
  • targeted cytotoxicity of various CD19-CAR NK92 on CD19-K562 cells E/T (Effector/Target) equals 5: 1; 1: 1 and 0.5: 1) demonstrates that NK cells expressing CAR constructs with 4-1-BB signaling domain, 2B4 signaling domain, and/or CD28 signaling domain exhibited targeted cytotoxicity against CD19-presenting K562 target cells.
  • WT-NK92 cell were used as unmodified control, CD19-K562 is K562 engineered with CD19 highly expressed. Referring to FIG.
  • non-specific cytotoxicity of CD19-CAR NK92 on K562 cells that are not engineered to express CD19 at a high level E/T (Effector/Target) equals 5: 1; 1: 1 and 0.5: 1) demonstrated lower degree of cytotoxicity, indicating that 4-1-BB, 2B4, and/or CD28 intracellular signaling domains are useful in designing various CAR constructs, e.g., for immunotherapies such as NK cell threapies.
  • Example 5 Engineered NK cells with a hypo-immunity regulator
  • cells of interest can be engineered to exhibit (i) reduced expression of one or more immune regulating polypeptides (e.g., one or more endogenous immune regulating polypeptides) and/or (ii) enhanced or introduced expression of one or more additional immune regulating polypeptides (e.g., one or more heterologous immune regulating polypeptides) .
  • immune regulating polypeptides e.g., one or more endogenous immune regulating polypeptides
  • additional immune regulating polypeptides e.g., one or more heterologous immune regulating polypeptides
  • Cells comprising (i) and/or (ii) as disclosed herein can exhibit enhanced function, such as a persistence level (or survival level) , hypo-immunity (e.g., resistance against immune rejection or cytotoxicity) , growth rate, cytotoxicity against a target cell (e.g., tumor cell) , etc.
  • a persistence level or survival level
  • hypo-immunity e.g., resistance against immune rejection or cytotoxicity
  • growth rate cytotoxicity against a target cell (e.g., tumor cell)
  • a target cell e.g., tumor cell
  • having the combination of (i) and (ii) can synergistically improve function of the cells, as compared to having either one of (i) and (ii) alone, or a combination of individual effects of (i) and (ii) , or none.
  • having reduced expression of two or more immune regulating polypeptides can synergistically improve function of the cells, as compared to having an individual member of the reduced expression of the two or more immune regulating polypeptides, or a combination of individual effects of such individual members.
  • having enhanced/introduced expression of two or more additional immune regulating polypeptides can synergistically improve function of the cells, as compared to having an individual member of the enhanced/introduced expression of the two or more additional immune regulating polypeptides, or a combination of individual effects of such individual members.
  • the cells of interest can be stem cells, such as isolated stem cells (e.g., embryonic stem cells) or induced stem cells (e.g., iPSCs) .
  • the cells of interest can be immune cells (e.g., NK cells) .
  • immune cells e.g., NK cells
  • Such immune cells can be derived from the stem cells as disclosed herein.
  • immune cells can be immune cell lines (e.g., NK cell lines) .
  • Table 2 illustrates example combinations of modified expression or activity of the plurality of immune regulator polypeptides.
  • a combination of modified expression or activity of the plurality of immune regulator polypeptides from Table 2 may be introduced in cells (e.g., engineered NK cells) to, for example, reduce or avoid immune response (e.g., immune attack, such as adaptive immune rejection) from a host’s body upon administration of the cells to the host’s body.
  • a combination of modified expression or activity of the plurality of immune regulator polypeptides from Table 2 may comprise (i) reduced expression or activity of one or more first immune regulator polypeptides (column 2) and (ii) enhanced expression or activity of one or more second immune regulator polypeptides (column 3) .
  • a combination of modified expression or activity of the plurality of immune regulator polypeptides from Table 8 may comprise (i) knock-out of one or more endogenous immune regulator polypeptide genes (column 2) and (ii) knock-in of one or more heterologous immune regulator polypeptide genes (column 3) .
  • NK cells can be engineered to carry certain transgenes and/or loss-of-function of genes of interest, such as the non-limiting exemplary guide RNA sequences are show in Table 9 below.
  • Human iPSC cells can be engineered by knocking in gene edits such as HLA-E, CD47, PDL2, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and/or CD59. Such engineered iPSC cells can be differentiated into NK cells. Alternatively, human peripheral blood (PB) -NK cells can be engineered with AAV system. Possible functional readouts to test the engineered NK cells for hypo-immunity include mixed lymphocyte reaction (MLR) , T cell activation assay, in vitro NK-cell-induced killing assay, and complement-dependent cytotoxicity.
  • MLR mixed lymphocyte reaction
  • iPSC can be edited with different knock-ins and knock-outs. Subsequently, these edited iPSC can be subjected for differentiation into iNK which can be further expanded into eNK.
  • iNK cells can be used for T cell proliferation assay, and eNK can be used for NK cytotoxicity test and hypoimmunity test.
  • edited iPSC cells can be differentiated into iEC cells (e.g., endothelial cells derived from iPSCs) which can be used for NK susceptibility assay.
  • RNP method is a method of electroporating target cells with pre-mixed ribonucleoprotein (RNP) containing Cas9 and sgRNA. After delivery to the cells, the RNP edits the genome region paired to the sgRNA.
  • Adenine base editor (ABE) method is a method where Cas proteins can be fused to an enzyme that can deaminate a DNA nucleoside.
  • clones 05, 07, 08, 104, 111, 112 were derived by electroporating human iPSC with RNP targeting B2M.
  • the Edit-1 clones were confirmed to be B2M knock-out by FACS analysis for MHC-I (see FIG. 14A) and by Sanger sequencing (FIG. 14B) .
  • clone 05 was sequenced to have an insertion of one nucleotide in both B2M alleles
  • clone 07 was sequenced to have an insertion of one nucleotide in one B2M allele and a deletion of two nucleotides in the other allele.
  • clones 03 and 06 were derived by electroporating human iPSC with RNP targeting CIITA, followed by FACS sorting for single cells.
  • the edit-2 clones were confirmed to be CIITA KO by sanger sequencing.
  • clone 03 was sequenced to have an insertion of one nucleotide in both CIITA alleles
  • clone 06 was sequenced to have an insertion of one nucleotide in one CIITA allele and a deletion of sixteen nucleotides in the other allele.
  • Edit-3 clones clones 04, 20, 25, 48, and 16, were derived by electroporating hiPSC with two RNPs, targeting B2M and CIITA, respectively.
  • the Edit-3 clones were confirmed to be B2M knock-out by FACS analysis for MHC-I (see FIG. 14D) and sanger sequencing was used to confirm the knock-out of B2M and CIITA at the genomic level (see FIG. 14E) .
  • Edit-4 clones clones 02 and 30, were derived by electroporating hiPSC with a construct overexpressing PD-L1, PD-L2, TGF- ⁇ , HLA-E, HLA-G, CD47, IL-10, CCL-21, CD46, CD55, CD59 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS sorting for B2M - ; PDL1 + ; CD47 + single cells.
  • the Edit-4 clones were confirmed to be B2M-by FACS analysis for MHC-I, and PDL1+, PD2+, CD47+, CD46+, CD55+ and CD59+ (see FIG. 14F) and sanger sequencing was used to confirm the knock-out of B2M and CIITA at the genomic level. For CIITA, only 1 allele was confirmed to be knock-out (see FIG. 14G) .
  • Edit-5 clones clones 01, 02, and 26, were derived by electroporating hiPSC with a construct overexpressing of PD-L1, HLA-E, CD47, IL-10, CCL-21 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS sorting for B2M-; PDL1+; CD47+ single cells.
  • the Edit-5 clones were confirmed to be B2M-by FACS analysis for MHC-I, and PDL1+, CD47+ (see FIG. 14H) and sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level (see FIG. 14I) .
  • Edit-6 clones clones 08, 13, 15, and 31, were derived by electroporating hiPSC with a construtct overexpressing PD-L1, HLA-E, CD47, CD46, CD55, CD59 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS sorting for B2M - ; PDL1+; CD47+ single cells.
  • the Edit-6 clones were confirmed to be B2M - by FACS analysis for MHC-I, and PDL1+, CD47+, CD46+, CD55+, CD59+ (see FIG. 14J) and sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level (see FIG. 14K) .
  • Edit-7 clones clones 32, 33, 39, and 42, were derived by electroporating hiPSC with a construct overexpressing PD-L1, HLA-E, CD47, CCL-21, CD55 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS sorting for B2M - ; PDL1+; CD47+ single cells.
  • the Edit-7 clones were confirmed to be B2M - by FACS analysis for MHC-I, and PDL1+, CD47+, HLA-E+, CD55+ (see FIG. 14L) and sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level (see FIG. 14M) .
  • Edit-8 clones clones 15, 36, 40, and 42, were derived by electroporating hiPSC with a construct overexpressing of CD47 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS sorting for B2M - ; CD47+ single cells.
  • the Edit-8 clones were confirmed to be B2M-by FACS analysis for MHC-I, and CD47+ (see FIG. 14N) and Sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level (see FIG. 14O) .
  • clones 03, 10, 22, 27, 34, 36, 37, and 63 were derived by electroporating hiPSC with a construct overexpressing PD-L1, PD-L2, TGF- ⁇ , HLA-E, HLA-G, CD47, IL-10, CCL-21, CD46, CD55, CD59, two RNPs, targeting B2M and CIITA, and 1 Base Editor plasmid overexpressing sgRNA targeting MICA, MICB and ULBP1, followed by FACS sorting for B2M - ; PDL1+; CD47+ single cells.
  • the Edit-9 clones were confirmed to be B2M - by FACS analysis for MHC-I, and CD47+, B2M+, HLA-E+, PDL1+, CD55+ , CD46+ and CD59+ (see FIG. 14P) and sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level.
  • the KO of MICA/MICB/ULBP1 were confirmed using next generation sequencing.
  • the symbol ⁇ represents the gene was knocked out successfully (see FIG. 14Q) .
  • the enhanced resistance to antibody-mediated complement cytotoxicity may be attributed to having enhanced or introduced expression of one or more of the following immune regulator polypeptides: PD-L2, TGF-beta, CD46, CD55, CD59, and HLA-G (e.g., at least one or more of PD-L2, TGF-beta, CD46, CD55, and CD59) .
  • cells comprising Edit-9 may also exhibit enhanced resistance to antibody-mediated complement cytotoxicity, as compared to cells with either Edit-5 or Edit-8.
  • iPSCs with Edit-4 or Edit-9 can exhibit enhance resistance to antibody-mediated complement cytotoxicity in vitro or in vivo, as compared to that of iPSCs with either Edit-5 or Edit-9.
  • differentiated cells e.g., endothelial cells, immune cells, etc.
  • differentiated cells derived from iPSCs comprising Edit-4 or Edit-9 can exhibit enhance resistance to antibody-mediated complement cytotoxicity in vitro or in vivo, as compared to that of comparably differentiated cells derived from iPSCs with either Edit-5 or Edit-9.
  • immune cells derived from iPSCs comprising Edit-4 or Edit-9 can exhibit enhance resistance to antibody-mediated complement cytotoxicity in vitro or in vivo, as compared to that of immune cells derived from iPSCs with either Edit-5 or Edit-9.
  • NK cells derived from iPSCs comprising Edit-4 or Edit-9 can exhibit enhance resistance to antibody-mediated complement cytotoxicity in vitro or in vivo, as compared to that of NK cells derived from iPSCs with either Edit-5 or Edit-9.
  • FIG. 16F shows single time killing against K562.
  • FIG. 16G shows serial killing against K562.
  • K562-EGFP cells were monitored by the imaging with a frequency of every 3h.
  • K562 was added daily for 6 days.
  • K562-GFP only sample was set as a control.
  • FIGs. 16F and 16G indicate that B2M/CIITA double Knock-Out eNK did not show any hyporesponsive phenotype against K562 cells, and most eNK with transgenes had comparable cytotoxicity to WT eNK, when tested against K562 cells. Without wishing to be bound by theory, the hyporesonsivitiy or cytotoxicity against other target cells can be different.
  • corresponding eNK was co-cultured with CFSE-labeled PBMC.
  • PBS was used a negative control while PHA as a positive control.
  • the co-cultured cells were stained for CD3, CD4 and CD8.
  • CD3 + CD8 + CFSE low were regarded as proliferating CD8 + T cells.
  • PBMC from different donors were tested in FIGs. 16H-16M. The data show that B2M/CIITA double Knock-Out NK with or without transgenes did not stimulate CD8+ T cell proliferation.
  • corresponding eNK was co-cultured with CFSE-labeled PBMC.
  • PBS was used a negative control while PHA as a positive control.
  • the co-cultured cells were stained for CD3, CD4 and CD8.
  • CD3 + CD4 + CFSE low were regarded as proliferating CD4 + T cells.
  • PBMC from different donors were tested in FIGs. 16N-16S. The data show that B2M/CIITA double Knock-Out NK with or without transgenes did not stimulate CD4+ T cell proliferation.
  • Example 6 Engineered NK cells with enhanced function in tumor microenvironment (TME)
  • cells of interest can be engineered to carry (i) one or more enhanced or introduced genes (e.g., introduced transgenes) and/or (ii) one or more reduced expression level of endogenous genes (e.g., loss-of-function of genes of interest) .
  • the reduced expression level of the endogenous genes can be induced by, e.g., CRISPP/Cas and one or more guide nucleic acid molecules, such as the non-limiting exemplary guide RNA sequences provided in TABLE 3.
  • the cells of interest can be stem cells, such as isolated stem cells (e.g., embryonic stem cells) or induced stem cells (e.g., iPSCs) .
  • the cells of interest can be immune cells (e.g., NK cells) .
  • immune cells e.g., NK cells
  • Such immune cells can be derived from the stem cells as disclosed herein.
  • immunes can be immune cell lines (e.g., NK cell lines) .
  • NK cells can be engineered to carry certain transgenes and/or loss-of-function of genes of interest, as shown in TABLE 11.
  • Exemplary optimized transgene sequences for KLRD1, CD96, CD244, CCR4, CCR9, CXCR6, CCR2, CXCR2, CX3CR1, KLRC2, TGFBR2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, MIR21, MIR181B1, MIR181A1, MIR144, and MIR150 are shown in SEQ ID NOs: 59-85, respectively.
  • Example 7 Anti-tumor activity of the engineered NK cells.
  • NOG mice were intravenously injected with 1x105 luciferase expressing Nalm6 cells at day -1 as shown in Figure 27A.
  • mice were given either intravenous injections of 1x107 CB-NK cells or QN-019a (anti-CD19 CAR) NK cells.
  • An additional negative control group was included that did not introduce any heterologous NK cells to the mice.
  • mice were given 1 ⁇ g per mouse (/mouse) of IL-15 once a day.
  • mice 100,000 IU/mouse of IL-2 every 2-3 days. Mice were imaged using In Vivo Imaging System (IVIS) weekly.
  • IVIS In Vivo Imaging System
  • Example 8 Treatment of autoimmune disease by using the engineered NK cell.
  • Example 9 Treatment of cancer by using the engineered NK cell alone or in combination with antibodyNOG mice were intravenously injected with 1x106 luciferase expressing Raji cells at day 0. On days 0, 3, and 6, mice were given intravenous injections of 1x107 QN-019a (anti-CD19 CAR) NK cells. An additional negative control group was included that did not introduce any heterologous NK cells to the mice. Mice were either left untreated or treated with QN-019a alone or in combination with 300ug Rituximab on day 1. Mice were imaged using In Vivo Imaging System (IVIS) weekly.
  • IVIS In Vivo Imaging System
  • compositions of matter including the engineered immune cells of the present disclosure may be utilized in the method section including methods of use and production disclosed herein, or vice versa.

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Abstract

Provided is a novel engineered immune cell (e.g., engineered natural killer (NK) cells)or a population thereof. The immune cells can be engineered to exhibit improved characteristics as compared to control cell (e.g., a non-engineered immune cell). Also provided is a method of preparing the cell and a use of the cell or a composition comprising the cell in the manufacture of a medicament for treating autoimmune diseases.

Description

A NOVEL IMMUNE CELL AND USE THEREOF FOR TREATING DISEASES FIELD OF INVENTION
The present invention relates to immunology, chimeric antigen receptors, and chimeric autoantigen receptors, specifically relates to a novel engineered immune cell (e.g., engineered natural killer (NK) cells) or a population thereof. The immune cells can be engineered to exhibit improved characteristics as compared to control cell (e.g., a non-engineered immune cell) . The present disclosure provides a method of preparing the cell as well as a method for treating autoimmune diseases by using the cell or a composition comprising the cell.
BACKGROUND
Cancer (e.g., neoplasm, tumor) is a leading cause of death worldwide, accounting for about 10 million deaths annually. Cancer continues to bring increasing health, economic, and emotional burden on individuals, families, communities, and countries. Increase understanding of cancer biology (e.g., specifically cancer immune biology) and genetic engineering has encouraged development of adoptive cell therapies (e.g., cellular immunotherapy) , with a goal to treat or control a number of different cancers.
Induced pluripotent stem cells (iPSCs) were originally developed by Japanese scientist Shinya Yamanaka in 2006 by transfering a combination of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) into adult somatic cells with a viral vector and obtaining a pluripotent stem cell similar to embryonic stem cells after a reprogramming process. Human induced pluripotent stem cells have the following advantages: no ethical issue is involved, sources of cells are easily acquired from adult cells (such as skin, blood, and the like) , the cells possess strong differentiation ability, are able to differentiate into different functional cells, can be infinitely expanded, are cost-effective, and have high cell consistency.
SUMMARY
The present disclosure provides a novel immune cell or a population thereof, a method of preparing the cell, use of the cell or a composition comprising the cell for the manufacture of a medicament for treating diseases. Some aspects of the present disclosure provides engineered immune cells (e.g., engineered natural killer (NK) cells) and methods and/or uses for treatment of diseases such as cancer by using the engineered immune cells or a composition or a kit comprising the engineered immune cells.
Specifically, the present disclosure provides the following technical solutions to solve the technical problems existing in the prior art:
1. A cell or a population thereof, wherein
a) the cell is (a) an induced pluripotent cell (iPSC) , a clonal iPSC, or an iPS cell line cell, or embryonic stem cell (ESC) ; (b) a derivative cell obtained from differentiating the cell of (a) ; or (c) an immune cell  derived from an animal body,
b) the cell comprises a component which comprising an ectodomain, a transmembrane domain, and an intracellular domain;
wherein the ectodomain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, and a full length or at least a portion of CD64 or its variant; and
wherein the transmembrane domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at least a portion of the native or modified transmembrane domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, CD25, CD122, CD132, CD127, CD218, CD360 and ICAM-1 polypeptide,
wherein the intracellular domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at least a portion of the native or modified intracellular domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, ICAM-1, CD25, CD122, CD132, CD127, CD218, CD360, and CD3ζ, and wherein the cell has enhanced or acquired ADCC (antibody-dependent cell-mediated cytotoxicity) or ADCP (antibody-dependent cell-mediated phagocytosis) in comparison to a control cell or population thereof without the component.
2. The cell or population thereof of item 1, wherein the component further comprises a full length or at least a portion of the native or modified costimulatory domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, and NKp80 polypeptide.
3. The cell or population thereof of item 1 or 2, wherein the cell is a hemopoietic cell, an NK cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, or the derivative thereof, preferably the NK cell is an iPSC NK cell, a PBNK cell, a CBNK cell, or an NK92 cell.
4. The cell or population thereof of any one of items 1-3, wherein:
when the ectodomain is a full length or at least a portion of CD16a or its variant, the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant; or
when the ectodomain is a full length or at least a portion of CD32a or its variant, the transmembrane  domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant; or when the ectodomain is a full length or at least a portion of CD64 or its variant, the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant.
5. The cell or population thereof of one of items 1-4, wherein:
when the ectodomain is CD16a with F176V or S197P, the transmembrane domain and the intracellular domain are selected from the group consisting of CD32a, CD64, and CD89; or
when the ectodomain is CD32a or CD32a with H131 variant, the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89; or
when the ectodomain is CD64, the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89.
6. The cell or population thereof of any one of items 1-5, wherein the component comprises or consists of a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of SEQ ID NOs: 1-10.
7. The cell or population thereof of one of items 1-6, wherein the cell further comprises at least one feature selected from the group consisting of:
(i) a chimeric antigen receptor (CAR) or a T cell receptor (TCR) , or the nucleotide coding sequence thereof;
(ii) a persistence component, or the nucleotide coding sequence thereof;
(iii) a hypo-immunity regulator, or the nucleotide coding sequence thereof;
(iv) an immune activity component, or the nucleotide coding sequence thereof;
(v) a safety switch component, or the nucleotide coding sequence thereof;
(vi) a gene modification to enhance or reduce the expression of one or more endogenous or heterologous genes; and/or
(vii) a tumor microenvironment component, or the nucleotide coding sequence thereof.
8. The cell or population thereof of item 7, wherein the CAR or the TCR specifically recognizes a tumor antigen selected from the group consisting of Adhesion G protein-coupled receptor E2 (ADGRE2) , Armadillo repeat-containing X-linked protein 3 (ARMCX3) , Carbonic Anhydrase IX (CA1X) , CCRI, CCR4, Carcinoembryonic Antigen (CEA) , CD3ζ, CD5, CD7, CD8, CD9, CD10, CD19, CD20, CD22, CD25, CD26, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD52, CD56, CD70,  CD74, CD82, CD99, CD123, CD133, CD138, CD200, CD269 (BCMA) , CD S, CLEC12A, Collectin Liver 1 (CLL1) , an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen) , Decoy Receptor 2 (DCR2) , Density Enhanced Phosphatase 1 (DEP1) , Dipeptidyl peptidase-4 (DPP4) , Dopamine Receptor D2 (DRD2) , ERM-binding phosphoprotein-50 (EBP50) , epithelial glycoprotein2 (EGP 2) , epithelial glycoprotein-40 (EGP-40) , epithelial cell adhesion molecule (EpCAM) , EGFRvIII, receptor tyrosine-protein kinases erb-B2, 3, 4, EGFIR, EGFRVIII, ERBB folate-binding protein (FBP) , fetal acetylcholine receptor (AChR) , folate receptor-a, Ganglioside G2 (GD2) , Ganglioside G3 (GD3) , gp100, gp120, gp160, G-protein coupled receptor 56 (GPR56) , human Epidermal Growth Factor Receptor 2 (HER-2) , human telomerase reverse transcriptase (hTERT) , ICAM-1, Integrin B7, interleukin 6 receptor (IL6R) , Interleukin-13 receptor subunit alpha-2 (IL-13Rα2) , κ-light chain, kinase insert domain receptor (KDR) , Kappa, Lewis A (CA19.9) , LanC-like protein 1 (LANCL1) , Lewis Y (LeY) , L1 cell adhesion molecule (L1-CAM) , LILRB2, MART-1, melanoma antigen family A 1 (MAGE-A1) , MICA/B, Mucin 1 (Muc-1) , Mucin 16 (Muc-16) , Mesothelin (MSLN) , NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, NY-ESO-2, oncofetal antigen (h5T4) , PRAIVIE, prostate stem cell antigen (PSCA) , PRAME, prostate-specific membrane antigen (PSMA) , Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) , Syntaxin 4 (STX4) , Tumor-Associated Glycoprotein 72 (TAG-72) , TIM-3, TRBCI, TRBC2, Trophoblast Cell-Surface Antigen 2 (Trop-2) , Urokinase Receptor (uPAR) , Vesicle Associated Membrane Protein 3 (VAMP3) , vascular endothelial growth factor R2 (VEGF-R2) , Wilms tumor protein (WT-1) , and pathogen antigen derived from a virus, bacteria, fungi, parasite and protozoa capable of causing diseases.
9. The cell or population thereof of item 8, wherein the pathogen antigen is derived from HIV, HBV, EBV, HPV, Lasse Virus, Influenza Virus, or Coronavirus.
10. The cell or population thereof of any one of items 7-9, wherein the CAR comprises at least one of the following domains:
a) a full length or at least a portion of the native or modified transmembrane domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, and ICAM-1 polypeptide;
b) a full length or at least a portion of the native or modified costimulatory domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, and NKp80 polypeptide; and
c) a full length or at least a portion of the native or modified signaling domain selected from CD3ζ, 2B4, 4-1BB, DAP10, DAP12, NKG2D, NKp30, NKp44, NKp46, DNAM1, and NKp80 polypeptide.
11. The cell or population thereof of item 10, wherein the CAR specifically binds to CD33 antigen and comprises at least one of a transmembrane domain of CD8, a costimulatory domain of 2B4, and a signaling domain of CD3ζ.
12. The cell or population thereof of item 10, wherein the transmembrane domain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 11-13, the costimulatory domain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 14-16, and/or the signaling domain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to SEQ ID NO: 17.
13. The cell or population thereof of any one of items 7-12, wherein
the CAR comprises or consists of (i) an scFv having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 18 and 20-21 or (ii) an scFv encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NO: 22.
14. The cell or population thereof of any one of items 7-13, wherein the CAR further comprises a hinge domain having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 23-24.
15. The cell or population thereof of any one of items 7-14, wherein the CAR comprises or consists of (i) a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 25-29; or (ii) an amino acid encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NO: 30.
16. The cell or population thereof of any one of items 7-15, wherein the persistence component is selected from the group consisting of:
a) a full length or at least a portion of IL15, preferably in secretion form;
b) a full length or at least a portion of IL15 fused with a full length or at least a portion of IL15Ra;
c) a transgene for p-STAT5 enhancement;
d) a modification to an endogenous gene for p-STAT5 enhancement, preferably a disruption of an endogenous gene;
e) a reduced expression of one or more genes selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, CD70; and
f) an enhanced expression of one or more genes selected from the group consisting of CD25, CD122, and NKG2C.
17. The cell or population thereof of any one of items 7-16, wherein the persistence component comprises or consists of (i) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting SEQ ID NOs: 31-37; (ii) an amino acid sequence encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 38-39.
18. The cell or population thereof of any one of items 7-17, wherein the hypo-immunity regulator comprises a reduced expression of one or more genes selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and a NKG2DL (e.g., ULBP1) , and/or an enhanced expression of one or more genes selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, CD59, CD3, CD4, CD80, 41BBL, and CD131, preferably selected from the group consisting of PD-L2, TGF-beta, CD46, CD55, and CD59, preferably the hypo-immunity regulator comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 57-58.
19. The cell or population thereof of any one of items 7-18, wherein the immune activity component comprises a reduced expression of one or more genes selected from the group consisting of TGFb receptor, TIGIT, PD1, PDL1, SIGLEC9, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, CD96, LAG3, and 2B4, and/or an enhanced expression of one or more genes selected from the group consisting of NCR, NKp30, NKp44, NKp46, NKG2D, NKp80, DNAM1, other NK activating receptors, chemokines or chemikine receptors.
20. The cell or population thereof of any one of items 7-19, wherein the safety switch component 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.
21. The cell or population thereof of any one of items 7-20, wherein the gene modification comprises a reduced or disrupted expression of FcRγ.
22. The cell or population thereof of any one of items 7-21, wherein the tumor microenvironment component comprises a reduced or an enhanced expression of one or more genes selected from the group consisting of KLRD1, CD96, CD244, CCR4, CCR9, CXCR6, CCR2, CXCR2, CX3CR1, KLRC2, TGFBR2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, MIR21, MIR181B1, MIR181A1 MIR144, and MIR150.
23. A method of preparing the cell or population thereof of any one of items 1-20, comprising:
a) providing a cell which is (a) an induced pluripotent cell (iPSC) , a clonal iPSC, or an iPS cell line cell, or embryonic stem cell (ESC) ; (b) a derivative cell obtained from differentiating the cell of (a) ; or (c) an immune cell derived from an animal body;
b) introducing the component as defined in any one of items 1-6 into the cell provided by step a) ; and
c) optionally introducing the at least one of the feature as defined in any one of items 7-22 into the cell provided by step b) .
24. A composition or a kit comprising the cell or population thereof of any one of items 1-22.
25. The composition or the kit of item 24, further comprising an antibody selected from the group consisting of: 20- (74) - (74) (milatuzumab; veltuzumab) , 20-2b-2b, 3F8, 74- (20) - (20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, zlintuzumab) , actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003/SIM-BD0801 (sevacizumab) , APX005M, arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also referred to as tocilizumab) , atorolimumab, Avelumab, B-701, bapineuzumab, basiliximab, bavituximab, BAY1129980, BAY1187982, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, Betalutin (177Lu-tetraxetan-tetulomab) , bevacizumab, BEVZ92 (bevacizumab biosimilar) , bezlotoxumab, BGB-A317, BHQ880, BI 836880, BI-505, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab) , CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, cidfusituzumab, cidtuzumab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, Cotara (iodine I-131 derlotuximab biotin) , cR6261, crenezumab, DA-3111 (trastuzumab biosimilar) , dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, Daratumumab Enhanze (daratumumab) , Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, Depatuxizumab, Depatuxizumab mafodotin, derlotuximab biotin, detumomab, DI-B4, dinutuximab, diridavumab, DKN-01, DMOT4039A, dorlimomab aritox, drozitumab, DS-1123,  DS-8895, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, Etanercept, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, femzumab, fezakinumab, FF-21101, FGFR2 AntibodyDrug Conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, FPA144, fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, Gerilimzumab, gevokizumab, girentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hu14.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (claudiximab) , imalumab, IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan) , IMMU-114, ImmuTune IMP701 Antagonist Antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, Ipafricept, IPH4102, ipilimumab, iratumumab, isatuximab, Istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2/L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L-DOS47, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (inebilizumab) , MEDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006/S80880, MGD007, MGD009, MGD011, milatuzumab, Milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, nolovizumab, NOV-10, numavizumab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, OX002/MEN1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, PankoMabGEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pecfusituzumab, pectuzumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab) ,  PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, quilizumab, racotumomab, radretumab, rafivirumab, ralivizumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/SAR439684, reslivizumab, reslizumab, resyvizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, RO7009789, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-CD19A, SGN-CD19B, SGNCD33A, SGN-CD70A, SGN-LIV1A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, SYD985, SYM004 (futuximab and modotuximab) , Sym015, TAB08, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, Tanibirumab, taplitumomab paptox, tarextumab, TB-403, tefibazumab, Teleukin, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, TG-1303, TGN1412, Thorium-227-Epratuzumab Conjugate, ticilimumab, tigatuzumab, tildrakizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH 011, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tucusituzumab, tuvirumab, U3-1565, U3-1784, ublituximab, ulocuplumab, umavizumab, urelumab, urtoxazumab, ustekinumab, Vadastuximab Talirine, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB-101, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.
26. A use of the cell or population thereof of any one of items 1-22 or the composition or the kit of item 24 or 25 in the manufacture of a medicament for treating a tumor, an infection disease, or an autoimmune disease.
27. The use of item 26, wherein the tumor is selected from the group consisting of: Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer,  Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma,  Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, Tcell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms'tumor.
28. The use of item 26, wherein the infection disease is selected from the group consisting of: a viral infection, a bacterial infection, and a parasitic infection.
29. The use of item 26, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE) , myasthenia gravis (MG) , multiple sclerosis, scleroderma, Addison's Disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS) , Waldenstrom's macroglobulinemia (WM) , chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) , Hashimoto's Encephalopathy (HE) , Hashimoto's Thyroiditis, Graves’ Disease, Wegener’s Granulomatosis, and antibody-mediated transplantation rejection (e.g., for tissue transplants such as renaltransplant) .
30. A use of the cell or population thereof of any one of items 1-22 or the composition or the kit of item 24 or 25 in the manufacture of a medicament for anti-aging.
INCORPORATION BY REFERENCE
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG. ” herein) , of which:
Figure 1 shows a diagram of chimeric Fc receptor designs;
Figure 2 shows mRNA level of chimeric receptor genes in transduced NK-92 cells evaluated by qPCR;
Figure 3A-3C show surface expression of chimeric receptors in transduced NK-92 cells evaluated by FACS;
Figure 4 shows ADCC of trastuzumab mediated by FcR-engineered NK92 cells against SKOV3 at the effector to target ratio of 1: 1 following incubation for 5 hours;
Figures 5A-5C show FcR5 (CD16/32) is superior to hnCD16 and CD64/16 to mediate ADCC;
Figure 6 shows that FcR-engineered NK92 cells were stimulated as indicated for 4 hours, and percentage (A) and MFI (B) of each FcR was determined by flow cytometry;
Figure 7A-7B show surface expression of chimeric receptors in engineered iPSC clones evaluated by FACS;
Figure 8 shows that the expression of TRA-181, a human pluripotent stem cell marker, was evaluated by FACS;
Figure 9 shows that iPSC expressing chimeric Fc receptors were differentiated into CD56+ NK cells;
Figures 10A-10B show that FcR-expressing iNK cells were stimulated as indicated for 4 hours, and percentage (A) and MFI (B) of each FcR was determined by flow cytometry;
Figure 11 shows that ADCC mediated by FcR-engineered iPSC-NK cells with different dose of Rituximab against Raji cells at the effector to target ratio of 1: 1 following incubation for 5 hours;
Figure 12 shows serial killing assay to examine the ADCC of engineered iPSC-NK cells with rituximab;
Figure 13A is a schematic of antibody-based CAR structure design of the present invention. ScFv stands for single chain variable fragment; TM stands for transmembrane domain; CSD stands for co-stimulatory domain; and SD stands for signaling domain. Figures 13B-13C shows targeted cytotoxicity of NK cells comprising one of the different chimeric receptor polypeptides, against CD19-presenting target cells. Figure 13D is a schematic of the binding mechanism of a T cell receptor (TCR) -based CAR of the present invention. TCR-like ScFv specifically recognizes peptide-major histocompatibility complex (pMHC) on the antigen presenting cells (APCs) . Figure 13E is a schematic of the binding mechanism of a pMHC-based CAR of the present invention. PMHC specifically recognizes TCR on the self-reactive T cells.
Figures 14B-14C illustrate engineered NK cells comprising heterologous human IL-15 as a persistency component;
Figure 15 illustrates the surface expression of IL-15 in iNK cells differentiated from hIL15-IL15Ra fused-1 iPSC clones detected by FACS with APC (allophycocyanin) conjugated anti-IL-15 antibody. PW15, PW18, and PW23 are clones expressing membrane-bound IL-15;
Figure 16 illustrates an in-vitro growth curve of eNK cells differentiated from mbIL-15-expressing iPSC clones (KB-15) cultured with or without IL-2;
Figures 17A-17E illustrate expression of CD56+, NKG2A+, NKp30+, NKp44+, and NKp46+ among WT iNK and iNK differentiated from different mbIL-15-iPSC clones. (FIG. 17A) the percentage of CD56+ cells in the total differentiated cells. (FIG. 17B) the percentage of NKG2A+ in the CD56+population. (FIG. 17C) the percentage of NKp30+ in the CD56+ population. (FIG. 17D) the percentage of NKp44+ in the CD56+ population. (FIG. 17E) the percentage of NKp46+ cells in the CD56+population;
FIG. 18A-18C illustrate properties of NK cells differentiated from iPSC clones expressing secreted IL-15.(FIG. 18A) the percentage of CD56+ cells in the total differentiated cells among WT iNK cells and iNK differentiated from different sIL15-iPSC clones. (FIG. 18B) the concentration of IL-15 in culture medium from WT iNK cells and iNK cells differentiated from iPSC clones expressing secreted IL-15 (KA08) . (FIG. 18C) an in-vitro growth curve of WT eNK cells and eNK cells differentiated from  secretory IL-15-expressing iPSC clones (OQ-20) , in absence of an exogenous cytokine;
Figures 19A-19C illustrate a screen of engineered NK cells for persistency. (FIG. 19A) method design for in-vitro screening of the NK persistency related genes comparing culturing with low or high cytokine. (FIG. 19B) the percentage of indel in FCER1G deficient editing when cultured with low or high cytokine. (FIG. 19C) the percentage of indel in PTPN2 deficient editing when cultured with low or high cytokine; Figures 20A-20G illustrate a screen of engineered NK cells for persistency. (FIG. 20A) the method design for in-vivo screening of the NK persistency related genes comparing in vitro culture and in-vivo growth. (FIG. 20B) the percentage of indel in STAT3 deficient editing in mouse liver versus being cultured with high cytokine. (FIG. 20C) the percentage of indel in STAT3 deficient editing in mouse spleen versus being cultured with high cytokine. (FIG. 20D) the percentage of indel in STAT3 deficient editing in mouse bone marrow (BM) versus being cultured with high cytokine. (FIG. 20E) the percentage of indel in PTPN2 deficient editing in mouse liver versus being cultured with high cytokine. (FIG. 20F) the percentage of indel in PTPN2 deficient editing in mouse spleen versus being cultured with high cytokine. (FIG. 20G) the percentage of indel in PTPN2 deficient editing in mouse bone marrow (BM) versus being cultured with high cytokine;
Figure 21 illustrates a testing scheme for introducing a hypoimmunity regulator via editing and differentiating iPSC;
Figures 22A-22Q illustrate confirmed establishment of edit-1 clones to edit-9 clones. FIG. 22A illustrates FACS analysis of edit-1 clones (hiPSC electroporated with pre-mixed ribonucleoprotein [RNP] targeting B2M) . FIG. 22B illustrates Sanger sequencing of edit-1 clones. FIG. 22C illustrates Sanger sequencing of edit-2 clones (hiPSC electroporated with RNP targeting CIITA) . FIG. 22D illustrates FACS analysis of edit-3 clones (hiPSC electroporated with two RNPs targeting B2M and CIITA) . FIG. 22E illustrates Sanger sequencing of edit-3 clones. FIG. 22F illustrates FACS analysis of edit-4 clones (hiPSC electroporated with a construct overexpressing PD-L1, PD-L2, TGF-β, HLA-E, HLA-G, CD47, IL-10, CCL-21, CD46, CD55, CD59 and two RNPs, targeting B2M and CIITA) . FIG. 22G illustrates Sanger sequencing of edit-4 clones. FIG. 22H illustrates FACS analysis of edit-5 clones (hiPSC electroporated with a construct overexpressing of PD-L1, HLA-E, CD47, IL-10, CCL-21 and two RNPs, targeting B2M and CIITA) . FIG. 22I illustrates Sanger sequencing of edit-5 clones. FIG. 22J illustrates FACS analysis of edit-6 clones (hiPSC electroporated with a construct overexpressing PD-L1, HLA-E, CD47, CD46, CD55, CD59 and two RNPs, targeting B2M and CIITA) . FIG. 22K illustrates Sanger sequencing of edit-6 clones. FIG. 22L illustrates FACS analysis of edit-7 clones (hiPSC electroporated with a construct overexpressing PD-L1, HLA-E, CD47, CCL-21, CD55 and two RNPs, targeting B2M and CIITA) . FIG. 22M illustrates Sanger sequencing of edit-7 clones. FIG. 22N illustrates FACS analysis of edit-8 clones (hiPSC electroporated with a construct overexpressing of CD47 and two RNPs, targeting B2M and CIITA) . FIG. 22O illustrates Sanger sequencing of edit-8 clones. FIG. 22P illustrates FACS analysis of edit-9 clones (hiPSC electroporated with a construct overexpressing PD-L1, PD-L2, TGF-β, HLA-E, HLA-G, CD47, IL-10, CCL-21, CD46,  CD55, CD59, two RNPs, targeting B2M and CIITA) ; FIG. 22Q illustrates Sanger sequencing of edit-9 clones;
Figure 23 is a summary of the gene edits from edit-1 through edit-9;
Figures 24A-24S illustrate functional properties of edit-1 clones to edit-9 clones. (FIG. 24A) cell lysis when different edited iPSC clones co-incubating with human complement. (FIG. 24B) cell lysis when different edited iPSC clones co-incubating with cord blood-derived natural killer (CBNK) . (FIG. 24C) cell counts of CD56+ cells among different iNK differentiated from corresponding edited iPSC. The iPSC clone number was shown in the parenthesis. (FIG. 24D) CD56+ percentage among different iNK differentiated from corresponding edited iPSC. The iPSC clone number was shown in the parenthesis. (FIG. 24E) NKG2A+ percentage among different iNK differentiated from corresponding edited iPSC. The iPSC clone number was shown in the parenthesis. (FIG. 24F) NK cell-mediated lysis of K562 cells when co-cultured with corresponding eNKs at a single time point. (FIG. 24G) NK cell-mediated lysis of K562 cells when co-cultured with corresponding eNKs over 6 days. (FIG. 24H) the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with peripheral blood mononuclear cell (PBMC) from donor 1. (FIG. 24I) the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 2. (FIG. 24J) the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 3. (FIG. 24K) the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 4. (FIG. 24L) the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 5. (FIG. 24M) the percentage of proliferating CD8+ T cells when iNK with different edits are co-cultured with PBMC from donor 6. (FIG. 24N) the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 1. (FIG. 24O) the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 2. (FIG. 24P) the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 3. (FIG. 24Q) the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 4. (FIG. 24R) the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 5. (FIG. 24S) the percentage of proliferating CD4+ T cells when iNK with different edits are co-cultured with PBMC from donor 6;
Figures 25A-25C illustrate enhanced NK cell activity conferred by aCD19-CAR;
Figures 26A-26B illustrate enhanced IL-15 signaling conferred by IL-15-RF;
Figures 27A-27C illustrate anti-tumor activity of QN-019a (anti-CD19 CAR NK) cells in Nalm6 NOG tumor mouse model; and
Figure 28A-B illustrates that QN-019a NK cells effectively inhibited tumor growth in the mice and QN-019a NK cell+Rituximab combined treatment showed lower tumor fluorescence values than the QN-019a NK cell only group.
DETAILED DESCRIPTION
While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
1. Definition
As used in the specification and claims, the singular forms “a, ” “an, ” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “achimeric transmembrane receptor” includes a plurality of chimeric transmembrane receptors.
The term “about” or “approximately” generally mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1%of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.
The use of the alternative (e.g., “or” ) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and/or” should be understood to mean either one, or both of the alternatives.
The term “cell” generally refers to a biological cell. A cell can be the basic structural, functional and/or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g. cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses) , an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like) , seaweeds (e.g., kelp) , a fungal cell (e.g., a yeast cell, a cell from a mushroom) , an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc. ) , a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal) , a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc. ) , and etcetera. Sometimes a cell is not originating from a natural organism (e.g., a cell can be a synthetically made,  sometimes termed an artificial cell) .
The term “reprogramming, ” “dedifferentiation, ” “increasing cell potency, ” or “increasing developmental potency, ” as used interchangeable herein, generally refers to a method of increasing the potency of a cell or dedifferentiating the cell to a less differentiated state. For example, a cell that has an increased cell potency has more developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state. In other words, a reprogrammed cell is one that is in a less differentiated state than the same cell in a non-reprogrammed state.
The term “differentiation” generally refers to a process by which an unspecialized ( “uncommitted” ) or less specialized cell acquires the features of a specialized cell such as, e.g., an immune cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized ( “committed” ) position within the lineage of a cell. The term “committed” generally refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
The term “pluripotent” generally refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper) . For example, embryonic stem cells are a type of pluripotent stem cells that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm. Pluripotency can be a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell) , which is unable to give rise to a complete organism to the more primitive, more pluripotent cell, which is able to give rise to a complete organism (e.g., an embryonic stem cell) .
The term “induced pluripotent stem cells” (iPSCs) generally refers to stem cells that are derived from differentiated cells (e.g., differentiated adult, neonatal, or fetal cells) that have been induced or changed (i.e., reprogrammed) into cells capable of differentiating into tissues of all three germ or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells as they are found in nature. In some cases, iPSCs can be engineered to differentiation directly into committed cells (e.g., natural killer (NK) cells. In some cases, iPSCs can be engineered to differentiate first into tissue-specific stem cells (e.g., hematopoietic stem cells (HSCs) ) , which can be further induced to differentiate into committed cells (e.g., NK cells) .
The term “embryonic stem cell” (ESCs) generally refers to naturally occurring pluripotent stem cells of the inner cell mass of the embryonic blastocyst. Embryonic stem cells are pluripotent and give rise during development to all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In some cases, ESCs can be engineered to differentiation directly into committed cells (e.g., NK cells) . In some cases, ESCs can be engineered to differentiate first into tissue-specific stem cells (e.g., HSCs) , which can be further induced to differentiate into committed cells (e.g., NK cells) .
The term “isolated stem cells” generally refers to any type of stem cells disclosed herein (e.g., ESCs, HSCs, mesenchymal stem cells (MSCs) , etc. ) that are isolated from a multicellular organism. For  example, HSCs can be isolated from a mammal’s body, such as a human body. In another example, an embryonic stem cells can be isolated from an embryo.
The term “isolated” generally refers to a cell or a population of cells, which has been separated from its original environment. For example, a new environment of the isolated cells is substantially free of at least one component as found in the environment in which the “un-isolated” reference cells exist. An isolated cell can be a cell that is removed from some or all components as it is found in its natural environment, for example, isolated from a tissue or biopsy sample. The term also includes a cell that is removed from at least one, some or all components as the cell is found in non-naturally occurring environments, for example, isolated form a cell culture or cell suspension. Therefore, an isolated cell is partly or completely separated from at least one component, including other substances, cells or cell populations, as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments.
The term “hematopoietic stem and progenitor cells, ” “hematopoietic stem cells, ” , “hematopoietic progenitor cells, ” or “hematopoietic precursor cells, ” as used interchangeably herein, generally refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation (e.g., into NK cells) and include, multipotent hematopoietic stem cells (hematoblasts) , myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stem and progenitor cells (HSCs) are multipotent stem cells that give rise to all the blood cell types including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) , and lymphoid lineages (T cells, B cells, NK cells) . In some cases, HSCs can be CD34+ hematopoietic cells capable of giving rise to both mature myeloid and lymphoid cell types including T cells, NK cells and B cells.
The term “immune cell” generally refers to a differentiated hematopoietic cell. Non-limiting examples of an immune cell can include an NK cell, a T cell, a monocyte, an innate lymphocyte, a tumor-infiltrating lymphocyte, a macrophage, a granulocyte, etc.
The term “NK cell” or “Natural Killer cell” generally refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3) . In some cases, NK cells that are phenotypically CD3-and CD56+, expressing at least one of NKG2C and CD57 (e.g., NKG2C, CD57, or both in same or different degrees) , and optionally, CD16, but lack expression of one or more of the following: PLZF, SYK, FceRγ, and EAT-2. In some cases, isolated subpopulations of CD56+ NK cells can exhibit expression of CD16, NKG2C, CD57, NKG2D, NCR ligands, NKp30, NKp40, NKp46, activating and inhibitory KIRs, NKG2A and/or DNAM-1.
The term “nucleotide, ” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) ) . The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP) , uridine triphosphate (UTP) , cytosine triphosphate (CTP) , guanosine  triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide may be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides may include but are not limited fluorescein, 5-carboxyfluorescein (FAM) , 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE) , rhodamine, 6-carboxyrhodamine (R6G) , N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) , 6-carboxy-X-rhodamine (ROX) , 4- (4′dimethylaminophenylazo) benzoic acid (DABCYL) , Cascade Blue, Oregon Green, Texas Red, Cyanine and 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) . Specific examples of fluorescently labeled nucleotides can include [R6G] dUTP, [TAMRA] dUTP, [R110] dCTP, [R6G] dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110] ddCTP, [TAMRA] ddGTP, [ROX] ddTTP, [dR6G] ddATP, [dR110] ddCTP, [dTAMRA] ddGTP, and [dROX] ddTTP available from Perkin Elmer, Foster City, Calif. FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein-15-dATP, Fluorescein-12-dUTP, Tetramethyl-rodamine-6-dUTP, IR770-9-dATP, Fluorescein-12-ddUTP, Fluorescein-12-UTP, and Fluorescein-15-2′-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically-modified single nucleotide can be biotin-dNTP. Some non-limiting examples of biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP) , biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP) , and biotin-dUTP (e.g. biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP) .
The term “polynucleotide, ” “oligonucleotide, ” or “nucleic acid, ” as used interchangeably herein, generally refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell-free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three dimensional structure, and can  perform any function, known or unknown. A polynucleotide can comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase) . If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (e.g. rhodamine or flurescein linked to the sugar) , thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA (tRNA) , ribosomal RNA (rRNA) , short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA) , nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.
The term “gene” generally refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5′and 3′ends. In some uses, the term encompasses the transcribed sequences, including 5′and 3′untranslated regions (5′-UTR and 3′-UTR) , exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region” ) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can refer to an “exogenous gene” or a non-native gene. A non-native gene can refer to a gene not normally found in the host organism but which is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene not in its natural location in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence) .
The term “expression” generally refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product. ” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a  eukaryotic cell. “Up-regulated, ” with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression in a wild-type state. Expression of a transfected gene can occur transiently or stably in a cell. During “transient expression” the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell.
The term “peptide, ” “polypeptide, ” or “protein, ” as used interchangeably herein, generally refers to a polymer of at least two amino acid residues joined by peptide bond (s) . This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and/or tertiary structure (e.g., domains) . The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids, ” as used herein, generally refer to natural and non-natural amino acids, including, but not limited to, modified amino acids and amino acid analogues. Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. Amino acid analogues can refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids.
The term “derivative, ” “variant, ” or “fragment, ” as used herein with reference to a polypeptide, generally refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary) , activity (e.g., enzymatic activity) and/or function. Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions) , truncations, modifications, or combinations thereof compared to a wild type polypeptide.
The term “engineered, ” “chimeric, ” or “recombinant, ” as used herein with respect to a polypeptide molecule (e.g., a protein) , generally refers to a polypeptide molecule having a heterologous amino acid sequence or an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the polypeptide molecule, as well as cells or organisms which express the polypeptide molecule. The term “engineered” or “recombinant, ” as used herein with  respect to a polynucleotide molecule (e.g., a DNA or RNA molecule) , generally refers to a polynucleotide molecule having a heterologous nucleic acid sequence or an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In some cases, an engineered or recombinant polynucleotide (e.g., a genomic DNA sequence) can be modified or altered by a gene editing moiety.
The term “gene editing moiety” generally refers to a moiety which can edit a nucleic acid sequence, whether exogenous or endogenous to a cell comprising the nucleic acid sequence. In some embodiments, a gene editing moiety regulates expression of a gene by editing a nucleic acid sequence. In some cases, a gene editing moiety can regulate expression of a gene by editing genomic DNA sequence. In some cases, a gene editing moiety can regulate expression of a gene by editing an mRNA template. Editing a nucleic acid sequence can, in some cases, alter the underlying template for gene expression.
Alternatively or in addition to, a gene editing moiety can be capable of regulating expression or activity of a gene by specifically binding to a target sequence operatively coupled to the gene (or a target sequence within the gene) , and regulating the production of mRNA from DNA, such as chromosomal DNA or cDNA. In some cases, a gene editing moiety can recruit or comprise at least one transcription factor that binds to a specific DNA sequence, thereby controlling the rate of transcription of genetic information from DNA to mRNA. A gene editing moiety can itself bind to DNA and regulate transcription by physical obstruction, for example preventing proteins such as RNA polymerase and other associated proteins from assembling on a DNA template. A gene editing moiety can regulate expression of a gene at the translation level, for example, by regulating the production of protein from mRNA template. In some cases, a gene editing moiety can regulate gene expression by affecting the stability of an mRNA transcript.
The term “antibody” generally refers to a proteinaceous binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies) , as well as derivatives, variants, and fragments thereof. Antibodies include, but are not limited to, immunoglobulins (Ig's ) of different classes (i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2, etc. ) . A derivative, variant or fragment thereof can refer to a functional derivative or fragment which retains the binding specificity (e.g., complete and/or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab′, F (ab′) 2, variable fragment (Fv) , single chain variable fragment (scFv) , minibodies, diabodies, and single-domain antibodies ( “sdAb” or “nanobodies” or “camelids” ) . The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity-matured antibodies. Examples of antibodies that have been engineered include Fc optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and  multispecific antibodies (e.g., bispecific antibodies) .
The term “chimeric polypeptide receptor” generally refers to a non-natural polypeptide receptor comprising one or more antigen binding moieties, each antigen binding moiety capable of binding to a specific antigen. A chimeric polypeptide receptor can be monospecific (i.e., capable of binding to one type of specific antigen) . Alternatively, a chimeric polypeptide receptor can be multi-specific (i.e., capable of binding to two or more different types of specific antigens) . A chimeric polypeptide receptor can be monovalent (i.e., comprising a single antigen binding moiety) . Alternatively, a chimeric polypeptide receptor can be multivalent (i.e., comprising a plurality of antigen binding moieties) . In some cases, a chimeric polypeptide receptor can comprise a T-cell receptor (TCR) fusion protein (TFP) or a chimeric antigen receptor (CAR) .
The term “antigen binding domain” generally refers to a construct exhibiting preferential binding to a specific target antigen. An antigen binding domain can be a polypeptide construct, such as an antibody, 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.
The term “designed ankyrin repeat protein” or “DARPin” 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. In some examples, the ankyrin repeat motif comprises of two anti-parallel α-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.
In some cases, 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. In another example, an ankyrin repeat motif can comprise the following sequence: DxxGxTPLHLAxxxGxxxVVxLLLxxGADVNAx (SEQ ID NO: 93) , wherein “x” denotes any amino acid. In another example, an ankyrin repeat motif can comprise the following sequence: DxxGxTPLHLAxxxGxxx|VxVLLxxGADVNAx (SEQ ID NO:  94) , wherein “x” denotes any amino acid.
In some cases, 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) .
Examples and details of DARPin constructs are provided in, for example, International Patent Application No. PCT/EP2001/010454, which is entirely incorporated herein by reference.
The term “safety switch” generally refers to an engineered polypeptide construct designed to prevent potential toxicity or otherwise adverse effects of a cell therapy. When expressed in a cell, the safety switch can induce death of the host cell, thereby inactivating activity of the cell in a host (e.g., in a subject’s body) . Thus, the safety switch can be a suicide moiety. In some cases, the cell can be programmed to express the suicide moiety at certain stage of its life-cycle (e.g., time-programmed) . In some cases, expression of the suicide moiety in a cell can be conditional or inducible. In some examples, conditional regulation (e.g., expression) of a suicide moiety can include control through a small molecule-mediated post-translational activation and tissue-specific and/or temporal transcriptional regulation. Thus, the safety switch can be an inducible suicide moiety. A safety switch can mediate induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional genetic regulation, and/or antibody-mediated depletion. In some cases, a safety switch can be activated by an exogenous molecule (e.g., a drug or a prodrug) that, when activated, triggers apoptosis and/or cell death of a cell (e.g., engineered NK cell as disclosed herein) .
The term “immune regulator polypeptide” generally refers to a polypeptide construct (e.g., protein, antibody, membrane-bound polypeptide, secretory polypeptide, cleavable polypeptide, non-cleavable polypeptide, etc. ) capable of regulating or controlling one or more attributes of an immune cell, such as a NK cell. One or more attributes of an immune cell can comprise differentiation of the immune cell, immune cell morphology, expression of a polynucleotide or polypeptide construct within the immune cell, or activity of the immune cell (e.g., cytotoxic activity of an engineered NK cell against a diseased cell, such as a cancer cell) . An immune regulator polypeptide can be endogenous to a host cell. Alternatively or in addition to, an immune regulator polypeptide can be heterologous to a host cell. In some cases, controlling the one or more attributes of the immune cell can be mediated by downregulating expression of the immune regulator polypeptide (e.g., suppression, knock-down or knock-out) . Alternatively or in addition to, controlling the one or more attributes of the immune cell can be mediated by upregulating expression of the immune regulator polypeptide (e.g., upregulation of an endogenous gene or knock-in of a heterologous gene encoding the immune regulator polypeptide) . Yet in another alternative or additionally, controlling the one or more attributes of the immune cell can be mediated by maintaining expression of the immune regulator polypeptide for time period that is longer than a natural or normal expression profile of the immune regulator polypeptide in a host cell. In some cases, an immune regulator polypeptide can comprise a hypo-immunity regulator. In some cases, an immune regulator polypeptide can comprise an immune checkpoint inhibitor.
The term “hypo-immunity regulator” generally refers to a polypeptide construct in a cell, wherein either enhanced expression (e.g., via knock-in of a heterologous gene) or reduced expression (e.g., via knock-out or knock-down of an endogenous gene) of the hypo-immunity regulator in the cell can help the cell to reduce or avoid immune response (e.g., immune attack, such as adaptive immune rejection) from a host’s body upon administration to the host’s body. In some cases, cells (e.g., engineered NK cells as disclosed herein) can be modified to exhibit either enhanced expression or reduced expression of the hypo-immunity regulator, such that the cells can evade the host immune attack upon second or further infusion of the cells into the host (i.e., recipient) . As such, the cells (i) would not be rejected by the host’s immune system (e.g., antibody-mediated complement cytotoxicity, or antibody-dependent cellular cytotoxicity (ADCC) ) and/or (ii) would be rejected at a slower rate by the host’s immune system as compared with a control cell without the enhanced expression or reduced expression of the hypo-immunity regulator. A cell exhibiting the enhanced expression or reduced expression of the hypo-immunity regulator can be referred to as exhibiting “hypo-immunity” or being “immune-privileged. ” For example, enhanced hypo-immunity (e.g., enhanced resistance against ADCC) of a population of engineered immune cells (e.g., a population of engineered NK cells) as disclosed herein can be ascertained in vitro (e.g., in the presence of human serum or human complement and an antibody (e.g., SSEA-4 antibody) ) or in vivo (e.g., upon administration to a subject’s bloodstream) .
In some cases, engineering of an immune cell (e.g., NK cells) as disclosed herein can enhance the immune cell’s resistance against immune rejection (e.g., ADCC) by at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95%, at least or up to about 100%, at least or up to about 150%, at least or up to about 200%, at least or up to about 300%, at least or up to about 400%, or at least or up to about 500%.
In some cases, the enhanced resistance against immune rejection (e.g., ADCC) can be ascertained in vitro in a medium comprising at least or up to about 5%, at least or up to about 10%, at least or up to about 15%, at least or up to about 20%, at least or up to about 25%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, or at least or up to about 80%, human complement.
The term “immune checkpoint inhibitor” generally refers to a group of molecules presented on a cell surface of an immune cell (e.g., T cells, myeloid cells, NK cells, B cells, etc. ) that can modulate immune response of the cell by down-regulating or inhibiting the immune response of the immune cell against a target cell, such as a cancer cell (i.e., anti-cancer or anti-tumor immune response) . The target cell can express a receptor or a ligand of the immune checkpoint inhibitor presented on the surface of the immune cell, to engage with the immune checkpoint inhibitor and down-regulate or inhibit the immune response of the immune cells against the target cell. As such, down-regulating or inhibiting expression of the  immune checkpoint inhibitor in the immune cell can, in some cases, enhance or prolong the immune response of the immune cell against a target cell.
The term “immune response” generally refers to T cell mediated and/or B cell mediated immune responses from a host’s immune system to an object (e.g., a foreign object) . An example of an immune response include T cell responses, e.g., cytokine production and cellular cytotoxicity. IN some cases, an immune response can be indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, such as macrophages.
The term “enhanced expression, ” “increased expression, ” or “upregulated expression” generally refers to production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is above a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) . The normal level of expression can be substantially zero (or null) or higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. The moiety of interest can comprise a heterologous gene or polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced expression of the polypeptide of interest in the host strain.
The term “enhanced activity, ” “increased activity, ” or “upregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is above a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) . The normal level of activity can be substantially zero (or null) or higher than zero. The moiety of interest can comprise a polypeptide construct of the host strain. The moiety of interest can comprise a heterologous polypeptide construct that is introduced to or into the host strain. For example, a heterologous gene encoding a polypeptide of interest can be knocked-in (KI) to a genome of the host strain for enhanced activity of the polypeptide of interest in the host strain.
The term “reduced expression, ” “decreased expression, ” or “downregulated expression” generally refers to a production of a moiety of interest (e.g., a polynucleotide or a polypeptide) to a level that is below a normal level of expression of the moiety of interest in a host strain (e.g., a host cell) . The normal level of expression is higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced expression of the moiety of interest can include a complete inhibition of such expression in the host strain.
The term “reduced activity, ” “decreased activity, ” or “downregulated activity” generally refers to activity of a moiety of interest (e.g., a polynucleotide or a polypeptide) that is modified to a level that is below a normal level of activity of the moiety of interest in a host strain (e.g., a host cell) . The normal level of activity is higher than zero. The moiety of interest can comprise an endogenous gene or polypeptide construct of the host strain. In some cases, the moiety of interest can be knocked-out or knocked-down in the host strain. In some examples, reduced activity of the moiety of interest can include a complete inhibition of such activity in the host strain.
The term “subject, ” “individual, ” or “patient, ” as used interchangeably herein, generally refers to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
The term “treatment” or “treating” generally refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. For example, a treatment can comprise administering a system or cell population disclosed herein. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
The term “effective amount” or “therapeutically effective amount” generally refers to the quantity of a composition, for example a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and/or NK cells) comprising a system of the present disclosure, that is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” generally refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.
2. The immune cell of the present disclosure comprising ADCC component
The present disclosure provides a cell or a population thereof, wherein a) the cell is (a) an induced pluripotent cell (iPSC) , a clonal iPSC, or an iPS cell line cell, or embryonic stem cell (ESC) ; (b) a derivative cell obtained from differentiating the cell of (a) ; or (c) an immune cell derived from an animal body, b) the cell comprises a component which comprising an ectodomain, a transmembrane domain, and an intracellular domain. In some cases, the ectodomain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, and a full length or at least a portion of CD64 or its variant. In some cases, the transmembrane domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at least a portion of the native or modified transmembrane domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, CD25, CD122, CD132, CD127, CD218, CD360, and ICAM-1 polypeptide. In some cases, the intracellular domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at  least a portion of the native or modified intracellular domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, ICAM-1, CD25, CD122, CD132, CD127, CD218, CD360, and CD3ζ. In some cases, the cell has enhanced or acquired ADCC (antibody-dependent cell-mediated cytotoxicity) or ADCP (antibody-dependent cell-mediated phagocytosis) in comparison to a control cell or population thereof without the component. In some cases, the component further comprises a full length or at least a portion of the native or modified costimulatory domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, and NKp80 polypeptide.
In some cases, the cell is an iPSC, a clonal iPSC, or an iPS cell-line cell. In some cases, the cell is an ESC, preferably a human ESC or a non-human animal ESC. In some cases, the cell is a derivative cell obtained from differentiating an iPSC, a clonal iPSC, or an iPS cell-line cell. In some cases, the cell is a derivative cell obtained from differentiating an ESC. In some cases, the cell is an immune cell derived from an animal body, preferably a human or a non-human animal. In some cases, the cell is a hemopoietic cell, an NK cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, or the derivative thereof. In some cases, the NK cell is an iPSC NK cell, a PBNK cell, a CBNK cell, or an NK92 cell. In some cases, when the ectodomain is a full length or at least a portion of CD16a or its variant, the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant. In some cases, when the ectodomain is a full length or at least a portion of CD32a or its variant, the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant. In some cases, when the ectodomain is a full length or at least a portion of CD64 or its variant, the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant.
In some cases, when the ectodomain is CD16a with F176V or S197P, the transmembrane domain and the intracellular domain are selected from the group consisting of CD32a, CD64, and CD89. In some cases, when the ectodomain is CD32a or CD32a with H131 variant, the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89. In some cases, when the ectodomain is CD64, the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89.
In some cases, the component comprises or consists of a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of SEQ ID NOs: 1-7.
Enhanced CD16 signaling (e.g., constitutively activated signaling of CD16) of the engineered immune  cell (e.g., engineered NK cell) as disclosed herein can be achieved by having non-cleavable CD16 variant in the subject cell. CD16 (e.g., CD16a) is a transmembrane protein expressed by immune cells (e.g., NK cells) , which binds monomeric IgG attached to target cells to activate the immune cells and facilitate antibody-dependent cell-mediated cytotoxicity (ADCC) . In a non-engineered immune cell, the binding between CD16 and the monomeric IgG can induce cleavage of the CD16 protein at a cleavage site near the transmembrane domain, to regulate the cell surface density of CD16 upon immune cell activation. Thus, the endogenous CD16 of the engineered immune cell can be modified to enhance its signaling. Alternatively, an enhanced signaling variant of CD16 can be artificially introduced to the engineered immune cell.
In some cases, the engineered immune cell’s endogenous gene encoding CD16 can be genetically modified in its ectodomain (e.g., F176V) via action of a gene editing moiety as disclosed herein, such that the modified CD16 exhibits higher binding affinity to its target (e.g., monomeric IgG) as compared to a natural CD16. In some cases, a heterologous gene encoding such modified CD16 can be introduced to the cell.
In some cases, the engineered immune cell’s endogenous gene encoding CD16 can be genetically modified via action of a gene editing moiety as disclosed herein, such that the modified CD16 is non-cleavable and can induce enhanced CD16 signaling. In some examples, the cleavage site (e.g., position 195-198) in the membrane-proximal region (position 189-212) of CD16 can be modified or eliminated (e.g., CD16 S197P variant as a non-cleavable CD16 variant) . In some cases, a heterologous gene encoding such modified CD16 can be introduced to the cell.
In some cases, the engineered immune cell comprises an ADCC component, the ADCC component is selected from the group consisting of: a) a full length or at least a portion of a high affinity non-cleavable CD16 (hnCD16) ; b) a full length or at least a portion of CD16a; c) a full length or at least a portion of CD32a; d) a full length or at least a portion of CD89; e) a full length or at least a portion of CD64; f) at least a portion of CD16a fused with at least a portion of CD32a or CD89 or CD64; g) at least a portion of CD32a fused with at least a portion of CD89 or CD64; and h) at least a portion of CD64 fused with at least a portion of CD89.
In some cases, the ADCC component comprises or consists of a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of SEQ ID NOs: 8-10.
In some cases, a heterologous gene encoding a heterologous CD16 variant that (i) exhibits higher binding affinity to its target (e.g., monomeric IgG) and (ii) is non-cleavable can be introduced to the cell (i.e., hnCD16) . In some examples, the heterologous CD16 variant can be a modified CD16 comprising, for example, F176V and S197P, as disclosed herein. In some examples, the heterologous CD variant can be a fusion receptor protein comprising (i) at least a portion of CD16 with an inactivated cleavage site and (ii) an ectodomain of a different cell surface protein, such as a glycoprotein (e.g., CD64) , that exhibits enhanced binding to the target (e.g., monomeric IgG) as compared to an unmodified CD16.
A heterologous gene as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein. Alternatively, the heterologous gene may not and need not be integrated into the genome of the engineered cell.
The enhanced CD16 signaling of the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., SHP-1) via Western blotting or (ii) expression of a downstream gene (e.g., CD25, IFN-gamma, TNF, etc. ) via Western blotting or PCR techniques.
In some cases, the CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be greater than CD16 signaling of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, enhanced CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be characterized by an increase in phosphorylation of a downstream signaling protein by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.
In some cases, enhanced CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be characterized by an increased expression of a downstream gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at  least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.
In some cases, the CD16 signaling of the engineered immune cell (e.g., the engineered NK cell comprising hnCD16) of the present disclosure can be more prolonged (e.g., a longer duration of time of activated CD16 signaling) than CD16 signaling of a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
The engineered immune cell (e.g., the engineered NK cell) can exhibit enhanced signaling of an endogenous signaling pathway that involves the heterologous cytokine as compared to a control cell, as disclosed herein.
3. Chimeric antigen receptor (CAR) and T cell receptor (TCR)
In some cases, the cell furhter comprises at least one feature selected from the group consisting of: (i) a chimeric antigen receptor (CAR) or a T cell receptor (TCR) , or the nucleotide coding sequence thereof; (ii) a persistence component, or the nucleotide coding sequence thereof; (iii) a hypo-immunity regulator, or the nucleotide coding sequence thereof; (iv) an immune activity component, or the nucleotide coding sequence thereof; (v) a safety switch component, or the nucleotide coding sequence thereof; (vi) a gene modification to enhance or reduce the expression of one or more endogenous or heterologous genes; and/or (vii) a tumor microenvironment component or the nucleotide coding sequence thereof. In some cases, the cell further comprises at least two features selected from the group consisting of: (i) - (vii) . In some cases, the cell further comprises at least three features selected from the group consisting of: (i) -(vii) . In some cases, the cell further comprises at least four features selected from the group consisting of: (i) - (vii) . In some cases, the cell further comprises at least five features selected from the group  consisting of: (i) - (vii) . In some cases, the cell further comprises all six features selected from the group consisting of: (i) - (vii) . In some cases, the cell further comprises (i) and (ii) . In some cases, in addition to (i) - (ii) , the cell additionally comprises at least one, at least two, at least three, at least four, or all five features selected from the group consisting of (iii) - (vii) . In some cases, the cell further comprises a gene modification to enhance or reduce the expression of one or more endogenous or heterologous genes. In some cases, the cell further comprises a gene modification to enhance the expression of one or more endogenous or heterologous genes, preferably an endogenous gene. In some cases, the cell further comprises a gene modification to reduce the expression of one or more endogenous or heterologous genes, preferably an endogenous gene. In some cases, the cell further comprises a gene modification to disrupt the expression of one or more endogenous or heterologous genes, preferably an endogenous gene. In some cases, the gene modification comprises a reduced or disrupted expression of FcRγ.
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) or a T cell receptor (e.g., at least 1, 2, 3, 4, 5, or more different types of T cell receptors) . The engineered immune cell can be engineered to express a chimeric polypeptide receptor or a T cell receptor transiently or permanently. In some cases, a recombinant chimeric polypeptide receptor or a recombinant T cell receptor can be delivered to the engineered immune cell via, e.g., a liposome, and be incorporated into the engineered immune cell via membrane fusion. In some cases, a heterologous polynucleotide construct (e.g., DNA or RNA) encoding the chimeric polypeptide receptor or the T cell receptor can be delivered to the engineered immune cell. The heterologous polynucleotide construct (i.e., a gene) encoding the heterologous polynucleotide construct can be incorporated into the chromosome of the engineered immune cell (i.e., chromosomal gene) or, alternatively, may not or need not be integrated into the chromosome of the engineered immune cell as disclosed herein.
A chimeric polypeptide receptor can comprises a T cell receptor fusion protein (TFP) . The term “T cell receptor fusion protein” or “TFP” generally refers to a recombinant polypeptide construct comprising (i) one or more antigen binding moieties (e.g., monospecific or multispecific) , (ii) at least a portion of TCR extracellular domain, (iii) at least a portion of TCR transmembrane domain, and (iv) at least a portion of TCR intracellular domain.
In some cases, an endogenous T cell receptor (TCR) of the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be inactivated. In some examples, a function of the endogenous TCR of the engineered immune cell can be inhibited by an inhibitor. In some examples, a gene encoding a subunit of the endogenous TCR can be inactivated (e.g., edited via action of the gene editing moiety as disclosed herein) such that the endogenous TCR is inactivated. The gene encoding the subunit of endogenous TCR can be one or more of: TCRα, TCRβ, CD3ε, CD3δ, CD3γ, and CD3ζ. A chimeric polypeptide receptor can comprises a chimeric antigen receptor (CAR) . The term “chimeric antigen receptor” or “CAR” generally refers to a recombinant polypeptide construct  comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as “an intracellular or intrinsic signaling domain” ) comprising a functional signaling domain derived from a stimulatory molecule. In some cases, the stimulatory molecule may be the zeta chain associated with the T cell receptor complex. In some cases, the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule. In some cases, the costimulatory molecule may comprise 4-1BB (i.e., CD137) , CD27, and/or CD28. In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (e.g., a scFv, DARPin, etc. ) during cellular processing and localization of the CAR to the cellular membrane.
A CAR may be a first-, second-, third-, or fourth-generation CAR system, a functional variant thereof, or any combination thereof. First-generation CARs (e.g., CD19R or CD19CAR) include an antigen binding domain with specificity for a particular antigen (e.g., an antibody or antigen-binding fragment thereof such as 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 CD3ζ receptor or FcεRIγ) . Second-generation CARs modify the first-generation CAR by addition of a co-stimulatory domain to the intracellular signaling domain portion of the CAR (e.g., derived from co-stimulatory receptors that act alongside T-cell receptors such as CD28, CD137/4-1BB, and CD134/OX40) , which abrogates the need for administration of a co-factor (e.g., IL-2) alongside a first-generation CAR. Third-generation CARs add multiple co-stimulatory domains to the intracellular signaling domain portion of the CAR (e.g., CD3ζ-CD28-OX40, or CD3ζ-CD28-41BB) . Fourth-generation CARs modify second-or third-generation CARs by the addition of an activating cytokine (e.g., IL-12, IL-23, or IL-27) to the intracellular signaling portion of the CAR (e.g., between one or more of the costimulatory domains and the CD3ζ ITAM domain) or under the control of a CAR-induced promoter (e.g., the NFAT/IL-2 minimal promoter) . In some cases, a CAR may be a new generation CAR system that is different than the first-, second-, third-, or fourth-generation CAR system as disclosed herein.
In some cases, the engineered immune cell comprises a CAR or a TCR, the CAR or the TCR specifically recognize a tumor antigen selected from the group consisting of Adhesion G protein-coupled receptor E2 (ADGRE2) , Armadillo repeat-containing X-linked protein 3 (ARMCX3) , Carbonic Anhydrase IX (CA1X) , CCRI, CCR4, Carcinoembryonic Antigen (CEA) , CD3ζ, CD5, CD7, CD8, CD9, CD10, CD19, CD20, CD22, CD25, CD26, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD52, CD56, CD70, CD74, CD82, CD99, CD123, CD133, CD138, CD200, CD269 (BCMA) , CD S, CLEC12A, Collectin Liver 1 (CLL1) , an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell  surface antigen) , Decoy Receptor 2 (DCR2) , Density Enhanced Phosphatase 1 (DEP1) , Dipeptidyl peptidase-4 (DPP4) , Dopamine Receptor D2 (DRD2) , ERM-binding phosphoprotein-50 (EBP50) , epithelial glycoprotein2 (EGP 2) , epithelial glycoprotein-40 (EGP-40) , epithelial cell adhesion molecule (EpCAM) , EGFRvIII, receptor tyrosine-protein kinases erb-B2, 3, 4, EGFIR, EGFRVIII, ERBB folate-binding protein (FBP) , fetal acetylcholine receptor (AChR) , folate receptor-a, Ganglioside G2 (GD2) , Ganglioside G3 (GD3) , gp100, gp120, gp160, G-protein coupled receptor 56 (GPR56) , human Epidermal Growth Factor Receptor 2 (HER-2) , human telomerase reverse transcriptase (hTERT) , ICAM-1, Integrin B7, interleukin 6 receptor (IL6R) , Interleukin-13 receptor subunit alpha-2 (IL-13Rα2) , κ-light chain, kinase insert domain receptor (KDR) , Kappa, Lewis A (CA19.9) , LanC-like protein 1 (LANCL1) , Lewis Y (LeY) , L1 cell adhesion molecule (L1-CAM) , LILRB2, MART-1, melanoma antigen family A 1 (MAGE-A1) , MICA/B, Mucin 1 (Muc-1) , Mucin 16 (Muc-16) , Mesothelin (MSLN) , NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, NY-ESO-2, oncofetal antigen (h5T4) , PRAIVIE, prostate stem cell antigen (PSCA) , PRAME, prostate-specific membrane antigen (PSMA) , Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) , Syntaxin 4 (STX4) , Tumor-Associated Glycoprotein 72 (TAG-72) , TIM-3, TRBCI, TRBC2, Trophoblast Cell-Surface Antigen 2 (Trop-2) , Urokinase Receptor (uPAR) , Vesicle Associated Membrane Protein 3 (VAMP3) , vascular endothelial growth factor R2 (VEGF-R2) , Wilms tumor protein (WT-1) . In some cases, the CAR or the TCR specifically recognize a pathogen antigen derived from a virus, bacteria, fungi, parasite and protozoa capable of causing diseases. In some cases, the CAR or the TCR specifically recognize a pathogen antigen derived from HIV, HBV, EBV, HPV, Lasse Virus, Influenza Virus, or Coronavirus. In some cases, the engineered immune cell comprises a CAR, the CAR specifically recognize a tumor antigen of CD19 or CD33.
In some cases, the engineered immune cell comprises a CAR, wherein the CAR comprises at least one of the following domains: a) a transmembrane domain, such as a full length or at least a portion of the native or modified transmembrane domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, and ICAM-1 polypeptide; b) a costimulatory domain, such as a full length or at least a portion of the native or modified costimulatory domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, and NKp80 polypeptide; and c) a signaling domain, such as a full length or at least a portion of the native or modified signaling domain selected from CD3ζ, 2B4, 4-1BB, DAP10, DAP12, NKG2D, NKp30, NKp44, NKp46, DNAM1, and NKp80 polypeptide. In some cases, the engineered immune cell comprises a CAR, wherein the CAR comprises at least one of a transmembrane domain of CD8, a costimulatory domain of 2B4, and a signaling domain of CD3ζ. In some cases, the transmembrane domain of CAR has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 11-13, the costimulatory domain of CAR has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 14-16, and/or the signaling domain of CD3ζhas at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NO:  17.
In some cases, the CAR further comprises: (i) an anti-CD33 scFv having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 18 and 20-21, or (ii) an anti-CD33 scFv encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NO: 22.
In some cases, the CAR further comprises a CD8 hinge domain having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 23-24.
In some cases, the CAR comprises or consists of (i) a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NOs: 25-29, or (ii) a sequence encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to SEQ ID NO: 30.
A hinge domain (e.g., the linker between the extracellular antigen binding domain and the transmembrane domain) of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.
A transmembrane domain of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of the native or modified transmembrane region of CD3D, CD3E, CD3G, CD3c CD4, CD8, CD8a, CD8b, CD27, CD28, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, 2B4, BTLA, CD16, IL7, IL12, IL15, KIR2DL4, KIR2DS1, NKp30, NKp44, NKp46, NKG2C, NKG2D, or T cell receptor polypeptide.
The hinge domain and the transmembrane domain of a CAR as disclosed herein (e.g., for the engineered immune cell, such as the engineered NK cell) can be derived from the same protein (e.g., CD8) . Alternatively, the hinge domain and the transmembrane domain of the CAR as disclosed herein can be derived from different proteins.
A signaling domain of a CAR can comprise at least or up to about 1 signaling domain, at least or up to about 2 signaling domains, at least or up to about 3 signaling domains, at least or up to about 4 signaling domains, at least or up to about 5 signaling domains, at least or up to about 6 signaling domains, at least or up to about 7 signaling domains, at least or up to about 8 signaling domains, at least or up to about 9 signaling domains, or at least or up to about 10 signaling domains.
A signaling domain (e.g., a signaling peptide of the intracellular signaling domain) of a CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at least a portion of a polypeptide of CD3ζ, 2B4, DAP10, DAP12, DNAM1, CD137 (41BB) , IL21, IL7, IL12, IL15, NKp30, NKp44, NKp46, NKG2C, NKG2D, or any combination thereof.
Alternatively or in addition to (i.e., a co-stimulatory domain) , the signaling domain CAR in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can comprise a full length or at  least a portion of a polypeptide of CD27, CD28, 4-1BB, OX40, ICOS, PD-1, LAG-3, 2B4, BTLA, DAP10, DAP12, CTLA-4, or NKG2D, or any combination thereof.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein comprises the chimeric polypeptide receptor (e.g., CAR) that comprises at least CD8 transmembrane domain and one or more of: (i) 2B4 signaling domain and (ii) DAP10 signaling domain. In some cases, the engineered cell (e.g., the engineered NK cell) as disclosed herein comprises the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises at least (i) CD8 transmembrane domain, (ii) 2B4 signaling domain, and (iii) DAP10 signaling domain. The 2B4 signaling domain can be flanked by the CD8 transmembrane domain and the DAP10 signaling domain. Alternatively, the DAP10 signaling domain can be flanked by the CD8 transmembrane domain and the 2B4 signaling domain. In some cases, the chimeric polypeptide receptor as disclosed herein can further comprise yet an additional signaling domain derived from CD3ζ.
An antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor (e.g., TFP or CAR) as disclosed herein can be a cell surface marker, a secreted marker, or an intracellular marker.
Non-limiting examples of an antigen (i.e., a target antigen) of an antigen binding moiety of a chimeric polypeptide receptor (e.g., TFP or CAR) or TCR as disclosed herein can include ADGRE2, carbonic anhydrase IX (CA1X) , CCRI, CCR4, carcinoembryonic antigen (CEA) , CD3ζ, CD5, CD8, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD49f, CD56, CD70, CD74, CD99, CD123, CD133, CD138, CD269 (BCMA) , CD S, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen) , epithelial glycoprotein2 (EGP 2) , epithelial glycoprotein-40 (EGP-40) , epithelial cell adhesion molecule (EpCAM) , EGFRvIII, receptor tyrosine-protein kinases erb-B2,3, 4, EGFIR, EGFR-VIII, ERBB folate-binding protein (FBP) , fetal acetylcholine receptor (AChR) , folate receptor-a, Ganglioside G2 (GD2) , Ganglioside G3 (GD3) , gp100, human Epidermal Growth Factor Receptor 2 (HER-2) , human telomerase reverse transcriptase (hTERT) , ICAM-1, Integrin B7, Interleukin-13 receptor subunit alpha-2 (IL-13Rα2) , κ-light chain, kinase insert domain receptor (KDR) , Kappa, Lewis A (CA19.9) , Lewis Y (LeY) , L1 cell adhesion molecule (L1-CAM) , LILRB2, MART-1, melanoma antigen family A 1 (MAGE-A1) , MICA/B, Mucin 1 (Muc-1) , Mucin 16 (Muc-16) , Mesothelin (MSLN) , NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, NY-ESO-2, oncofetal antigen (h5T4) , PRAIVIE, prostate stem cell antigen (PSCA) , PRAME prostate-specific membrane antigen (PSMA) , ROR1, tumor-associated glycoprotein 72 (TAG-72) , TIM-3, TRBCI, TRBC2, vascular endothelial growth factor R2 (VEGF-R2) , Wilms tumor protein (WT-1) , and various pathogen antigen (e.g., pathogen antigens derived from a virus, bacteria, fungi, parasite and protozoa capable of causing diseases) . In some examples, a pathogen antigen is derived from HIV, HBV, EBV, HPV, Lasse Virus, Influenza Virus, or Coronavirus.
Additional examples of the antigen of the antigen binding moiety of the chimeric polypeptide receptor or TCR as disclosed herein can include 1-40-β-amyloid, 4-1BB, 5AC, 5T4, activin receptor-like kinase  1, ACVR2B, adenocarcinoma antigen, AGS-22M6, alpha-fetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1) , B7-H3, Bacillus anthracis anthrax, BAFF, beta-amyloid, B-lymphoma cell, C242 antigen, C5, CA-125, Canis lupus familiaris IL31, carbonic anhydrase 9 (CA-IX) , cardiac myosin, CCL11 (eotaxin-1) , CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin) , CD15, CD152, CD154 (CD40L) , CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor) , CD25 (αchain of IL-2receptor) , CD27, CD274, CD28, CD3, CD3 epsilon, CD30, CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-related antigen, CFD, ch4D5, CLDN18.2, Clostridium difficile, clumping factor A, CSF1R, CSF2, CTLA-4, C-X-C chemokine receptor type 4, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli shiga toxin type-1, E. coli shiga toxin type-2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, Escherichia coli, F protein of respiratory syncytial virus, FAP, fibrin II beta chain, fibronectin extra domain-B, folate hydrolase, folate receptor 1, folate receptor alpha, Frizzled receptor, ganglioside GD2, GD2, GD3 ganglioside, glypican 3, GMCSF receptor α-chain, GPNMB, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1, HER2/neu, HER3, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, Hsp90, human scatter factor receptor kinase, human TNF, human beta-amyloid, ICAM-1 (CD54) , IFN-α, IFN-γ, IgE, IgE Fc region, IGF-1 receptor, IGF-1, IGHE, IL17A, IL17F, IL20, IL-12, IL-13, IL-17, IL-1β, IL-22, IL-23, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, ILGF2, influenza A hemagglutinin, influenza A virus hemagglutinin, insulin-like growth factor I receptor, integrin α4β7, integrin α4, integrin α5β1, integrin α7 β7, integrin αIIbβ3, integrin αvβ3, interferon α/β receptor, interferon gamma-induced protein, ITGA2, ITGB2 (CD18) , KIR2D, Lewis-Y antigen, LFA-1 (CD11a) , LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L) , LTA, MCP-1, mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen) , neural apoptosis-regulated proteinase 1, NGF, N-glycolylneuraminic acid, NOGO-A, Notch receptor, NRP1, Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-R α, phosphate-sodium co-transporter, phosphatidylserine, platelet-derived growth factor receptor beta, prostatic carcinoma cells, Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, Rhesus factor, RON, RTN4, sclerostin, SDC1, selectin P, SLAMF7, SOST, sphingosine-1-phosphate, Staphylococcus aureus, STEAP1, TAG-72, T-cell receptor, TEM1, tenascin C, TFPI, TGF-β 1, TGF-β2, TGF-β, TNF-α, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, tumor specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75) , VEGFA, VEGFR1, VEGFR2, vimentin, and VWF.
Additional examples of the antigen of the antigen binding moiety of the chimeric polypeptide receptor or TCR 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, CD23, CD24, CD30, CD33, CD38, CD44v7/8, CDC27, CDK- 4, CEA, CLCA2, Cyp-B, DAM-10, DAM-6, DEK-CAN, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6/AML, FBP, fetal acetylcholine receptor, FGF-5, FN, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6) , HST-2/neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR/FUT, Lewis Y, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, Malic enzyme, Mammaglobin-A, MART-1/Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, Myosin, NA88-A, Neo-PAP, NKG2D, NPM/ALK, N-RAS, NY-ESO-1, OA1, OGT, oncofetal antigen (h5T4) , OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, Survivin, Survivin-2B, SYT/SSX, TAG-72, TEL/AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2/INT2, TRP-2-6b, Tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ-light chain.
Additional examples of the antigen of the antigen binding moiety of the chimeric polypeptide receptor or TCR as disclosed herein can include an antibody, a fragment thereof, or a variant thereof. Such antibody can be a natural antibody (e.g., naturally secreted by a subject’s immune cell, such as B cells) , a synthetic antibody, or a modified antibody. In some cases, he antigen of the antigen binding moiety of the chimeric polypeptide receptor as disclosed herein can include an Fc domain of an antibody from the group comprising 20- (74) - (74) (milatuzumab; veltuzumab) , 20-2b-2b, 3F8, 74- (20) - (20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, zlintuzumab) , actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003/SIM-BD0801 (sevacizumab) , APX005M, arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also referred to as tocilizumab) , atorolimumab, Avelumab, B-701, bapineuzumab, basiliximab, bavituximab, BAY1129980, BAY1187982, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, Betalutin (177Lu-tetraxetan-tetulomab) , bevacizumab, BEVZ92 (bevacizumab biosimilar) , bezlotoxumab, BGB-A317, BHQ880, BI 836880, BI-505, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab) , CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch. 14.18, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab  ravtansine, conatumumab, concizumab, Cotara (iodine I-131 derlotuximab biotin) , cR6261, crenezumab, DA-3111 (trastuzumab biosimilar) , dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, Daratumumab Enhanze (daratumumab) , Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, Depatuxizumab, Depatuxizumab mafodotin, derlotuximab biotin, detumomab, DI-B4, dinutuximab, diridavumab, DKN-01, DMOT4039A, dorlimomab aritox, drozitumab, DS-1123, DS-8895, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, FF-21101, FGFR2 Antibody-Drug Conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, FPA144, fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, Gerilimzumab, gevokizumab, girentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hu14.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (claudiximab) , imalumab, IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan) , IMMU-114, ImmuTune IMP701 Antagonist Antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, Ipafricept, IPH4102, ipilimumab, iratumumab, isatuximab, Istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2/L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L-DOS47, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (inebilizumab) , MEDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006/S80880, MGD007, MGD009, MGD011, milatuzumab, Milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, NOV-10, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab,  OX002/MEN1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, PankoMab-GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab) , PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/SAR439684, reslizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, RO7009789, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-CD19A, SGN-CD19B, SGN-CD33A, SGN-CD70A, SGN-LIV1A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, SYD985, SYM004 (futuximab and modotuximab) , Sym015, TAB08, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, Tanibirumab, taplitumomab paptox, tarextumab, TB-403, tefibazumab, Teleukin, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, TG-1303, TGN1412, Thorium-227-Epratuzumab Conjugate, ticilimumab, tigatuzumab, tildrakizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH 011, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tuvirumab, U3-1565, U3-1784, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, Vadastuximab Talirine, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB-101, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to an antigen comprising one or more (e.g., 1, 2, 3, 4, or more) members selected from the group comprising BCMA, CD20, CD22, CD30, CD33, CD38, CD70, CD123, Kappa, Lewis Y, NKG2D ligand, ROR1, NY-ESO-1, NY-ESO-2, MART-1, and gp100. Non-limiting examples of the NKG2D ligand comprises one or more (e.g., 1, 2, 3, 4, or more) members selected from the group comprising of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain capable of specifically binding an antigen of a target cell, and the engineered immune cell can  exhibit reduced expression or activity of an endogenous gene encoding the same antigen of the chimeric polypeptide receptor. As such, a population of the engineered immune cells can avoid targeting and killing each other, e.g., upon administration to a subject in need thereof.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD23. In some cases, the engineered immune cell’s endogenous gene encoding CD23 can be modified to effect reduced expression or activity of the endogenous CD23.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD123. In some cases, the engineered immune cell’s endogenous gene encoding CD123 can be modified to effect reduced expression or activity of the endogenous CD123.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38. In some cases, the engineered immune cell’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38. In some cases, the subject engineered immune cells comprising the chimeric polypeptide receptor against CD38 can be capable of targeting and effecting death (or degradation) of plasma cells.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD38. In some examples, the engineered immune cell is an engineered NK cell that is derived from an isolated ESC or an induced stem cell (e.g., iPSC) . In some cases, the engineered immune cell’s endogenous gene encoding CD38 can be modified to effect reduced expression or activity of the endogenous CD38.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD7. In some cases, the engineered immune cell’s endogenous gene encoding CD7 can be modified to effect reduced expression or activity of the endogenous CD7.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the chimeric polypeptide receptor (e.g., TFP or CAR) that comprises the antigen binding domain, and the antigen binding domain can be capable of binding specifically and preferentially to CD19 and/or CD33. Furthermore, the CAR comprises at least one of a transmembrane domain of CD8, a costimulatory domain of 2B4, and a signaling domain of CD3ζ.
4. Features comprised in the immune cell
The present disclosure describes systems and methods for immunotherapies. Immune cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered immune cell) . Immune cells can be engineered to exhibit enhanced proliferation as compared to a control cell. Immune cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered Immune cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo. The engineered Immune cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) . The engineered Immune cells can be autologous to the subject. Alternatively, the engineered immune cells can be allogeneic to the subject.
In some cases, engineered immune cells (e.g., engineered NK cells) disclosed herein can be derived from an isolated stem cell (e.g., isolated ESCs) . In some cases, engineered immune cells disclosed herein can be derived from induced stem cells (e.g., iPSCs) .
In some cases, the stem cell disclosed herein (e.g., isolated stem cell, induced stem cell) can be an autologous cell or derived from the autologous cell. The autologous cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the autologous cell can be obtained from the subject before the subject is found to have the condition. In some cases, the autologous cell can be an allogeneic cell, e.g., a universal stem cell with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs. The autologous cell can be obtained from a healthy donor.
In some cases, the engineered immune cell (e.g., engineered T cell or NK cell) can be an autologous cell. The engineered immune cell can be obtained from a subject having a condition or is suspected of having the condition. Alternatively, the engineered immune cell can be obtained from the subject before the subject is found to have the condition. In some cases, the engineered immune cell can be an allogeneic cell, e.g., for a universal allogenic immunotherapy with reduced immunogenicity and with reduced amount or no need for immunosuppressive drugs. The engineered immune cell can be obtained from a healthy donor.
In some aspects, T cells can be engineered to exhibit enhanced half-life as compared to control cell (e.g., a non-engineered T cell) . T cells can be engineered to exhibit enhanced proliferation as compared to a control cell. T cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered T cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo. The engineered T cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) . The engineered T cells can be autologous to the subject. Alternatively, the engineered T cells can be allogeneic to the subject.
In some aspects, NK cells can be engineered to exhibit enhanced half-life as compared to control cell  (e.g., a non-engineered NK cell) . NK cells can be engineered to exhibit enhanced proliferation as compared to a control cell. NK cells can be engineered to effectively and specifically target diseased cells (e.g., cancer cells) that a control cell otherwise is insufficient or unable to target. The engineered NK cells disclosed herein can be engineered ex vivo, in vitro, and in some cases, in vivo. The engineered NK cells that are prepared ex vivo or in vitro can be administered to a subject in need thereof to treat a disease (e.g., myeloma or solid tumors) . The engineered NK cells can be autologous to the subject. Alternatively, the engineered NK cells can be allogeneic to the subject.
In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell. In some cases, the heterologous cytokine can comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) . In some examples, the antigen is not CD19. Thus, the antigen binding moiety may not and need not exhibit any specific binding to CD19, but rather a specific binding to an antigen (e.g., one or more antigens) that is not CD19.
The engineered immune cell (e.g., an engineered NK cell) as disclosed herein can comprise a heterologous receptor that is a respective receptor of the heterologous cytokine as disclosed herein (e.g., heterologous IL-15 receptor (IL-15R, such as IL-15α or IL-15β) for heterologous IL-15) . Alternatively, the engineered immune cell may not and need not comprise any heterologous receptor that is a respective receptor of the heterologous cytokine. For example, the engineered immune cell comprising a heterologous IL (e.g., IL-15) lacks a heterologous receptor of the heterologous IL (e.g., IL-15R) . In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a cytokine (e.g., a secretory cytokine) that is heterologous to the immune cell. The heterologous cytokine can further comprise a heterologous interleukin (IL) (e.g., a heterologous secretory IL-15) . The engineered immune cell may and need not comprise a heterologous receptor that is a respective receptor of the heterologous cytokine (e.g., a heterologous IL-15R) .
The heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be of the same species as that of the engineered immune cell (e.g., the engineered NK cell) . For example, both the heterologous cytokine and the engineered immune cell can be of human origin. Alternatively, the heterologous cytokine can be of a different species than that of the engineered immune cell.
A heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be introduced to the engineered immune cell (e.g., engineered NK cell) by contacting a heterologous polynucleotide encoding the heterologous cytokine to the engineered immune cell. The heterologous polynucleotide can be integrated into the engineered immune cell’s chromosome (e.g., nuclear chromosome) . Alternatively, the heterologous polynucleotide may not and need not be integrated into the chromosome of the engineered immune cell. In an example, an mRNA encoding a heterologous cytokine can be introduced (or inserted into) the engineered immune cell.
In some cases, the heterologous cytokine as disclosed herein can be a heterologous IL. A heterologous IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of heterologous ILs.  A heterologous IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of heterologous ILs. Alternatively, the heterologous IL can be a single type of heterologous IL. Non-limiting examples of the heterologous IL can include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36. In some examples, the heterologous IL can comprise one or more members selected from the group consisting of IL2, IL4, IL6, IL7, IL9, IL10, IL11, IL12, IL15, IL18, IL21, and functional modifications thereof. For example, the engineered immune cell (e.g., an engineered NK cell) as disclosed herein can comprise at least a portion of heterologous human IL-15 (or a gene encoding thereof) .
The heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be a secretory cytokine. Alternatively, the heterologous cytokine may not and need not be secreted by the engineered immune cell. In such a case, for example, the heterologous cytokine can be bound to a cell surface of the engineered immune cell.
In some cases, the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be a secretory cytokine. An expression cassette encoding the heterologous cytokine can be introduced to the engineered immune cell. The expression cassette can further encode an additional heterologous polypeptide, e.g., a heterologous receptor. Within the expression cassette, a first polynucleotide sequence encoding the heterologous cytokine and a second polynucleotide sequence encoding the additional heterologous polypeptide (e.g., the heterologous receptor) can be coupled to each other via a polynucleotide linker encoding a cleavage linker. In some examples, the heterologous receptor can be a respective receptor of the heterologous cytokine (e.g., heterologous IL-15α or IL-15β for heterologous IL-15) . Alternatively, the expression cassette may not and need not encode any additional heterologous polypeptide other than the heterologous cytokine.
A cleavable linker as disclosed herein can comprise a self-cleaving peptide, such as a self-cleaving 2A peptide. Self-cleaving peptides can be found in members of the Picornaviridae virus family, including aphthoviruses such as foot-and-mouth disease virus (FMDV) , equine rhinitis A virus (ERAV) , Thosea asigna virus (TaV) and porcine tescho virus-1 (PTV-I) , and cardioviruses such as Theilovirus (e.g., Theiler's murine encephalomyelitis) and encephalomyocarditis viruses. Non-limiting examples of the self-cleaving 2A peptide can include “F2A” , “E2A” , “P2A” , “T2A” , and functional variants thereof. In some cases, the heterologous cytokine (e.g., the heterologous IL) as disclosed herein can be bound to a cell surface the engineered immune cell (e.g., the engineered NK cell) . In some examples, the engineered immune cell can be genetically modified such that a heterologous polynucleotide sequence encoding the heterologous cytokine is coupled to a gene encoding an endogenous transmembrane protein of the engineered immune cell. In an example, the endogenous transmembrane protein can be a respective receptor of the heterologous cytokine (e.g., heterologous IL-15α or IL-15β for heterologous IL-15) . In some examples, an expression cassette encoding a heterologous fusion polypeptide comprising (i) the heterologous cytokine that is coupled to (ii) a heterologous receptor can be introduced  to the engineered immune cell. Here, the heterologous cytokine may not and need not be cleavable from the heterologous receptor. Non-limiting examples of the heterologous receptor can include a respective receptor of the heterologous cytokine (e.g., heterologous IL-15α or IL-15β for heterologous IL-15) , or a different receptor such as a common gamma chain (γC) receptor or a modification thereof. An expression cassette as disclosed herein can be integrated into the genome of the engineered cell (e.g., the engineered NK cell) via action of a gene editing moiety as disclosed herein. Alternatively, the expression cassette may not and need not be integrated into the genome of the engineered cell.
The engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit enhanced signaling of an endogenous signaling pathway that involves the heterologous cytokine (e.g., the heterologous IL, such as the heterologous IL-15) and/or the heterologous receptor (e.g., the heterologous IL receptor, such as the heterologous IL-15R) as disclosed herein. The enhanced signaling of the endogenous signaling pathway as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., JAK3, STAT3, STAT5, etc. for IL-15/IL-15R) or (ii) expression of a downstream gene (e.g., Mcl1, Cdk4/6, Mki67, Tnf, Gzmb, Gzmc, Ifng, etc. for IL-15/IL-15R) via Western blotting or polymerase chain reaction (PCR) techniques. In some cases, enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor (e.g., induced by the heterologous cytokine and/or heterologous receptor, such as IL-15/IL-15R as disclosed herein) in the engineered immune cell of the present disclosure can be characterized by an increase in phosphorylation of a downstream signaling protein by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.
In some cases, enhanced signaling of the endogenous signaling pathway that is induced by the heterologous cytokine and/or the heterologous receptor (e.g., induced by the heterologous cytokine and/or heterologous receptor, such as IL-15/IL-15R as disclosed herein) in the engineered immune cell of the present disclosure can be characterized by an increased expression of a downstream gene by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1- fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell. In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a heterologous cytokine as disclosed herein, wherein the heterologous cytokine is bound to a membrane (e.g., plasma membrane) of the engineered immune cell. In some cases, the heterologous cytokine can comprise a heterologous IL as disclosed herein (e.g., a heterologous IL-15) . The engineered immune cell can further comprise one, two, or all of: (a) a different heterologous cytokine (e.g., a heterologous cytokine as disclosed herein, other than the one that is bound to the membrane of the subject cell) , (b) reduced expression or activity of an endogenous immune regulator polypeptide, and (c) a safety switch. In some examples, the endogenous immune regulator polypeptide is not B2M. Thus, the endogenous immune regulator can be, for example, a polypeptide other than B2M.
The engineered immune cell (e.g., an engineered NK cell) can comprise the different heterologous cytokine and one or both of (b) the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) and (c) the safety switch. The engineered immune cell comprise the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) and one or both of (a) the different heterologous cytokine and (c) the safety switch. The engineered immune cell comprise the safety switch and one or both of (a) the different heterologous cytokine and (b) the reduced expression or activity of an endogenous immune regulator polypeptide (e.g., a non-B2M polypeptide) . The engineered immune cell comprise all of (a) , (b) , and (c) .
The expression or activity of the endogenous immune regulator polypeptide can be reduced in the engineered immune cell (e.g., the engineered NK cell) , for example, via action of a gene editing moiety as disclosed herein.
The reduced expression or activity of the endogenous immune regulator polypeptide in the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation or dephosphorylation of a downstream signaling protein (e.g., SHP2, Igα/β, Syk, etc. for PD1/PDL1 signaling) or (ii) expression of the endogenous immune regulator polypeptide (e.g., PD1) via Western blotting or PCR techniques. In some cases, reduced expression of the endogenous immune regulator polypeptide in the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can be characterized by a decrease in the expression of the endogenous immune regulator polypeptide (e.g., PD1) by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at  least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.
In some cases, reduced activity of the endogenous immune regulator polypeptide in the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can be characterized by a decrease in phosphorylation of a downstream signaling protein (e.g., SHP2 for PD1/PDL1 signaling) by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.
In some cases, reduced activity of the endogenous immune regulator polypeptide in the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can be characterized by an increase in phosphorylation of a downstream target signaling protein (e.g., Igα/β or Syk for PD1/PDL1 signaling) by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell. The downstream target signaling protein may be a protein that is normally inhibited by action of a functional signaling pathway of the endogenous immune regulator polypeptide.
In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a CD16 variant as disclosed herein for enhanced CD16 signaling in the engineered NK cell. In some cases, the CD16 variant (e.g., a heterologous CD16 variant) can comprise at least a portion of CD16 and at least a portion of CD64 (e.g., hnCD16 as disclosed herein) . The engineered immune cell can further comprise reduced expression or activity of an endogenous immune regulator polypeptide as compared to a control cell, as disclosed herein.
The engineered immune cell (e.g., the engineered NK cell) can exhibit enhanced CD16 signaling as compared to a control cell, as disclosed herein.
In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) . The engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) . Alternatively, the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
In some cases, the engineered NK cell can be treated with inhibitors (e.g., small molecule inhibitors) of the endogenous cytokine signaling. In some cases, the engineered NK cell can comprise reduced expression of endogenous IL-17 or endogenous receptor thereof (e.g., via indel or transgene mutation, via transient or permanent suppression, etc. ) . In some cases, the engineered NK cell can comprise reduced expression of endogenous IL-17. In some cases, the engineered NK cell can comprise reduced expression of endogenous IL-17R. In some cases, the engineered NK cell can comprise reduced expression of endogenous IL-17 and endogenous IL-17R.
In some cases, the endogenous cytokine as disclosed herein can be an endogenous IL. An endogenous IL as disclosed herein can comprise at least 1, 2, 3, 4, 5, or more different types of endogenous ILs. An endogenous IL as disclosed herein can comprise at most 5, 4, 3, or 2 different type of endogenous ILs. Alternatively, the endogenous IL can be a single type of endogenous IL. Non-limiting examples of the endogenous IL can include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and IL-36. In some examples, the endogenous IL can be IL-17. Non-limiting examples of endogenous Il-17 can include IL-17A, IL-17F, and natural mutations thereof. For example, the engineered immune cell (e.g., an engineered NK cell) as disclosed herein can exhibit reduced expression or activity of IL-17A or IL-17F. In some cases, an endogenous gene encoding the endogenous cytokine (e.g., an endogenous IL, such as IL-17) as disclosed herein can be modified via action of a gene editing moiety as disclosed herein.
The endogenous receptor can be a respective receptor of any cytokine as disclosed herein (e.g., a respective receptor of any IL as disclosed herein) . In some cases, the endogenous receptor can be a respective receptor of IL (e.g., IL-17R for IL-7 signaling) . Non-limiting examples of IL-17R can include IL-17RA, IL-17RB, IL-17RC, IL-17RD, IL-17RE, and variants thereof. In an example, the endogenous IL-17R comprises IL-17RA.
In some cases, the reduced expression or activity of the endogenous cytokine (e.g., an endogenous IL, such as IL-17) or endogenous receptor thereof as disclosed herein can be ascertained by a number of methods, including, but are not limited to, (i) phosphorylation of a downstream signaling protein (e.g., PI3K, Act1, MAP3K, MEK1/2, MKK3/6, MKK4/7, MKK3/6, ERK, p38, JNK, etc. for IL-17) or (ii) expression of a downstream gene via Western blotting or PCT techniques. In some examples, a downstream gene of IL cytokine, such as IL-17, can include a chemokine (e.g., CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CCL2, CCL20, etc. ) , a cytokine (e.g., IL-6, TNFa, G-CSF, GM-CSF, etc. ) , an acute phase response molecule (e.g., SAA, CRP, lipocalin 2/24p3, etc. ) , and/or an enzyme (e.g., a metalloproteinase, such as MMP1, MMP3, MMP9, MMP13) .
In some cases, reduced expression or activity of the endogenous cytokine (e.g., the endogenous IL, such as IL-17) or endogenous receptor thereof in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can be characterized by a decrease in phosphorylation of a downstream signaling protein of the endogenous cytokine by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.
In some cases, reduced expression or activity of the endogenous cytokine (e.g., the endogenous IL, such as IL-17) or endogenous receptor thereof in the engineered immune cell (e.g., engineered NK cell) as disclosed herein can be characterized by a decrease in the expression of a downstream gene of the endogenous cytokine by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as compared to a control cell.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit enhanced expression profile of a specific cell marker for a committed immune cell (e.g., a NK cell marker) as compared to a control cell that does not exhibit the reduced activity of the endogenous cytokine signaling (e.g., endogenous IL signaling, such as endogenous IL-17 signaling) as disclosed herein. For example, non-limiting examples of a specific cell marker for committed NK cells can include CD57 or killer immunoglobulin-like receptors (KIR) . KIR can comprise KIR2D and/or KIR3D. KIR2D can comprise KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and/or KIR2DS5. KIR3D can comprise KIR3DL1, KIR3DL2, KIR3DL3, and/or KIR3DS1.
The enhanced expression profile of the specific cell marker for the committed immune cell (e.g., CD57 or KIR for NK cells) as disclosed herein can be ascertained by a number of methods, including, but are not limited to, Western blotting or PCR techniques.
In some cases, the expression of the specific cell marker for a committed immune cell (e.g., CD57 or KIR or NK cells) in the engineered immune cell of the present disclosure can be greater than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold. In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise one or both of: (i) a heterologous transcription factor (e.g., a heterologous STAT) , (ii) reduced activity of endogenous cytokine signaling (e.g., endogenous IL signaling as disclosed herein) , and (iii) reduced expression or activity of endogenous enzyme (e.g., a ligase, such as CBL-B) . The engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) . Alternatively, the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
The heterologous transcription factor can comprise at least 1, 2, 3, 4, 5, or more different types of heterologous transcription factor. The heterologous transcription factor can comprise at most 5, 4, 3, or 2 different types of transcription factor. Alternatively, the heterologous transcription factor can have a single type of transcription factor. The transcription factor can be involved in the engineered immune cell’s immune activity, proliferation, apoptosis, and/or differentiation. In some cases, the heterologous transcription factor for the engineered immune cell (e.g., the engineered NK cell) can be STAT. Non-limiting examples of STAT can include STAT1, STAT2, STAT3, STAT4, STAT3, STAT4, STAT5A,  STAT5B, STAT6, and modifications thereof. In an example, STAT can comprise STAT3. In another example, STAT can comprise STAT5B.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can exhibit enhanced survival in the presence of tumor cells as compared to a control cell without (i) the heterologous transcription factor (e.g., the heterologous STAT) or (ii) the reduced activity of endogenous cytokine signaling (e.g., endogenous IL-17 signaling) . In some cases, the engineered immune cell can, in the presence of tumor cells, survive longer than the control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of a specific endogenous cell marker for a committed immune cell as disclosed herein (e.g., a NK cell marker, such as KIR) as compared to a control cell. In some examples, the specific endogenous cell marker is KIR. The engineered immune cell can further comprise one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein, (b) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein, (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell, wherein the CD16 variant is heterologous to the engineered NK cell, as disclosed herein, (d) an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell, and (e) reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein. The engineered immune cell can comprise the chimeric polypeptide receptor and one or more of (e.g., 1, 2, 3, or 4 of) : (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
The engineered immune cell can comprise the heterologous cytokine and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (c) the CD16 variant for enhanced CD16 signaling, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
The engineered immune cell can comprise the CD16 variant for enhanced CD16 signaling and one or  more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (d) the heterologous immune regulator, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
The engineered immune cell can comprise the heterologous immune regulator and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, and (e) the reduced expression or activity of an endogenous immune regulator polypeptide.
The engineered immune cell can comprise the reduced expression or activity of an endogenous immune regulator polypeptide and one or more of (e.g., 1, 2, 3, or 4 of) : (a) the chimeric polypeptide receptor, (b) the heterologous cytokine, (c) the CD16 variant for enhanced CD16 signaling, and (d) the heterologous immune regulator.
The reduced expression or activity of the specific endogenous cell marker for the committed immune cell (e.g., KIR for NK cells) as disclosed herein can be ascertained by a number of methods, including, but are not limited to, Western blotting or PCR techniques.
In some cases, the expression of the specific endogenous cell marker for a committed immune cell (e.g., KIR or NK cells) in the engineered immune cell of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold. In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise a heterologous cytokine (e.g., a heterologous IL) , as disclosed herein that is not IL-15. In some examples, the heterologous cytokine comprises IL-21 or variants thereof. The engineered immune cell can be derived from an induced stem cell (e.g., iPSC) .
In some cases, a control cell can be a cell can be an immune cell, such as a NK cell, used for comparison purposes. In some cases, a control cell can be a cell that does not comprise a heterologous cytokine (e.g., IL-15) . In some cases, a control cell can be a cell that does not comprise a CD16 variant for enhanced CD16 signaling. In some cases, a control cell can be a cell that a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen. In some cases, a control cell can be a cell that comprises a heterologous IL-15R. In some cases, a control cell can be a cell that does not comprise a membrane bound heterologous cytokine (e.g., IL-15) . In some cases, a  control cell can be a cell that does not exhibit reduced expression or activity of an endogenous immune regulator polypeptide. In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of an endogenous cytokine (e.g., IL-17) or a receptor thereof (e.g., IL-17R) . In some cases, a control cell can be a cell that does not comprise a heterologous transcription factor (e.g., STAT) . In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of a specific endogenous cell marker for a committed immune cell (e.g., a NK cell marker, such as KIR) . In some cases, a control cell can be a cell that does not comprise a heterologous immune regulator polypeptide. In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of one or more (e.g., 1, 2, 3, or 4) of: endogenous CD94, endogenous CD96, endogenous TGF beta receptor, or endogenous SHIP2. In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of one or more (e.g., 1, 2, 3, 4, or 5) of: endogenous CD80, endogenous CD86, endogenous ICOSL, endogenous CD40L, endogenous MICA or MICB, or endogenous NKG2DL. In some cases, a control cell can be a cell that does not exhibit reduced expression or activity of ICAM1. In some cases, a control cell can be a cell that does not comprise a heterologous PDL2 or heterologous TGF beta. In some cases, a control cell can be a cell that does not comprise one or more (e.g., 1, 2, 3, 4, or 5) of: heterologous CCL21, heterologous IL-10, heterologous CD46, heterologous CD55, or heterologous CD59. In some cases, a control cell can be a cell that does not comprise heterologous IL-21. In some cases, a control cell can be a cell that is not derived from a cell line. In some cases, a control cell can be a cell that is not derived from an isolated ESC. In some cases, a control cell can be a cell that is not derived from an iPSC.
In some embodiments, the present disclosure provides a population of engineered immune cells comprising any one of the engineered immune cells as disclosed herein (e.g., a population of engineered NK cells comprising any one of the engineered NK cells as disclosed herein) . An engineered immune cell (e.g., an engineered NK cell) of the population of immune cells (e.g., the population of NK cells) can comprise a heterologous polypeptide, wherein the heterologous polypeptide comprises a heterologous IL-15 (e.g., heterologous secretory IL-15, and/or membrane-bound IL-15, such as IL15-IL15 receptor fusion, e.g., IL15-IL15 receptor alpha fusion) .
In some cases, the engineered immune cell comprises a persistence component, the persistence component is selected from the group consisting of: a) , wherein the persistence component is selected from the group consisting of: a) a full length or at least a portion of IL15, preferably in secretion form; b) a full length or at least a portion of IL15 fused with a full length or at least a portion of IL15Ra; c) a transgene for p-STAT5 enhancement; d) a modification to an endogenous gene for p-STAT5 enhancement, preferably a disruption of an endogenous gene; e) a reduced expression of one or more genes selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, CD70; and f) an enhanced expression of one or more genes selected from the group consisting of CD25, CD122, and NKG2C.
In some cases, the persistence component comprises or consists of (i) an amino acid sequence having at  least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting SEQ ID NOs: 31-37; or (ii) an amino acid sequence encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 38-39.
An activity level (e.g., persistence level) of the population of engineered immune cells in an environment that is substantially free of an exogenous interleukin (e.g., IL-2, IL-15, etc. ) can be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, at least about or more greater than a control persistence level of a comparable population of immune cells (e.g., engineered to comprise a comparable heterologous IL-15 as disclosed herein) in a control environment comprising the exogenous interleukin. Such persistence level may ascertained after the population of immune cells are in the environment for at least or up to about 1 day, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 11 days, at least or up to about 12 days, at least or up to about 13 days, at least or up to about 14 days, at least or up to about 15 days, at least or up to about 16 days, at least or up to about 17 days, at least or up to about 18 days, at least or up to about 19 days, at least or up to about 20 days, at least or up to about 21 days, at least or up to about 22 days, at least or up to about 23 days, at least or up to about 24 days, at least or up to about 25 days, at least or up to about 26 days, at least or up to about 27 days, at least or up to about 28 days, at least or up to about 5 weeks, at least or up to about 6 weeks, or at least or up to about 8 weeks. The amount of the exogenous interleukin (e.g., IL-2, IL-15, etc. ) in the environment can be at least or up to about 1 unit per milliliter (U/ml) , at least or up to about 5 U/mL, at least or up to about 10 U/mL, at least or up to about 15 U/mL, at least or up to about 20 U/mL, at least or up to about 30 U/mL, at least or up to about 40 U/mL, at least or up to about 50 U/mL, at least or up to about 60 U/mL, at least or up to about 80 U/mL, at least or up to about 100 U/mL, at least or up to about 150 U/mL, at least or up to about 200 U/mL, at least or up to about 300 U/mL, at least or up to about 400 U/mL, or at least or up to about 500 U/mL. The environment can be in vitro, ex vivo, or in vivo.
The population of engineered NK cells as disclosed herein can exhibit at least or up to about 10%, at least or up to about 20%, at least or up to about 30%, at least or up to about 40%, at least or up to about 50%, at least or up to about 60%, at least or up to about 70%, at least or up to about 80%, at least or up to about 90%, at least or up to about 95%, or more persistence (or survival rate) after being in the environment that is substantially free of the exogenous interleukin (e.g., IL-2) for at least or up to about 1 day, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 11 days, at least or up to about 12 days, at least or up to about 13 days, at least or up to about 14 days, at least or up  to about 3 weeks, or at least or up to about 4 weeks.
For example., the population of engineered NK cells as disclosed herein can exhibit at least about 50%survival after at least about 5 days (50%, after 9 days, 25%after 15 days) in the environment that is substantially free of the exogenous interleukin (e.g., IL-2) . For example, the population of engineered NK cells as disclosed herein can exhibit at least about 50%survival after at least about 9 days in the environment that is substantially free of the exogenous interleukin (e.g., IL-2) . For example, the population of engineered NK cells as disclosed herein can exhibit at least about 25%survival after at least about 15 days in the environment that is substantially free of the exogenous interleukin (e.g., IL-2) .
The population of engineered NK cells as disclosed herein can exhibit enhanced persistence by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 3-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 200-fold, at least or up to about 300-fold, at least or up to about 400-fold, or at least or up to about 500-fold, after being in the environment substantially free of an exogenous interleukin (e.g., IL-2, IL-15, etc. ) for at least or up to about 1 day, at least or up to about 2 days, at least or up to about 3 days, at least or up to about 4 days, at least or up to about 5 days, at least or up to about 6 days, at least or up to about 7 days, at least or up to about 8 days, at least or up to about 9 days, at least or up to about 10 days, at least or up to about 11 days, at least or up to about 12 days, at least or up to about 13 days, at least or up to about 14 days, at least or up to about 2 weeks, at least or up to about 3 weeks, at least or up to about 4 weeks, at least or up to about 6 weeks, or at least or up to about 8 weeks, as compared to that of a comparable population of NK cells lacking the heterologous polypeptide comprising the heterologous IL-15 (e.g., lacking a heterologous membrane-bound IL-15) . For example, the population of engineered NK cells as disclosed herein can exhibit enhanced persistence by at least about 7-fold after at least about 5 days in the environment, as compared to that of a comparable population of NK cells lacking the heterologous IL-15.
Without wishing to be bound by theory, having such enhanced persistence level by having the heterologous polypeptide comprising the enhanced IL-15 signaling (e.g., via comprising a heterologous polypeptide that comprises heterologous IL-15) can reduce the amount of exogenous proteins (e.g., IL-2) required for the production of the engineered immune cells, enhance the efficiency of producing the engineered immune cells, and/or reduce overall cost of the immune cell therapy. Any enhanced level of persistence ascertained in vitro can be translated to an event in vivo (e.g., in the blood stream of a subject in need thereof) .
An exogenous interleukin, as disclosed herein, can comprise an interleukin that is not secreted by the cell or the population of cells (e.g., the engineered immune cell (s) , such as the engineered NK cells as disclosed herein) , and can be artificially added to the environment. In an in vitro environment, an exogenous interleukin may comprise a recombinant interleukin protein that is added to a medium. In an in vivo environment (e.g., in plasma) , an exogenous interleukin may comprise a recombinant interleukin protein that is administered to a subject in need thereof.
The term “persistence” as used herein may generally refer to a presence of at least a portion of a population of cells (e.g., a population of engineered immune cells, such as a population of engineered NK cells as disclosed herein) remaining in an environment after introducing the population of cells to the environment (e.g., in an in vitro medium, in the serum after intravenous (IV) administration, etc. ) . A persistence may be ascertained by a duration of time that at least a portion of the population of cells remain in the environment at a detectable level. In some examples, persistence of a population of cells may correlate to the half-life of the population of cells in the environment (e.g., medium, blood stream, etc. ) . In some cases, a population of cells of interest (e.g., a population of engineered immune cells, such as a population of engineered NK cells) may not grow (e.g., proliferate) over time, and a persistence level of the population of cells as disclosed herein can be ascertained by measuring a decrease (or a rate thereof) in a size of the population of cells over time. For example, a population of cells having a greater persistence level (e.g., at least 5%greater) in an environment after a period of time (e.g., after at least about 5 days) than a control population of cells in a comparable environment after a comparable period of time may indicate that a greater proportion (e.g., at least 5%greater) of the size of the population of cells have survived as compared to the control population of cells.
In some cases, the engineered immune cell comprises a gene modification to enhance or reduce the expression of one or more endogenous or heterologous genes, wherein the gene modification comprises a reduced or disrupted expression of endogenous CD33 and/or CD38.
In some cases, the reduced or disrupted expression of endogenous CD33 and/or CD38 does not impact growth and/or function of the cell, preferably does not impact differentiation of the cell.
In some cases, the reduced or disrupted expression of endogenous CD33 and/or CD38 is achieved by using RNAi or a gene editing system.
In some cases, the expression of endogenous CD33 and/or CD38 is reduced by RNAi selected from the group consisting of siRNA-, shRNA-, micoRNA-, and circular RNA-mediated RNA interferences. In some cases, the expression of endogenous CD33 and/or CD38 is disrupted by the gene editing system selected from the group of CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these systems, preferably by CRISPR-Cas9 system.
In some cases, the CRISPR-Cas9 system comprises an sgRNA having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of SEQ ID NOs: 40-45 for the disruption of CD33 and/or an sgRNA having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of  SEQ ID NOs: 46-51 for the disruption of CD38. In some cases, the CRISPR-Cas9 system comprises an sgRNA of any one of SEQ ID NOs: 40-45 for the disruption of CD33 and/or an sgRNA of any one of SEQ ID NOs: 46-51 for the disruption of CD38.
5. Additional aspects of the engineered immune cells
In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein, a heterologous gene can be operatively coupled to (e.g., for knock-in) a constitutive, inducible, temporal, tissue-specific, and/or cell type-specific promoter. Non-limiting examples of a promoter of interest can include CMV, EF1a, PGK, CAG, and UBC. Non-limiting examples of an insertion site can include AAVS1, CCR5, ROSA26, collagen, HTRP, H11, B2M, GAPDH, TCR, RUNX1, TAP1, TAP2, tapasin, NLRC5, CIITA, RFXANK, CIITA, RFX5, RFXAP, TCR a or b constant region, NKG2A, NKG2D, CD38, CIS, CBL-B, SOCS2, PD1, CTLA4, LAG3, TIM3, and TIGIT.
In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit reduced expression or activity of one or more of the following endogenous genes for enhancing function of the engineered immune cell in a tumor microenvironment (i.e., tumor microenvironment gene or “TME” ) . In some cases, having reduced expression or activity of a TME can enhance the engineered immune cell’s immune activity against a target cell. In some cases, a TME gene may be an immune checkpoint inhibitor. Non-limiting examples of the TME can include: NKG2A, NKG2D, PD1, CTLA4, LAG3, TIM3, TIGIT, KIR2D, CD94, CD96, TGF beta receptor, 2B4, and SHIP2.
In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit one or more heterologous genes (e.g., knocked-in) for, e.g., enhanced function: CD137, CD80, CD86, DAP10 (e.g., with or without point mutation) .
In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit reduced expression or activity of one or more of the following endogenous genes for, e.g., hypo-immunity: B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and a NKG2DL (e.g., ULBP1) .
In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit one or more heterologous genes (e.g., knocked-in) for, e.g., hypo-immunity: HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and CD59. In some embodiments of any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can exhibit one or more heterologous genes (e.g., knocked-in) : CD3, CD4, CD80, 41BBL, and CD131.
In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein. In some cases, the endogenous immune regulator polypeptide comprise one or more (e.g., 1, 2, 3, 4, 5, or more) hypo-immunity regulators. In some cases, the  engineered immune cell exhibits reduced expression or activity of one or more (e.g., 1, 2, 3, 4, 5, or more) hypo-immunity regulators from: (i) endogenous CD80, (ii) endogenous CD86, (iii) endogenous ICOSL, (iv) endogenous CD40L, (v) endogenous MICA or MICB, or (vi) endogenous NKG2DL. The engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) . Alternatively, the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
In some cases, the engineered immune cell comprises a hypo-immunity regulator, the hypo-immunity regulator comprises a reduced expression of one or more genes selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and a NKG2DL (e.g., ULBP1) , and/or an enhanced expression of one or more genes selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, CD59, CD3, CD4, CD80, 41BBL, and CD131, preferably selected from the group consisting of PD-L2, TGF-beta, CD46, CD55, and CD59. In some cases, the hypo-immunity regulator comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 57-58.
In some cases, the reduced expression or activity of the endogenous hypo-immunity regulator (e.g., CD80, CD86, ICOSL, CD40L, MICA, MICB, NKG2DL, etc. ) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as ascertained by Western blotting or PCT techniques, as disclosed herein.
In some cases, the reduced expression or activity of the endogenous CD80 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about  20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the reduced expression or activity of the endogenous CD86 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the reduced expression or activity of the endogenous ICOSL in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the reduced expression or activity of the endogenous hypo-immunity regulator CD40L in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about  50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the reduced expression or activity of the endogenous MICA or MICB in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the reduced expression or activity of the endogenous NKG2DL in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein. In some cases, the endogenous immune regulator polypeptide comprise a hypo-immunity regulator (e.g., ICAM1) . The engineered immune cell further comprises one or more of: (a) a chimeric polypeptide receptor comprising an antigen binding moiety capable of binding to an antigen, as disclosed herein, (b) a heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein, and (c) a CD16 variant for enhanced CD16 signaling as compared to a control cell. In some cases, the engineered immune cell (e.g., the engineered NK cell) comprises a chimeric polypeptide receptor as disclosed herein and one or both of: (b) the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein and (c) the CD16 variant for enhanced CD16 signaling.
In some cases, the engineered immune cell (e.g., the engineered NK cell) comprise the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein and one or both of: (a) the chimeric polypeptide receptor as disclosed herein and (c) the CD16 variant for enhanced CD16 signaling. In some cases, the engineered immune cell (e.g., the engineered NK cell) comprises the CD16 variant for enhanced CD16 signaling and one or both of: (a) the chimeric polypeptide receptor as disclosed herein and (b) the heterologous cytokine (e.g., a heterologous IL, such as IL-15) , as disclosed herein. In some cases, the reduced expression or activity of the endogenous ICAM1 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold, as ascertained by Western blotting or PCT techniques, as disclosed herein.
In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of an endogenous immune regulator polypeptide, as disclosed herein. In some cases, the endogenous immune regulator polypeptide comprise a hypo-immunity regulator (e.g., ICAM1) . The engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
In some cases, the reduced expression or activity of the endogenous ICAM1 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell, as disclosed herein.
In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell. In some cases, the immune regulator polypeptide comprises a hypo-immunity regulator. The hypo-immunity regulator can be PDL2. Alternatively or in addition to, the hypo-immunity regulator can be TGF-beta. In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can comprise an immune regulator polypeptide as disclosed herein, wherein the immune regulator polypeptide is heterologous to the engineered immune cell. In some cases, the immune regulator polypeptide comprises a hypo-immunity regulator. The hypo-immunity regulator can comprise one or more (e.g., 1, 2, 3, 4, or more) members from: (i) a heterologous CCL21,  (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59. The engineered immune cell can be derived from an isolated stem cell (e.g., an isolated ESC) . Alternatively, the engineered immune cell can be derived from an induced stem cell (e.g., an iPSC) .
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous CCL21 and one or more of (e.g., 1, 2, 3, or all of) : (ii) a heterologous IL-10, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous IL-10 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (iii) a heterologous CD46, (iv) a heterologous CD55, and (v) a heterologous CD59.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous CD46 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iv) a heterologous CD55, and (v) a heterologous CD59.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous CD55 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, and (v) a heterologous CD59.
In some cases, the engineered immune cell (e.g., the engineered NK cell) as disclosed herein can comprise the heterologous CD59 and one or more of (e.g., 1, 2, 3, or all of) : (i) a heterologous CCL21, (ii) a heterologous IL-10, (iii) a heterologous CD46, and (iv) a heterologous CD55.
In one aspect, the present disclosure provides an engineered immune cell (e.g., an engineered NK cell) . The engineered immune cell can exhibit reduced expression or activity of one or more (e.g., 1, 2, 3, 4, 5, or more) endogenous immune checkpoint inhibitors (e.g., CD94, CD96, TGF beta receptor, SHIP2, etc. ) . In some cases, the engineered immune cell can exhibit reduced expression or activity of one or more (e.g., 1, 2, 3, 4, 5, or more) of: (i) endogenous CD94, (ii) endogenous CD96, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .
In some cases, the engineered immune cell can exhibit reduced expression or activity of endogenous CD94 and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (ii) endogenous CD96, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .
In some cases, the engineered immune cell can exhibit reduced expression or activity of endogenous CD96 and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (iii) endogenous TGF beta receptor, and (iv) endogenous SHIP (e.g., SHIP2) .
In some cases, the engineered immune cell can exhibit reduced expression or activity of endogenous TGF beta receptor and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (ii) endogenous CD96, and (iv) endogenous SHIP (e.g., SHIP2) .
In some cases, the engineered immune cell can exhibit reduced expression or activity of endogenous SHIP (e.g., SHIP2) and also reduced expression or activity of one or more of (e.g., 1, 2, or all of) : (i) endogenous CD94, (ii) endogenous CD96, and (iii) endogenous TGF beta receptor.
In some cases, the reduced expression or activity of the immune checkpoint inhibitor (e.g., CD94, CD96,  TGF beta receptor, SHIP2, etc. ) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the reduced expression or activity of the endogenous CD94 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the reduced expression or activity of the endogenous CD96 in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the reduced expression or activity of the endogenous TGF beta receptor in the engineered  immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the reduced expression or activity of the endogenous SHIP (e.g., SHIP2) in the engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be less than expression of the same by a control cell by at least or up to about 0.1-fold, at least or up to about 0.2-fold, at least or up to about 0.3-fold, at least or up to about 0.4-fold, at least or up to about 0.5-fold, at least or up to about 0.6-fold, at least or up to about 0.7-fold, at least or up to about 0.8-fold, at least or up to about 0.9-fold, at least or up to about 1-fold, at least or up to about 2-fold, at least or up to about 4-fold, at least or up to about 4-fold, at least or up to about 5-fold, at least or up to about 6-fold, at least or up to about 7-fold, at least or up to about 8-fold, at least or up to about 9-fold, at least or up to about 10-fold, at least or up to about 20-fold, at least or up to about 30-fold, at least or up to about 40-fold, at least or up to about 50-fold, at least or up to about 60-fold, at least or up to about 70-fold, at least or up to about 80-fold, at least or up to about 90-fold, at least or up to about 100-fold, at least or up to about 500-fold, at least or up to about 1,000-fold, at least or up to about 5,000-fold, or at least or up to about 10,000-fold.
In some cases, the engineered immune cell further comprises an immune activity component, the immune activity component comprises a reduced expression of one or more genes selected from the group consisting of TGFb receptor, TIGIT, PD1, PDL1, SIGLEC9, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, CD96, LAG3, and 2B4, and/or an enhanced expression of one or more genes selected from the group consisting of NCR, NKp30, NKp44, NKp46, NKG2D, NKp80, DNAM1, other NK activating receptors, chemokines or chemikine receptors.
In some cases, the engineered immune cell further comprises a safety switch component, the safety switch component comprise one or more members selected from the group consisting of caspase (e.g., caspase 3, 7, or 9) , thymidine kinase, cytosine deaminase, modified EGFR, B-cell CD20, and functional variants thereof.
In some cases, the engineered immune cell further comprises a tumor microenvironment component, the tumor microenvironment component comprises a reduced or an enhanced expression of one or more genes selected from the group consisting of KLRD1, CD96, CD244, CCR4, CCR9, CXCR6, CCR2, CXCR2, CX3CR1, KLRC2, TGFBR2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5,  KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, MIR21, MIR181B1, MIR181A1 MIR144, and MIR150.
6. Stem cells
Any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein can be derived from an isolated stem cell (e.g., an ESC) or an induced stem cell (iPSC) . The isolated stem cell or the induced stem cell can be modified (e.g., genetically modified) to generate the engineered immune cell. In some cases, pluripotency of stem cells (e.g., ESCs or iPSCs) can be determined, in part, by assessing pluripotency characteristics of the cells. Pluripotency characteristics can include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) expression of pluripotent stem cell markers including, but not limited to SSEA1 (mouse only) , SSEA3/4, SSEA5, TRA1-60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30 and/or CD50; (iv) ability to differentiate to all three somatic lineages (ectoderm, mesoderm and endoderm) ; (v) teratoma formation consisting of the three somatic lineages; and (vi) formation of embryoid bodies consisting of cells from the three somatic lineages.
In some cases, stem cells (e.g., ESCs or iPSCs) can be genetically modified to generate (e.g., induce differentiation into) CD34+ hematopoietic stem cells. The stem cells can be genetically modified to express any one of the heterologous polypeptides (e.g., cytokines, receptors, etc. ) as disclosed herein prior to, subsequent to, or during the induced hematopoietic stem cell differentiation. The stem cells can be genetically modified to reduce expression or activity of any one of the endogenous genes or polypeptides (e.g., cytokines, receptors, etc. ) as disclosed herein prior to, subsequent to, or during the induced hematopoietic stem cell differentiation. In some cases, such genetically modified CD34+hematopoietic stem cell is or is a source of any one of the engineered immune cell of the present disclosure.
In some examples, stem cells as disclosed herein can be cultured in APEL media with ROCKi (Y-27632) (e.g., at about 10 micromolar (μM) ) , SCF (e.g., at about 40 nanograms per milliner (ng/mL) of media) , VEGF (e.g., at about 20 ng/mL of media) , and BMP-4 (e.g., at about 20 ng/mL of media) to differentiate into CD34+ hematopoietic stem cells.
In some cases, the CD34+ hematopoietic stem cells (e.g., genetically modified with one or more features of any one of the engineered immune cell of the present disclosure) can be induced to differentiate in to a committed immune cell, such as T cells or NK cells. As such, in some cases, the induced differentiation process generates any one of the engineered NK cell of the present disclosure. In some examples, genetically modified CD34+ hematopoietic stem cells are cultured in the presence of IL-3 (e.g., about 5 ng/mL) , IL-7 (e.g., about 20 ng/mL) , IL-15 (e.g., about 10 ng/mL) , SCF (e.g., about 20 ng/mL) , and Flt3L (e.g., about 10 ng/mL) to differentiate into CD45+ NK cells.
In some cases, the CD45+ NK cells can be expanded in culture, e.g., in a media comprising IL-2, mbIL- 21 aAPC using Gas Permeable Rapid Expansion (G-Rex) platform.
In some cases, iPSC-derived NK cells as disclosed herein can be cultured with one or more heterologous cytokines comprising Il-2, IL-15, or IL-21. In some cases, iPSC-derived NK cells as disclosed herein can be cultured with (e.g., for cell expansion) one or more heterologous cytokines selected from the group consisting of Il-2, IL-15, and IL-21. In some cases, iPSC-derived NK cells as disclosed herein can be cultured with two or more heterologous cytokines selected from the group consisting of Il-2, IL-15, and IL-21 (e.g., IL-2 and IL-15, IL-2 and IL-21, or IL-15 and IL-21) , either simultaneously or sequentially in any order. In some cases, iPSC-derived NK cells as disclosed herein can be cultured with all of Il-2, IL-15, and IL-21, either simultaneous or sequentially in any order.
7. Gene editing or genetic material delivery
The gene editing moiety as disclosed herein can comprise a CRISPR-associated polypeptide (Cas) , zinc finger nuclease (ZFN) , zinc finger associate gene regulation polypeptides, transcription activator-like effector nuclease (TALEN) , transcription activator-like effector associated gene regulation polypeptides, meganuclease, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP) , an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof. In some embodiments, the actuator moiety comprises a Cas protein, and the system further comprises a guide RNA (gRNA) which complexes with the Cas protein. In some embodiments, the actuator moiety comprises an RBP complexed with a gRNA which is able to form a complex with a Cas protein. In some embodiments, the gRNA comprises a targeting segment which exhibits at least 80%sequence identity to a target polynucleotide. In some embodiments, the Cas protein substantially lacks DNA cleavage activity.
In some cases, a suitable gene editing moiety comprises CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN) ; transcription activator-like effector nucleases (TALEN) ; meganucleases; RNA-binding proteins (RBP) ; CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo) , archaeal Argonaute (aAgo) , and eukaryotic Argonaute (eAgo) ) ; any derivative thereof, any variant thereof; and any fragment thereof.
Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD) , Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12) , Cas10, Cas10d, Cas1O, Cas1Od, CasF, CasG, CasH, Cpf1, Csy1, Csy2, Csy3, Cse1 (CasA) , Cse2 (CasB) , Cse3 (CasE) , Cse4 (CasC) , Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.
In some cases, the gene editing moiety as disclosed herein can be fused with an additional functional moiety (e.g., to form a fusion moiety) , and non-limiting examples of a function of the additional functional moiety can include methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodelling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, and demyristoylation activity. For example, a fusion protein can be a fusion in a Cas protein and an effector or repressor functional moiety.
Alternatively or in addition to, gene editing (e.g., knock in) or delivery of heterologous genetic material can be achieved other viral and non-viral based gene transfer methods can be used to introduce nucleic acids in host cells (e.g., stem cells, hematopoietic stem cells, etc. as disclosed herein) . Such methods can be used to administer nucleic acids encoding polypeptide molecules of the present disclosure to cells in culture (or in a host organism) . Viral vector delivery systems can include DNA and RNA viruses, which can have either episomal or integrated genomes after delivery to the cell. Non-viral vector delivery systems can include DNA plasmids, RNA (e.g. a transcript of a vector described herein) , naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
RNA or DNA viral based systems can be used to target specific cells and traffick the viral payload to the nucleus of the cell. Viral vectors can be used to treat cells in vitro, and the modified cells can optionally be administered (ex vivo) . Alternatively, viral vectors can be administered directly (in vivo) to the subject. Viral based systems can include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome can occur with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, which can result in long term expression of the inserted transgene.
Methods of non-viral delivery of nucleic acids can include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides can be used.
Alternatively or in addition to, antisense oligonucleotides can be utilized to suppress or silence a target gene expression. Non-limiting examples of antisense oligonucleotides can include short hairpin RNA (shRNA) , microRNA (miRNA) , and small interfering RNA (siRNA) .
8. Characteristics of the engineered immune cell
The engineered immune cell or the population thereof has enhanced or acquired ADCC and optionally  at least one of the following characteristics: (ii) improved persistency and/or survival; (iii) reduced fratricide; (iv) improved tumor microenvironment; (v) reduced immunogenicity, and (vi) improved safety, in comparison to a control cell (e.g., non-engineered immune cell or immune cell lacking the relevant feature (s) or component (s) ) or population thereof.
In some cases, the engineered immune cell has an improved persistency and/or survival in at least one of following circumstances: a) in combination with antibodies targeting CD38; and b) under high oxidative stress conditions.
In some cases, the engineered immune cell has a reduced fratricide in at least one of following circumstances: a) reduced antigen expression in the cell when the cell comprise a CAR targeting the antigen; b) reduced relevant receptor expression when its ligand is expressed in the cell; and c) reduced ligand expression when its receptor is expressed in the cell.
In some cases, the engineered immune cell has a reduced immunogenicity in at least one of following circumstances: a) in combination with a monoclonal antibody to reduce immune cells including T cell, B cell, NK cell, and/or microphage of a host of the cell; b) in combination with a therapeutic agent to reduce immune cells including T cell, B cell, NK cell, and/or microphage of a host of the cell; and c) in combination with a therapeutic agent to reduce the activity of a host immune cell.
In some cases, the engineered immune cell has an improved tumor microenvironment in at least one of following circumstances: a) with therapeutic agents for microenvironment improvement; and b) in combination with chemokine antagonist or agonist to improve cell infiltration.
9. Method of preparing the engineered immune cell
In some cases, the present disclosure provides a method of preparing the engineered immune cell or population thereof, the method comprises the following steps: a) providing a cell which is (a) an induced pluripotent cell (iPSC) , a clonal iPSC, or an iPS cell line cell, or embryonic stem cell (ESC) ; (b) a derivative cell obtained from differentiating the cell of (a) ; or (c) an immune cell derived from an animal body; b) introducing the component as defined in any one of items 1-6 into the cell provided by step a) ; and c) optionally introducing at least one of the following features into the cell provided by step b) : (i) a chimeric antigen receptor (CAR) ; (ii) a persistence related component; (iii) a hypo-immunity regulator; (iv) an immune activity component; (v) a safety switch component; (vi) a gene modification to enhance or reduce the expression of one or more endogenous or heterologous genes; and (vii) a tumor microenvironment component.
10. Co-therapy, composition, or kit
The engineered immune cell (e.g., the engineered NK cell) of the present disclosure can be combined with a co-therapeutic agent to treat a subject in need thereof. In some cases, the engineered immune cell can be administered to the subject prior to, concurrent with, or subsequent to administration of the co-therapeutic agent to the subject.
In one aspect, the present disclosure provides a composition comprising (a) any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein and (b) a co-therapeutic agent (i.e., a separate therapeutic agent) (e.g., an antibody, such as anti-CD20 antibody or anti-PD1 antibody) . In some cases, the co-therapeutic agent comprises an anti-CD20 antibody.
In some cases, the present disclosure provides a composition or a kit comprising the engineered immune cell or population thereof. In some cases, the composition or a kit further comprises one or more therapeutic agents. In some cases, the one or more therapeutic agents are selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA) , mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD) .
In some cases, the checkpoint inhibitor comprised in the composition or the kit comprises: (a) one or more antagonists checkpoint molecules comprising PD-1, PDL-1, TIM-3, TIGIT, LAG-3, CTLA-4, 2B4, 4-1BB, 4-1BBL, A2aR, BATE, BTLA, CD39, CD47, CD73, CD94, CD96, CD160, CD200, CD200R, CD274, CEACAM1, CSF-1R, Foxpl, GARP, HVEM, IDO, EDO, TDO, LAIR-1, MICA/B, NR4A2, MAFB, OCT-2, Rara (retinoic acid receptor alpha) , TLR3, VISTA, NKG2A/HLA-E, or inhibitory KIR; (b) one or more of atezolizumab, avelumab, durvalumab, ipilimumab, IPH4102, IPH43, IPH33, lirimumab, monalizumab, nivolumab, pembrolizumab, and their derivatives or functional equivalents; or (c) at least one of atezolizumab, nivolumab, and pembrolizumab.
In some cases, the therapeutic agents comprised in the composition or the kit comprise one or more of azacytidine, venetoclax, MG132, decitabine, dasatinib, cytarabine, pomalidomide, and the derivatives thereof.
In some cases, the antibody comprised in the composition or the kit comprises: (a) anti-CD20, anti-HER2, anti-CD52, anti-EGFR, anti-CD 123, anti-GD2, anti-PDL1, and/or anti-CD38 antibody; (b) one or more of rituximab, veltuzumab, ofatumumab, ublituximab, ocaratuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, certuximab, dinutuximab, avelumab, daratumumab, isatuximab, MOR202, 7G3, CSL362, elotuzumab, and their humanized or Fc modified variants or fragments and their functional equivalents and biosimilars.
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., GLEEVECTM (imatinib mesylate) ) , a COX-2 inhibitor (e.g., celecoxib) , interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR-β, BlyS, APRIL, BCMA receptor (s) , TRAIL/Apo2, other bioactive and organic chemical agents, and the like.
In some cases, cytotoxic agent generally refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. Non-limiting examples of a cytotoxic agent can include  radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and radioactive isotopes of Lu) , chemotherapeutic agents, e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide) , doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin. Non-limiting examples of a chemotherapeutic agent can include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone) ; delta-9-tetrahydrocannabinol (dronabinol, ) ; beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecanCPT-11 (irinotecan, ) , acetylcamptothecin, scopolectin, and 9-aminocamptothecin) ; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues) ; podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8) ; dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1) ; eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores) , aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin) , epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU) ; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aidophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;  lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, Oreg. ) ; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2′, 2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) ; urethan; vindesine dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ) ; thiotepa; taxoids, for example taxanes includingpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N. J. ) , ABRAXANETM Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill. ) , anddocetaxel (Rorer, Antony, France) ; chloranbucil; gemcitabine6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastineplatinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristineoxaliplatin; leucovovin; vinorelbinenovantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO) ; retinoids such as retinoic acid; capecitabinepharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin. Additional chemotherapeutic agents include the cytotoxic agents useful as antibody drug conjugates, such as maytansinoids (DM1, for example) and the auristatins MMAE and MMAF, for example.
Examples of a chemotherapeutic agent can also include “anti-hormonal agents” or “endocrine therapeutics” that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs) , including, for example, tamoxifen (includingtamoxifen) , raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, 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 asand ELIGARD) leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, andanastrozole. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, or) , etidronate, NE-58095, zoledronic acid/zoledronate, alendronate,  pamidronate, tiludronate, orrisedronate; as well as troxacitabine (a1, 3-dioxolane nucleoside cytosine analog) ; antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGFR) ; vaccines such as vaccine and gene therapy vaccines, for example, vaccine, vaccine, andvaccine; topoisomerase 1 inhibitor; rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016) ; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Examples of a chemotherapeutic agent can also include antibodies such as alemtuzumab (Campath) , bevacizumab (Genentech) ; cetuximab (Imclone) ; panitumumab (Amgen) , rituximab (Genentech/Biogen Idec) , pertuzumab (2C4, Genentech) , trastuzumab (Genentech) , tositumomab (Bexxar, Corixia) , and the antibody drug conjugate, gemtuzumab ozogamicin (Wyeth) . Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, feMzumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) which is a recombinant exclusively human-sequence, full-length IgG1λ antibody genetically modified to recognize interleukin-12 p40 protein.
Examples of a chemotherapeutic agent can also include “tyrosine kinase inhibitors” such as an EGFR-targeting agent (e.g., small molecule, antibody, etc. ) ; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724, 714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI) ; dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline) , an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis) ; pan-HER inhibitors such as canertinib (CI-1033; Pharmacia) ; Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate (available from Glaxo SmithKline) ; multi-targeted tyrosine kinase inhibitors such as sunitinib (available  from Pfizer) ; VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787/ZK222584, available from Novartis/Schering AG) ; MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia) ; quinazolines, such as PD 153035, 4- (3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4- (phenylamino) -7H-pyrrolo [2, 3-d] pyrimidines; curcumin (diferuloyl methane, 4, 5-bis (4-fluoroanilino) phthalimide) ; tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber) ; antisense molecules (e.g., those that bind to HER-encoding nucleic acid) ; quinoxalines (U.S. Pat. No. 5,804,396) ; tryphostins (U.S. Pat. No. 5,804,396) ; ZD6474 (Astra Zeneca) ; PTK-787 (Novartis/Schering AG) ; pan-HER inhibitors such as CI-1033 (Pfizer) ; Affinitac (ISIS 3521; Isis/Lilly) ; imatinib mesylatePKI 166 (Novartis) ; GW2016 (Glaxo SmithKline) ; CI-1033 (Pfizer) ; EKB-569 (Wyeth) ; Semaxinib (Pfizer) ; ZD6474 (AstraZeneca) ; PTK-787 (Novartis/Schering AG) ; INC-1C11 (Imclone) ; and rapamycin (sirolimus, ) .
Examples of a chemotherapeutic agent can also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.
Examples of a chemotherapeutic agent can also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate: immune selective anti-inflammatory peptides (ImSAIDs) such as phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG) (IMULAN BioTherapeutics, LLC) ; anti-rheumatic drugs such as azathioprine, ciclosporin (cyclosporine A) , D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanerceptinfliximab adalimumabcertolizumab pegolgolimumab Interleukin 1 (IL-1) blockers such as anakinraT-cell costimulation blockers such as abataceptInterleukin 6 (IL-6) blockers such as tocilizumab Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon alpha (IFN) blockers such as rontalizumab; beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as  Anti-M1 prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTa/β2 blockers such as Anti-lymphotoxin alpha (LTa) ; miscellaneous investigational agents such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or famesyl transferase inhibitors (L-739749, L-744832) ; polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, ) ; beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin) ; podophyllotoxin; tegafurbexarotenebisphosphonates such as clodronate (for example, or ) , etidronateNE-58095, zoledronic acid/zoledronatealendronatepamidronatetiludronateor risedronate and epidermal growth factor receptor (EGF-R) ; vaccines such asvaccine; perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib) , proteosome inhibitor (e.g., PS341) ; CCI-779; tipifamib (R11577) ; orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium pixantrone; famesyltransferase inhibitors such as lonafamib (SCH 6636, SARASARTM) ; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.
In some cases, “growth inhibitory agent” generally refers to a compound or composition which inhibits growth and/or proliferation of a cell (e.g., a cell whose growth is dependent on PD-L1 expression) either in vitro or in vivo. The growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase. Non-limiting examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase) , such as agents that induce G1 arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine) , taxanes, and topoisomerase II inhibitors such as the anthracycline antibiotic doxorubicin ( (8S-cis) -10- [ (3-amino-2, 3, 6-trideoxy-α-L-lyxo-hexapyranosyl) oxy] -7, 8, 9, 10-tetrahydro-6, 8, 11-trihydroxy-8- (hydroxyacetyl) -1-methoxy-5, 12-naphthacenedione) , epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (Rhone-Poulenc Rorer) , derived from the European yew, is a semisynthetic analogue of paclitaxel (Bristol-Myers Squibb) . Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
In some cases, the composition or the kit of the present invention comprises the cell or population thereof of any one of items 1-16. In some cases, the composition or the kit further comprises an antibody selected from the group consisting of: 20- (74) - (74) (milatuzumab; veltuzumab) , 20-2b-2b, 3F8, 74- (20) - (20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, zlintuzumab) , actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab,  afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003/SIM-BD0801 (sevacizumab) , APX005M, arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also referred to as tocilizumab) , atorolimumab, Avelumab, B-701, bapineuzumab, basiliximab, bavituximab, BAY1129980, BAY1187982, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, Betalutin (177Lu-tetraxetan-tetulomab) , bevacizumab, BEVZ92 (bevacizumab biosimilar) , bezlotoxumab, BGB-A317, BHQ880, BI 836880, BI-505, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab) , CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch. 14.18, cidfusituzumab, cidtuzumab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, Cotara (iodine I-131 derlotuximab biotin) , cR6261, crenezumab, DA-3111 (trastuzumab biosimilar) , dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, Daratumumab Enhanze (daratumumab) , Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, Depatuxizumab, Depatuxizumab mafodotin, derlotuximab biotin, detumomab, DI-B4, dinutuximab, diridavumab, DKN-01, DMOT4039A, dorlimomab aritox, drozitumab, DS-1123, DS-8895, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, Etanercept, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, femzumab, fezakinumab, FF-21101, FGFR2 AntibodyDrug Conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, FPA144, fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, Gerilimzumab, gevokizumab, girentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hu14.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (claudiximab) , imalumab, IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan) , IMMU-114, ImmuTune IMP701 Antagonist  Antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, Ipafricept, IPH4102, ipilimumab, iratumumab, isatuximab, Istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2/L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L-DOS47, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (inebilizumab) , MEDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006/S80880, MGD007, MGD009, MGD011, milatuzumab, Milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, nolovizumab, NOV-10, numavizumab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, OX002/MEN1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, PankoMabGEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pecfusituzumab, pectuzumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab) , PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, quilizumab, racotumomab, radretumab, rafivirumab, ralivizumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/SAR439684, reslivizumab, reslizumab, resyvizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, RO7009789, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-CD19A, SGN-CD19B, SGNCD33A, SGN-CD70A, SGN-LIV1A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, SYD985, SYM004 (futuximab and modotuximab) , Sym015, TAB08, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, Tanibirumab, taplitumomab paptox, tarextumab, TB-403, tefibazumab, Teleukin, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab,  tesidolumab, tetulomab, TG-1303, TGN1412, Thorium-227-Epratuzumab Conjugate, ticilimumab, tigatuzumab, tildrakizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH 011, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tucusituzumab, tuvirumab, U3-1565, U3-1784, ublituximab, ulocuplumab, umavizumab, urelumab, urtoxazumab, ustekinumab, Vadastuximab Talirine, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB-101, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.
11. Methods for treating diseases and uses
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, infection disease, autoimmune disease, tissue degeneration, fibrosis, etc. ) . A cell (e.g., a stem cell or a committed adult cell) can be obtained from the subject, and such cell can be cultured ex vivo and genetically modified to generate any subject engineered immune cell (e.g., any engineered NK cell) as disclosed herein. Subsequently, the engineered immune cell can be administered to the subject for adaptive immunotherapy.
The subject can be treated (e.g., administered with) a population of engineered immune cells (e.g., engineered NK cells) of the present disclosure for at least or up to about 1 dose, at least or up to about 2 doses, at least or up to about 3 doses, at least or up to about 4 doses, at least or up to about 5 doses, at least or up to about 6 doses, at least or up to about 7 doses, at least or up to about 8 doses, at least or up to about 9 doses, or at least or up to about 10 doses.
In one aspect, the present disclosure provides a method comprising (a) obtaining a cell from a subject; and (b) generating, from the cell, any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein. In some cases, the cell obtained from the subject is ESC. In some cases, the cell (e.g., a fibroblast, such as an adult skin fibroblast) obtained from the subject is modified and transformed into an iPSC.
In one aspect, the present disclosure provides a method comprising administering to a subject in need thereof a population of NK cells comprising any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein. In some cases, the method can further comprise administering to the subject a co-therapeutic agent (e.g., a chemotherapeutic agent, anti-CD20 antibody, etc. ) .
In one aspect, the present disclosure provides a method comprising administering to a subject in need thereof any one of the composition disclosed herein. In some cases, the composition can comprise (i) any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein and (ii) a co-therapeutic agent (e.g., a chemotherapeutic agent, anti-CD20 antibody, etc. ) .
Any one of the methods disclosed herein can be utilized to treat a target cell, a target tissue, a target  condition, a target disease of a subject, or can be used for anti-aging.
A target disease can be a viral, bacterial, and/or parasitic infection; inflammatory and/or autoimmune disease; or neoplasm such as a cancer and/or tumor.
A target cell can be a diseased cell. A diseased cell can have altered metabolic, gene expression, and/or morphologic features. A diseased cell can be a cancer cell, a diabetic cell, and an apoptotic cell. A diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.
A variety of target cells can be killed using any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein. A target cell can include a wide variety of cell types. A target cell can be in vitro. A target cell can be in vivo. A target cell can be ex vivo. A target cell can be an isolated cell. A target cell can be a cell inside of an organism. A target cell can be an organism. A target cell can be a cell in a cell culture. A target cell can be one of a collection of cells. A target cell can be a mammalian cell or derived from a mammalian cell. A target cell can be a rodent cell or derived from a rodent cell. A target cell can be a human cell or derived from a human cell. A target cell can be a prokaryotic cell or derived from a prokaryotic cell. A target cell can be a bacterial cell or can be derived from a bacterial cell. A target cell can be an archaeal cell or derived from an archaeal cell. A target cell can be a eukaryotic cell or derived from a eukaryotic cell. A target cell can be a pluripotent stem cell. A target cell can be a plant cell or derived from a plant cell. A target cell can be an animal cell or derived from an animal cell. A target cell can be an invertebrate cell or derived from an invertebrate cell. A target cell can be a vertebrate cell or derived from a vertebrate cell. A target cell can be a microbe cell or derived from a microbe cell. A target cell can be a fungi cell or derived from a fungi cell. A target cell can be from a specific organ or tissue.
A target cell can be a stem cell or progenitor cell. Target cells can include stem cells (e.g., adult stem cells, embryonic stem cells, induced pluripotent stem (iPS) cells) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc. ) . Target cells can include mammalian stem cells and progenitor cells, including rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, etc. Clonal cells can comprise the progeny of a cell. A target cell can comprise a target nucleic acid. A target cell can be in a living organism. A target cell can be a genetically modified cell. A target cell can be a host cell.
A target cell can be a totipotent stem cell, however, in some embodiments of this disclosure, the term “cell” may be used but may not refer to a totipotent stem cell. A target cell can be a plant cell, but in some embodiments of this disclosure, the term “cell” may be used but may not refer to a plant cell. A target cell can be a pluripotent cell. For example, a target cell can be a pluripotent hematopoietic cell that can differentiate into other cells in the hematopoietic cell lineage but may not be able to differentiate into any other non-hematopoietic cell. A target cell may be able to develop into a whole organism. A target cell may or may not be able to develop into a whole organism. A target cell may be a whole  organism.
A target cell can be a primary cell. For example, cultures of primary cells can be passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, 15 times or more. Cells can be unicellular organisms. Cells can be grown in culture.
A target cell can be a diseased cell. A diseased cell can have altered metabolic, gene expression, and/or morphologic features. A diseased cell can be a cancer cell, a diabetic cell, and a apoptotic cell. A diseased cell can be a cell from a diseased subject. Exemplary diseases can include blood disorders, cancers, metabolic disorders, eye disorders, organ disorders, musculoskeletal disorders, cardiac disease, and the like.
If the target cells are primary cells, they may be harvested from an individual by any method. For example, leukocytes may be harvested by apheresis, leukocytapheresis, density gradient separation, etc. Cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be harvested by biopsy. An appropriate solution may be used for dispersion or suspension of the harvested cells. Such solution can generally be a balanced salt solution, (e.g. normal saline, phosphate-buffered saline (PBS) , Hank's balanced salt solution, etc. ) , conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration. Buffers can include HEPES, phosphate buffers, lactate buffers, etc. Cells may be used immediately, or they may be stored (e.g., by freezing) . Frozen cells can be thawed and can be capable of being reused. Cells can be frozen in a DMSO, serum, medium buffer (e.g., 10%DMSO, 50%serum, 40%buffered medium) , and/or some other such common solution used to preserve cells at freezing temperatures.
Non-limiting examples of cells which can be target cells include, but are not limited to, lymphoid cells, such as B cell, T cell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, T helper cell) , Natural killer cell, cytokine induced killer (CIK) cells (see e.g. US20080241194) ; myeloid cells, such as granulocytes (Basophil granulocyte, Eosinophil granulocyte, Neutrophil granulocyte/Hypersegmented neutrophil) , Monocyte/Macrophage, Red blood cell (Reticulocyte) , Mast cell, Thrombocyte/Megakaryocyte, Dendritic cell; cells from the endocrine system, including thyroid (Thyroid epithelial cell, Parafollicular cell) , parathyroid (Parathyroid chief cell, Oxyphil cell) , adrenal (Chromaffin cell) , pineal (Pinealocyte) cells; cells of the nervous system, including glial cells (Astrocyte, Microglia) , Magnocellular neurosecretory cell, Stellate cell, Boettcher cell, and pituitary (Gonadotrope, Corticotrope, Thyrotrope, Somatotrope, Lactotroph) ; cells of the Respiratory system, including Pneumocyte (Type I pneumocyte, Type II pneumocyte) , Clara cell, Goblet cell, Dust cell; cells of the circulatory system, including Myocardiocyte, Pericyte; cells of the digestive system, including stomach (Gastric chief cell, Parietal cell) , Goblet cell, Paneth cell, G cells, D cells, ECL cells, I cells, K cells, S cells; enteroendocrine cells, including enterochromaffm cell, APUD cell, liver (Hepatocyte, Kupffer cell) , Cartilage/bone/muscle; bone cells, including Osteoblast, Osteocyte, Osteoclast, teeth (Cementoblast, Ameloblast) ; cartilage cells, including Chondroblast, Chondrocyte; skin cells, including  Trichocyte, Keratinocyte, Melanocyte (Nevus cell) ; muscle cells, including Myocyte; urinary system cells, including Podocyte, Juxtaglomerular cell, Intraglomerular mesangial cell/Extraglomerular mesangial cell, Kidney proximal tubule brush border cell, Macula densa cell; reproductive system cells, including Spermatozoon, Sertoli cell, Leydig cell, Ovum; and other cells, including Adipocyte, Fibroblast, Tendon cell, Epidermal keratinocyte (differentiating epidermal cell) , Epidermal basal cell (stem cell) , Keratinocyte of fingernails and toenails, Nail bed basal cell (stem cell) , Medullary hair shaft cell, Cortical hair shaft cell, Cuticular hair shaft cell, Cuticular hair root sheath cell, Hair root sheath cell of Huxley's layer, Hair root sheath cell of Henle's layer, External hair root sheath cell, Hair matrix cell (stem cell) , Wet stratified barrier epithelial cells, Surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, Urinary epithelium cell (lining urinary bladder and urinary ducts) , Exocrine secretory epithelial cells, Salivary gland mucous cell (polysaccharide-rich secretion) , Salivary gland serous cell (glycoprotein enzyme-rich secretion) , Von Ebner's gland cell in tongue (washes taste buds) , Mammary gland cell (milk secretion) , Lacrimal gland cell (tear secretion) , Ceruminous gland cell in ear (wax secretion) , Eccrine sweat gland dark cell (glycoprotein secretion) , Eccrine sweat gland clear cell (small molecule secretion) . Apocrine sweat gland cell (odoriferous secretion, sex-hormone sensitive) , Gland of Moll cell in eyelid (specialized sweat gland) , Sebaceous gland cell (lipid-rich sebum secretion) , Bowman's gland cell in nose (washes olfactory epithelium) , Brunner's gland cell in duodenum (enzymes and alkaline mucus) , Seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm) , Prostate gland cell (secretes seminal fluid components) , Bulbourethral gland cell (mucus secretion) , Bartholin's gland cell (vaginal lubricant secretion) , Gland of Littre cell (mucus secretion) , Uterus endometrium cell (carbohydrate secretion) , Isolated goblet cell of respiratory and digestive tracts (mucus secretion) , Stomach lining mucous cell (mucus secretion) , Gastric gland zymogenic cell (pepsinogen secretion) , Gastric gland oxyntic cell (hydrochloric acid secretion) , Pancreatic acinar cell (bicarbonate and digestive enzyme secretion) , Paneth cell of small intestine (lysozyme secretion) , Type II pneumocyte of lung (surfactant secretion) , Clara cell of lung, Hormone secreting cells, Anterior pituitary cells, Somatotropes, Lactotropes, Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary cell, Magnocellular neurosecretory cells, Gut and respiratory tract cells, Thyroid gland cells, thyroid epithelial cell, parafollicular cell, Parathyroid gland cells, Parathyroid chief cell, Oxyphil cell, Adrenal gland cells, chromaffin cells, Ley dig cell of testes, Theca interna cell of ovarian follicle, Corpus luteum cell of ruptured ovarian follicle, Granulosa lutein cells, Theca lutein cells, Juxtaglomerular cell (renin secretion) , Macula densa cell of kidney, Metabolism and storage cells, Barrier function cells (Lung, Gut, Exocrine Glands and Urogenital Tract) , Kidney, Type I pneumocyte (lining air space of lung) , Pancreatic duct cell (centroacinar cell) , Nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc. ) , Duct cell (of seminal vesicle, prostate gland, etc. ) , Epithelial cells lining closed internal body cavities, Ciliated cells with propulsive function, Extracellular matrix secretion cells, Contractile  cells; Skeletal muscle cells, stem cell, Heart muscle cells, Blood and immune system cells, Erythrocyte (red blood cell) , Megakaryocyte (platelet precursor) , Monocyte, Connective tissue macrophage (various types) , Epidermal Langerhans cell, Osteoclast (in bone) , Dendritic cell (in lymphoid tissues) , Microglial cell (in central nervous system) , Neutrophil granulocyte, Eosinophil granulocyte, Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell, Cytotoxic T cell, Natural Killer T cell, B cell, Natural killer cell, Reticulocyte, Stem cells and committed progenitors for the blood and immune system (various types) , Pluripotent stem cells, Totipotent stem cells, Induced pluripotent stem cells, adult stem cells, Sensory transducer cells, Autonomic neuron cells, Sense organ and peripheral neuron supporting cells, Central nervous system neurons and glial cells, Lens cells, Pigment cells, Melanocyte, Retinal pigmented epithelial cell, Germ cells, Oogonium/Oocyte, Spermatid, Spermatocyte, Spermatogonium cell (stem cell for spermatocyte) , Spermatozoon, Nurse cells, Ovarian follicle cell, Sertoli cell (in testis) , Thymus epithelial cell, Interstitial cells, and Interstitial kidney cells.
Of particular interest are cancer cells. In some embodiments, the target cell is a cancer cell. Non-limiting examples of cancer cells include cells of cancers including Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer,  Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor,  Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, Wilms'tumor, and combinations thereof. In some embodiments, the targeted cancer cell represents a subpopulation within a cancer cell population, such as a cancer stem cell. In some embodiments, the cancer is of a hematopoietic lineage, such as a lymphoma. The antigen can be a tumor associated antigen.
In some cases, the subject being treated with any one of the engineered immune cell (e.g., engineered NK cell) of the present disclosure can have or can be suspected of having an autoimmune disease. Non-limiting examples of an autoimmune disease can include acute disseminated encephalomyelitis (ADEM) , acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, antibody-mediated transplantation rejection, anti-GBM/Anti-TBM nephritis, antiphospholipid syndrome (APS) , autoimmune angioedema, autoimmune aplastic anemia, autoimmune dysautonomia, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED) , autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP) , autoimmune thyroid disease, autoimmune urticaria, axonal &neuronal neuropathies, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castleman disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP) , chronic recurrent multifocal ostomyelitis (CRMO) , Churg-Strauss syndrome, cicatricial pemphigoid/benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST disease, essential mixed  cryoglobulinemia, demyelinating neuropathies, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica) , discoid lupus, Dressler's syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis) , glomerulonephritis, goodpasture's syndrome, granulomatosis with polyangiitis (GPA) , Graves'disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia, hypergammaglobulinemia, idiopathic thrombocytopenic purpura (ITP) , IgA nephropathy, IgG4-related sclerosing disease, immunoregulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes (type 1) , interstitial cystitis, juvenile arthritis, juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD) , lupus (SLE) , lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD) , monoclonal gammopathy of undetermined significance (MGUS) , Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's ) , neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus) , paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH) , Parry Romberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheral uveitis) , pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, type I, II, &III autoimmune polyglandular syndromes, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynauds phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm &testicular autoimmunity, stiff person syndrome, subacute bacterial endocarditis (SBE) , Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis/Giant cell arteritis, thrombocytopenic purpura (TTP) , Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD) , uveitis, vasculitis, vesiculobullous dermatosis, vitiligo, Waldenstrom's macroglobulinemia (WM) , and Wegener's granulomatosis (Granulomatosis with Polyangiitis (GPA) ) .
In some cases, the autoimmune disease comprises one or more members selected from the group comprising rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE) , myasthenia gravis, multiple sclerosis, scleroderma, Addison's Disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS) , Waldenstrom's macroglobulinemia (WM) , chronic inflammatory demyelinating  polyradiculoneuropathy (CIDP) , Hashimoto's Encephalopathy (HE) , Hashimoto's Thyroiditis, Graves'Disease, Wegener's Granulomatosis, and antibody-mediated transplantation rejection (e.g., for tissue transplants such as renal transplant) . In examples, the autoimmune disease can be type 1 diabetes, lupus, or rheumatoid arthritis.
In some cases, the target disease is T cell leukemia (TCL) , such as T-cell acute lymphoblastic leukemia (T-ALL) . For example, any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein that comprises one or more of: (i) a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to CD7 as disclosed herein, (ii) a heterologous cytokine (e.g., IL-15) as disclosed herein, and (iii) reduced expression or activity of endogenous gene encoding the same cytokine as the heterologous cytokine (e.g., endogenous CD7) can be administered to a subject in need thereof to treat TCL.
In some cases the target disease is acute myeloid leukemia (AML) . For example, any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein that comprises one or more of:
(i) a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to an antigen (e.g., CD33 and/or CD123) as disclosed herein, (ii) a heterologous cytokine (e.g., IL-15) as disclosed herein, and (iii) a CD16 variant for enhanced CD16 signaling as disclosed herein can be administered to a subject in need thereof to treat AML.
In some cases, the target disease is non-Hodgkin’s lymphoma (NHL) .
In some cases, the target disease is chronic lymphocytic leukemia (CLL) .
In some cases, the target disease is B-cell leukemia (BCL) . For example, any one of the engineered immune cell (e.g., the engineered NK cell) disclosed herein that comprises one or more of: (i) a chimeric polypeptide receptor comprising an antigen binding domain capable of binding to CD19 as disclosed herein, (ii) a heterologous cytokine (e.g., IL-15) as disclosed herein, and (iii) a CD16 variant for enhanced CD16 signaling as disclosed herein can be administered to a subject in need thereof to treat BCL.
In some cases, the target disease is non-small-cell lung carcinoma (NSCLC) .
In some cases, the target cells form a tumor (i.e., a solid tumor) . A tumor treated with the methods herein can result in stabilized tumor growth (e.g., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20%in size, and/or do not metastasize) . In some cases, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some cases, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some cases, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. In some cases, the size of a tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more. In some cases, the tumor is completely eliminated, or reduced below a level of detection. In some cases, a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following treatment. In some cases, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following  treatment. In some cases, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.
In some cases, the present disclosure provide a method for treating disease in a subject suitable for adoptive cell therapy by using the engineered immune cell or population thereof or the composition or the kit of the present disclosure, and/or a use of the engineered immune cell or population thereof or the composition or the kit of the present disclosure in the manufacture of a medicament for treating a disease in a subject suitable for adoptive cell therapy, wherein the disease is selected from the group consisting of an autoimmune disorder; a hematological malignancy; a solid tumor; cancer, or a virus infection. In some cases, the present disclosure provide a method for reducing or preventing allorejection by using the engineered immune cell or population thereof or the composition or the kit of the present disclosure, and/or a use of the engineered immune cell or population thereof or the composition or the kit of the present disclosure in the manufacture of a medicament for reducing or preventing allorejection. In some cases, the present disclosure provide a method for treating acute myeloid leukemia in a subject by using the engineered immune cell or population thereof in combination with an anti-CD38 monoclonal antibody and/or azacytidine, and/or a use of the engineered immune cell or population thereof in combination with an anti-CD38 monoclonal antibody and/or azacytidine in the manufacture of a medicament for treating acute myeloid leukemia.
In some cases, the present disclosure provide a method for treating disease in a subject by using a composition or a kit comprising an immune cell derived from an animal body and one or more therapeutic agents, and/or a use of a composition or a kit comprising an immune cell derived from an animal body and one or more therapeutic agents in the manufacture of a medicament for treating a disease in a subject, wherein the disease is selected from the group consisting of an autoimmune disorder; a hematological malignancy, preferably acute myeloid leukemia; a solid tumor; cancer, or a virus infection. In some cases, the one or more therapeutic agents are selected from the group consisting of a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA) , mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent or a radioactive moiety, or an immunomodulatory drug (IMiD) .
EXAMPLES
Example 1: Engineered NK cells
Table 1 illustrates examples of engineered NK cells with or without genetic modifications, along with possible functions, and therapeutic indications. Examples of therapeutic indications can include acute myeloid leukemia (AML) , multiple myeloma (MM) , Myelodysplastic syndrome (MDS) , B cell leukemia, T cell leukemia, solid tumor, blood cancer, infection disease, autoimmune diseases, and/or aging.
TABLE 1
Example 2: Component for enhancing ADCC
For improved adaptive immunotherapy, NK cells can be engineered to exhibit enhanced ADCC. Exemplary amino acid sequences are:
SEQ ID NO. 1 (Ectodomain of CD32a with H131 -variant +Transmembrane domain of CD16+Intracellular domain of CD16)
SEQ ID NO. 2 (Ectodomain of CD16 with F158V+Transmembrane domain of CD32a+Intracellular domain of CD32a)
SEQ ID NO. 3 (Ectodomain of CD64+Transmembrane domain of CD32a+Intracellular domain of CD32a)
SEQ ID NO: 4 (Ectodomain of CD32a with H131 variant +Transmembrane domain of CD32a+Intracellular domain of CD32a)
SEQ ID NO: 5 (Ectodomain of CD16 with F158V+Transmembrane domain of CD89+Intracellular  domain of CD89)
SEQ ID NO: 6 (Ectodomain of CD64+Transmembrane domain of CD89+Intracellular domain of CD89)
SEQ ID NO: 7 (Ectodomain of CD32a with H131 variant +Transmembrane domain of CD89+Intracellular domain of CD89)
2.1 FcγR fusion designs to enhance ADCC
The following FcγR fusions are designed and constructed to enhance ADCC (see Table 2) .
TABLE 2. Structures of chimeric Fc receptors for ADCC

2.2 Transgene validation
(1) Copy number of chimeric receptor genes in transduced NK-92 cells are shown in Table 3.
Table 3. Copy number of chimeric receptor genes in transduced NK-92 cells
(2) mRNA level of chimeric receptor genes in transduced NK-92 cells evaluated by qPCR (see Figure 2) ;
(3) Surface expression of chimeric receptors in transduced NK-92 cells evaluated by FACS (see Figures 3A-3C) .
2.3 FcR5 (CD16/32) and FcR6 (CD64/32) can mediate ADCC comparable to hnCD16 and CD64/16 ADCC of trastuzumab mediated by FcR-engineered NK92 cells against SKOV3 at the effector to target ratio of 1: 1 following incubation for 5 hours (see Figure 4) .
2.4 FcR5 (CD16/32) is superior to hnCD16 and CD64/16 to mediate ADCC
(1) ADCC mediated by FcR-engineered NK92 cells with different dose of trastuzumab against SKOV3 at the effector to target ratio of 1: 1 following incubation for 5 hours (see Figures 5A-5B) .
(2) Comparison of the ADCC mediated by different FcR edits on NK92 cells (see Figure 5C) .
2.5 All chimeric FcRs except FcR1 (wt CD16) exhibited stable surface expression after activation. FcR-engineered NK92 cells were stimulated as indicated for 4 hours, and percentage (A) and MFI (B) of each FcR was determined by flow cytometry (see Figure 6) .
2.6 Summary of ADCC edits on NK92
The summary of ADCC edits on NK92 are shown in Table 4.
Table 4. Summary of ADCC edits on NK92

2.7 Chimeric Fc receptors were overexpressed on iPSC by piggybac system
(1) Copy number of chimeric receptor genes in engineered iPSC clones is shown in Table 5.
Table 5. Copy number of chimeric receptor genes in engineered iPSC clones
(2) Surface expression of chimeric receptors in engineered iPSC clones evaluated by FACS (see Figures 7A-7B) .
(3) The expression of TRA-181, a human pluripotent stem cell marker, was evaluated by FACS (see Figure 8) .
2.8 iPSC expressing chimeric Fc receptors were successfully differentiated to NK cells.
(1) iPSC expressing chimeric Fc receptors were differentiated into CD56+ NK cells (see Figure 9) .
2.9 CD64/32 showed devoid activation-induced cleavage, and enhanced expression under nonspecific stimulation
(1) FcR-expressing iNK cells were stimulated as indicated for 4 hours, and percentage (A) and MFI (B) of each FcR was determined by flow cytometry (see FIG. 18A-10B) .
2.10 CD64/32 expressed on iPSC-NK demonstrated robust ADCC comparable to hnCD16 and CD64/16
(1) ADCC mediated by FcR-engineered iPSC-NK cells with different dose of Rituximab against Raji cells at the effector to target ratio of 1: 1 following incubation for 5 hours (see Figure 11) .
(2) Serial killing assay to examine the ADCC of engineered iPSC-NK cells with rituximab. NK cells were re-challenged with Raji cells at the effector to target ratio of 1: 1 (see Figure 12) .
Example 3: Engineered NK cells with enhanced survival and/or persistency
For enhanced persistence of adaptive immunotherapy, NK cells can be engineered to comprise at least (i) a heterologous transcription factor (e.g., STAT) and (ii) reduced expression or activity of an endogenous cytokine receptor (e.g., endogenous IL receptor, such as IL-17R) . Having the combination of (i) and (ii) in the engineered NK cell can synergistically improve persistence of the engineered NK cells as compared to having either one of (i) and (ii) alone, or none.
Engineered NK cells:
NK cells are generated from isolated ESCs or iPSCs. The NK cells are engineered to express a heterologous STAT (e.g., STAT3 and/or STAT5B) . A gene encoding the heterologous STAT is incorporated into the NK cell’s genome via either viral transduction or via action of a gene editing moiety as disclosed herein. The NK cells are also engineered to exhibit reduced expression or activity of endogenous IL-17R (i.e., STAT3+IL-17R-NK cells) . NK cells with either one of (i) the heterologous STAT and (ii) reduced expression or activity of IL-17R, or non-engineered NK cells are used as a control.
In vitro survival and persistency:
The engineered STAT3+IL-17R-NK cells can be cultured in vitro to assess viability and growth (or proliferative capacity) of the engineered STAT3+IL-17R-NK cells in absence of an exogenous cytokine. The NK cells are cultured in culture medium without the addition of exogenous cytokines for 3-6 weeks. The engineered STAT3+IL-17R-NK cells exhibit a significantly higher number of NK cells as compared to the control cells, indicating the enhanced survival and persistency of the engineered STAT3+IL-17R-NK cells in vitro.
In vivo pharmacokinetics (PK) :
The engineered STAT3+IL-17R-NK cells can be administered in NCG mice having a Raji xenograft model. NCG mice are triple immunodeficient and lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function to host the xenograft model. The engineered STAT3+IL-17R-NK cells and the control cells are each administered into the respective Raji xenograft model mice via intravenous (IV) tail vein injection, at a dose of about 1 × 106 cells per animal. Mice injected with the engineered STAT3+IL-17R-NK cells exhibit higher NK cell concentrations in the peripheral blood from about 7 days to about 28 days post-infusion, demonstrating the enhanced survival and persistency of the engineered STAT3+IL-17R-NK cells in vivo.
Generation of other engineered NK cells:
Cells of interest (e.g., stem cells, immune cells, such as NK cells, etc. ) can be engineered with (i) reduced expression of at least one endogenous gene (e.g., loss-of-function of one or more immune regulating polypeptides) and/or (ii) enhanced or introduced expression of at least one additional gene (e.g., at least one transgene encoding one or more additional immune regulating polypeptides) . For example, NK cells (e.g., cord blood NK (CBNK) cells, NK cells derived from iPSCs, etc. ) may have edits with loss-of-function of BCL3 transcription coactivator (BCL3) ; loss-of-function of Cbl proto-oncogene B (CBLB) ; loss-of-function of cyclin dependent kinase 8 (CDK8) ; loss-of-function of Fc fragment of IgE receptor  Ig (FCER1G) ; loss-of-function of interleukin 17A (IL17A) ; loss-of-function of interleukin 17F (IL17F) ; loss-of-function of inositol polyphosphate-5-phosphatase D (INPP5D/SHIP1) ; loss-of-function of suppressor of cytokine signaling 1 (SOCS1) ; loss-of-function of suppressor of cytokine signaling 2 (SOCS2) ; loss-of-function of suppressor of cytokine signaling 3 (SOCS3) ; loss-of-function of signal transducer and activator of transcription 3 (STAT3) ; loss-of-function of tet methylcytosine dioxygenase 2 (TET2) ; loss-of-function of protein tyrosine phosphatase non-receptor type 2 (PTPN2) ; loss-of-function of protein tyrosine phosphatase non-receptor type 6 (PTPN6) ; loss-of-function of CD70 molecule (CD70) ; loss-of-function of CD38 molecule (CD38) ; loss-of-function of cytokine inducible SH2 containing protein (CISH) ; and/or a transgene for killer cell lectin like receptor C2 (KLRC2/NKG2C) .
In some cases, the loss-of function gene editing can be fulfilled by one or more gene editing moieties as disclosed herein, such as CRIPSR/Cas9 system. Briefly, NK cells (e.g., CBNK cells) are recovered from cryopreservation and cultured using the medium (e.g., Lymphocyte Serum-Free Medium KBM 581(Corning) , 10%Human male AB serum, 1%MEM Non-Essential Amino Acids Solution (100X, Gibco) , 1%L-Glutamine (200 mM) (Gibco) , 0.02%Vitamin C, 200U/mL IL-2) . For each editing, cells (e.g., about 1x107 cells) are isolated and transfected with guide RNA/Cas9 protein complex (RNP) (e.g., using Lonza Nucleofector 4D and P3 Primary Cell 4D-NucleofectorTM X Kit L) . The guide RNA and Cas9 are transfected, e.g., with the ratio of 2: 1 (guide RNA 75 picomole (pmol) , Cas9 protein 150 pmol) . The transfected cells are recovered (e.g., using the medium with doubled Human male AB serum (20%) ) before downstream assays. The editing efficiency is analyzed by fragment analysis or NGS.
The sequences of guide RNAs and primers for editing efficiency evaluation are listed in TABLE 6 and TABLE 7 below.
TABLE 6: Sequences of guide RNAs for NK cells with enhanced survival and persistency

TABLE 7: Primer sequences for editing efficiency evaluation
Optimized sequence of transgene for killer cell lectin like receptor C2 (KLRC2/NKG2C) (SEQ ID NO: 52) :
In vitro screening for cell persistence (e.g., NK cell persistence) related genes
NK cells can be utilized as a model population of cells to examine persistence enhancing gene edits. A  population of NK cells can be engineered comprise (i) reduced expression of at least one endogenous immune regulating polypeptide comprising one or more members selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, and CD70, and/or (ii) the enhanced or introduced expression of NKG2C. In a mixture of cells comprising such population of engineered NK cells and other cells (e.g., other NK cells) that do not comprise (i) and/or (ii) , the persistence level of the population of engineered NK cells in such mixture can be characterized by (i) an enrichment level of the population of engineered NK cells within the mixture in a sub-optimal environment that is greater than (ii) an enrichment level of the population of engineered NK cells within the mixture in an optimal environment. The sub-optimal environment can comprise a lower amount (or concentration) of exogenous cytokine (e.g., exogenous IL, such as IL-2) . For example, the sub-optimal environment can comprise an amount of the exogenous cytokine that is at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more lower than that in the optimal environment. The sub-optimal environment can be an in vitro medium, and the optimal environment can be an in vitro medium. The sub-optimal environment can be an in vivo environment (e.g., blood stream of a subject) , and the optimal environment can be an in vitro medium. The enrichment level of the population of engineered NK cells within such mixture can be ascertained by identifying an amount (or proportion) of cells exhibiting (i) the reduced expression of at least one endogenous immune regulating polypeptide comprising one or more members selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, and CD70, and/or (ii) the enhanced or introduced expression of NKG2C.
Without wishing to be bound by theory, NK cells with reduced expression or activity level of a gene of interest (e.g., loss-of-function of FCER1G) may survive or persist longer in a sub-optimal environment (e.g., low cytokine) . In a more optimal environment (e.g., high cytokine) , NKs cells may exhibit high survival rate or persistence, regardless of whether the NK cells comprise the reduced expression or activity level of such gene of interest (e.g., FCER1G) . Thus, exhibiting a higher enrichment (e.g., INDEL%) in a mixture with different types of cells in the sub-optimal (or more challenging) environment can suggest that such loss-of-function gene may induce enhanced survival or persistence to the cell.
For example, to find the gene related to NK persistency, one or more of such population of engineered NK cells (e.g., eighteen kinds of gene-edited NK cells, such as gene-edited CBNK cells) were mixed in a mixture as disclosed herein and cultured using high cytokine (200 U/mL IL-2) or low cytokine (10 U/mL IL-2) . Editing percentage (%INDEL) of each gene was analyzed by NGS after 8 days culturing. Two mixtures were prepared using two individual electroporation of each gene, three replicates were set for each culturing condition. A flowchart showing the method design is depicted in FIG. 11A. Persistency with low or high cytokines (or in sub-optimal environment or a more optimal environment) By comparing the editing percentage of low-cytokine-cultured NK cells with high-cytokine-cultured  NK cells, FCER1G deficient editing showed increased percentage when cultured with low cytokines, which indicated that FCER1G deficient NK cells had better persistency in low cytokine conditions, while PTPN2 had no significant difference in the same assay, as illustrated in FIG. 11B and FIG. 11C, respectively.
Thus, having reduced expression or activity level of an immune regulator polypeptide that is not PTPN2 (e.g., FCER1G) in a cell (e.g., stem cell, immune cell, such as NK cell, etc. ) can induce enhanced persistence or survival rate in the cell.
In vivo screening of the NK persistency related genes
Similar to the in vitro screening for cell persistence, one or more of such population of engineered NK cells (e.g., eighteen kinds of gene-edited NK cells, such as gene-edited CBNK cells) were mixed in a mixture as disclosed herein and cultured using high cytokine (200U/mL IL-2) (more optimal environment) or injected to hIL-15-NCG mice (sub-optimal environment) , each mixed NK cells were injected to 5 mice with an amount (e.g., an amount of 1X107 NK cells /mouse) . Over time (e.g., after 8 days) , in vitro cultured NK cells or mouse tissue are harvested to extract the genome. Editing percentage (%INDEL) of each gene is analyzed by NGS. Two mixtures were prepared using two individual electroporation of each gene, three replicates were set for in vitro culturing, five mice were used for each mixed NK cells injection. A flowchart showing the method design is depicted in FIG. 12A.
Persistency in vivo
As shown in FIG. 12B-FIG. 12G, by comparing the editing percentage of NK cells in mouse tissues with high cytokine cultured NK cells, STAT3 deficient editing shown increased percentage in mouse tissue compared to cultured using high cytokine, which indicated that STAT3 deficient NK cells have better survival and persistency in vivo, while PTPN2 had no significant difference in the same assay. Thus, having reduced expression or activity level of an immune regulator polypeptide that is not PTPN2 (e.g., STAT3) in a cell (e.g., stem cell, immune cell, such as NK cell, etc. ) can induce enhanced persistence or survival rate in the cell.
Engineered NK Cells with hIL15
For improved cell therapy (e.g., stem cell therapy, adaptive immunotherapy, etc. ) , cells of interest can be engineered to exhibit enhanced cytokine signaling (e.g., enhanced IL-15 signaling by enhanced or introduced IL-15, such as heterologous secretory IL-15, heterologous membrane-bound IL-15, heterologous IL-15 cytokine-IL15 receptor fusion, etc. ) . The cells of interest can be stem cells, such as isolated stem cells (e.g., embryonic stem cells) or induced stem cells (e.g., iPSCs) . The cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. Alternatively, such immune cells can be immune cell lines (e.g., NK cell lines) . Engineered NK cells:
NK-92 cells were engineered with (i) hIL-15 knock in or (ii) hIL-15-hIL15R fusion polypeptide knock in. Two variants of the hIL-15-hIL15R fusion polypeptide were tested. The first variant (i.e., hIL15-IL15Ra fused-1 or “fus1” ) was designed with a linker between hIL-15 and hIL15R, which linker comprising one  or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “GGGGS” (SEQ ID NO. 53) , e.g., “GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS” (SEQ ID NO. 54) . The second variant (i.e., hIL15-IL15Ra fused-2 or “fus2” ) was designed with a linker between hIL-15 and hIL15R, which linker comprising one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “GGGGS” (SEQ ID NO. 53) and one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, or more repeats) of “EGKSSGSGSESKST” (SEQ ID NO. 55) , e.g., “EGKSSGSGSESKSTEGKSSGSGSESKSTGGGGS” (SEQ ID NO. 56) . NK-92 cells with either of the hIL-15-hIL15R fusion polypeptide variant knocked-in were positive for hIL-15.
In addition, the engineered NK-92 cells expressing either variant of the hIL-15-hIL15R fusion polypeptide for enhanced IL-15 signaling exhibited longer persistency as compared to control NK-92 cells engineered express secretory form of IL-15. Western blotting analysis revealed increased phosphorylation of IL-15-stimulated STAT5 in the NK-92 cells expressing either hIL15-IL15Ra fused-1 (fus1) or hIL15-IL15Ra fused-2 (fus2) , as compared to the secretory IL-15 (IL15) .
hIL15-IL15Ra fused-1 sequence (SEQ ID NO. 31) :
hIL15-IL15Ra fused-2 sequence (SEQ ID NO. 32) :
Membrane-bound IL-15 expression in iNK cells differentiated from hIL15-IL15Ra fused-1 iPSC clones The expression of IL15 was confirmed in several iNK cells differentiated from hIL15-IL15Ra fused-1 iPSC clones, PW15, PW18, and PW23. As shown in FIGURE 15, Fluorescence-activated Cell Sorting (FACS) was performed to quantify the surface expression of IL-15 in clones expressing membrane-bound IL-15 in comparison with controls, wild-type (wt) iNK cells and isotype. Thus, the clones were validated for expected overexpression level of membrane-bound IL-15.
In-vitro growth of eNK cells differentiated from engineered iPSC
KB-15 cells were eNK cells (e.g., NK cells differentiated from iPSCs and subsequently expanded) differentiated from iPSC clones expressing hIL15-IL15Ra, aCD19 CAR, and a CD16 variant for  enhanced CD16 signaling. The in-vitro growth of 2x107 KB-15 cultured with or without IL-2 (100U/mL) was monitored for 30 days. The cultured cells was collected and counted every 3 to 4 days, and the medium was renewed with corresponding medium meanwhile. As shown in Figure 16, KB-15 cells were able to grow in the absence of exogenous cytokines as vigorously as the ones in the presence of exogenous cytokines.
For the engineered NK cells comprising enhanced IL-15 signaling (e.g., KB-15 NK cells comprising hIL15-IL15Ra) , the engineered NK cells cultured in a medium substantially free of exogenous IL-2 exhibited enhanced persistence (e.g., on day 5, day 9, day 12, day 16, day 23, day 26, etc. ) than the engineered NK cells cultured in a medium comprising exogenous IL-2.
Without wishing to be bound by theory, adding any exogenous IL-2 to the engineered NK cells comprising enhanced IL-15 signaling can interfere with the auto-activation of IL-15 or auto-activated IL-15 signaling pathway, thereby reducing the persistence or survival level of the engineered NK cells. For example, membrane-bound IL-15 can have a capability to induce downstream IL signaling (e.g., STAT pathway) to a higher degree than secreted IL (e.g., IL-2, IL-15) , but the secreted IL at sufficient amount can act as a competitor for the same membrane receptor to reduce the chance of binding between the membrane-bound IL-15 and the respective receptor (e.g., IL-15R) and the signaling thereof. In contrast, for wild type NK cells (wt eNK in Figure 16) , absence of exogenous IL-2 in the medium resulted in reduced persistence level because the wild type NK cells did not have any membrane-bound IL-15 (e.g., IL15-IL15R fusion) to promote self-activation of the IL signaling pathway for persistence. Thus, when the engineered NK cells comprising enhanced IL-15 signaling is administered in vivo, where an amount of cytokines such as IL-2 in the blood stream or in a tissue of interest may be low (or sub-optimal) , the engineered NK cells can exhibit optimal persistence or survival via self-induced activation of IL-15.
IPSC expressing membrane-bound IL-15 differentiated into iNK
The engineered iPSC were subjected for iNK differentiation. Specifically, 5x105 culture containing cells was collected and used for staining with NKG2A-PE, NKp30-PE, NKp44-PE, NKp46-PE and CD56-APC antibody. As shown in FIGs. 9A-9E respectively, CD56+ cells could be detected in the total differentiated cells, and NKG2A+, NKp30+, NKp44+, and NKp46+ cells could be detected in the CD56+population among three exemplified clones, PW15, PW18 and PW23, which expressed membrane-bound IL-15. Therefore, iPSC expressing membrane-bound IL-15were proven to be able to differentiate into iNK cells.
iPSC expressing secretory IL-15 differentiated into iNK
The engineered iPSC were subjected for iNK differentiation. Three clones expressing secretory IL-15, PX27, PX33, and PX39, were tested. Specifically, 1x105 culture containing cells was collected and used for staining with CD56-APC antibody. FIG. 10A with the percentage of CD56+ cells in the total differentiated cells illustrates that iPSC expressing secretory IL-15 could differentiate into iNK.
Validation of secretory IL-15 in culture medium
1x106 iNK cells differentiated from iPSC clones expressing secreted IL-15, aCD19 CAR, and a variant  for enhanced CD16 signaling (KA08) were cultured for 2 days without renewing the medium, and the supernatant was collected and diluted 10-fold to measure human IL-15 the Human IL-15 ELISA kit.
FIG. 10B showing the concentration of IL-15 in iNK culture medium proves that the iNK cells were validated for secreting human IL-15 into culture medium.
In-vitro growth of eNK cells expressing secretory IL-15
FIG. 10C shows the in-vitro growth curve of 5x106 eNK cells differentiated from iPSC clones expressing secretory IL-15, aCD19 CAR, and a variant for enhanced CD16 signaling (OQ-20) . The cultured cells were collected and counted every 4 days, and the medium in absence of exogenous cytokines was renewed with corresponding medium meanwhile. The growth of the cells within 16 days was recorded and plotted as curves. Therefore, it has been proven that eNK cells expressing secretory IL-15 facilitated in-vitro growth without exogenous cytokines.
Example 4: Engineered NK Cells with CAR
For improved cell therapy (e.g., adaptive immunotherapy, etc. ) , cells of interest can be engineered to comprise a heterologous chimeric polypeptide (e.g., a chimeric antigen receptor) comprising an antigen binding moiety against a specific antibody of a target cell (e.g., a cancer or tumor cell) , to exhibit enhanced cytotoxicity against such target cell. The cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. For example, for NK cells derived from stem cells, one or more genetic modifications as disclosed herein can be introduced at (A) the stem cell state (e.g., iPSC state) , (B) the hematopoietic stem cell state, and/or (C) the NK cell state. Alternatively, such immune cells can be immune cell lines (e.g., NK cell lines) .
NK cells expressing CD33-CAR
NK cells expressing a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to CD33 were generated. Targeted cytotoxicity of NK92 cells with CD33-CAR integration on KG1 cells, a tumor cell line with high expression of CD33, with an E/T (Effector/Target) ratio of 1: 1 was tested. WT-NK92 cell were used as unmodified control. The targeted cytotoxicity of CD33-CAR on KG1 cells were greatly improved compared to control. NK92 cells were engineered to express anti-CD33 CAR, then cultured in the presence of CD33+ KG1 cells to assess targeting of the Raji cells by the engineered anti-CD33 NK cells. Wild type (WT) NK92 cells were used as control. The anti-CD33 CAR NK cells exhibited enhanced cytotoxicity against the KG1 cells (as ascertained by a reduced number of alive Raji cells) as compared to the control.
NK cells expressing CD19-CAR
NK cells (e.g., NK-92 cells) were engineered to express anti-CD19 CAR, then cultured in the presence of CD19+ Raji cells to assess targeting of the Raji cells by the engineered anti-CD19 NK cells. Wild type (WT) NK-92 cells were used as control. The anti-CD19 CAR NK cells exhibited enhanced cytotoxicity against the Raji cells (as ascertained by a reduced number of alive Raji cells) as compared to the control. In addition, when cultured in the presence of the Raji cells, the anti-CD19 CAR NK  cells exhibited enhanced expression of endogenous CD107a (indicative of cytotoxic granule release) as compared to the control. Furthermore, when cultured in the presence of the Raji cells, the anti-CD19 CAR NK cells exhibited enhanced cytokine production (e.g., IFN-gamma and/or TNF-alpha production) as compared to the control.
NK cells expressing BCMA-CAR
NK cells expressing a chimeric polypeptide receptor comprising an antigen binding moiety capable of specifically binding to BCMA was generated. Targeted cytotoxicity, CD107a and INF-r expression of NK92 cells with BCMA-CAR integration on RPMI8826 cell, a tumor cell line with high expression of BCMA. E/T (Effector/Target) ratios used were 1: 1; 1: 5 and 1: 10. WT-NK92 cell were used as unmodified control.
Different chimeric polypeptide receptor design constructs
FIG. 5A illustrates different chimeric polypeptide receptor (e.g., CAR) constructs. FIG. 5A, Top schematically illustrates CD19 CAR (2B4) structure design. TM short for Transmembrane domain; SCFV short for single chain variable fragment. FIG. 5A, Middle schematically illustrates CD19 CAR (4-1-BB) structure design. TM short for Transmembrane domain; SCFV short for single chain variable fragment. FIG. 5A, bottom schematically illustrates CD19 CAR (CD28) structure design. TM short for Transmembrane domain; SCFV short for single chain variable fragment.
FIGs. 5B and 5C shows targeted cytotoxicity against target cells by NK cells expressing one of the chimeric polypeptide receptor design shown in FIG. 5A. Referring to FIG. 5B, targeted cytotoxicity of various CD19-CAR NK92 on CD19-K562 cells (E/T (Effector/Target) equals 5: 1; 1: 1 and 0.5: 1) demonstrates that NK cells expressing CAR constructs with 4-1-BB signaling domain, 2B4 signaling domain, and/or CD28 signaling domain exhibited targeted cytotoxicity against CD19-presenting K562 target cells. WT-NK92 cell were used as unmodified control, CD19-K562 is K562 engineered with CD19 highly expressed. Referring to FIG. 5C, non-specific cytotoxicity of CD19-CAR NK92 on K562 cells that are not engineered to express CD19 at a high level (E/T (Effector/Target) equals 5: 1; 1: 1 and 0.5: 1) demonstrated lower degree of cytotoxicity, indicating that 4-1-BB, 2B4, and/or CD28 intracellular signaling domains are useful in designing various CAR constructs, e.g., for immunotherapies such as NK cell threapies.
Example 5: Engineered NK cells with a hypo-immunity regulator
For improved cell therapy (e.g., stem cell therapy, adaptive immunotherapy, etc. ) , cells of interest can be engineered to exhibit (i) reduced expression of one or more immune regulating polypeptides (e.g., one or more endogenous immune regulating polypeptides) and/or (ii) enhanced or introduced expression of one or more additional immune regulating polypeptides (e.g., one or more heterologous immune regulating polypeptides) . Cells comprising (i) and/or (ii) as disclosed herein can exhibit enhanced function, such as a persistence level (or survival level) , hypo-immunity (e.g., resistance against immune rejection or cytotoxicity) , growth rate, cytotoxicity against a target cell (e.g., tumor cell) , etc. In some cases, having the combination of (i) and (ii) can synergistically improve function of the cells, as compared to having either one of (i) and (ii) alone, or a combination of individual effects of (i) and (ii) , or none. In some cases, having reduced expression of two or more immune regulating polypeptides can synergistically improve function of the cells, as compared to having an individual member of the reduced expression of the two or more immune regulating polypeptides, or a combination of individual effects of such individual members. In some cases, having enhanced/introduced expression of two or more additional immune regulating polypeptides can synergistically improve function of the cells, as compared to having an individual member of the enhanced/introduced expression of the two or more additional immune regulating polypeptides, or a combination of individual effects of such individual members.
The cells of interest can be stem cells, such as isolated stem cells (e.g., embryonic stem cells) or induced stem cells (e.g., iPSCs) . The cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. Alternatively, such immune cells can be immune cell lines (e.g., NK cell lines) .
Engineered cells:
Table 2 illustrates example combinations of modified expression or activity of the plurality of immune regulator polypeptides. A combination of modified expression or activity of the plurality of immune regulator polypeptides from Table 2 may be introduced in cells (e.g., engineered NK cells) to, for example, reduce or avoid immune response (e.g., immune attack, such as adaptive immune rejection) from a host’s body upon administration of the cells to the host’s body. A combination of modified expression or activity of the plurality of immune regulator polypeptides from Table 2 may comprise (i) reduced expression or activity of one or more first immune regulator polypeptides (column 2) and (ii) enhanced expression or activity of one or more second immune regulator polypeptides (column 3) . In some cases, a combination of modified expression or activity of the plurality of immune regulator polypeptides from Table 8 may comprise (i) knock-out of one or more endogenous immune regulator polypeptide genes (column 2) and (ii) knock-in of one or more heterologous immune regulator polypeptide genes (column 3) .
TABLE 8

Generation of modified NK cells
For achieving hypo-immunity, NK cells can be engineered to carry certain transgenes and/or loss-of-function of genes of interest, such as the non-limiting exemplary guide RNA sequences are show in Table 9 below.
TABLE 9: Guide RNA sequences for generating engineered NK cells with hypo-immunity

Optimized transgene sequence for NECTIN3/CD113 (SEQ ID NO. 109)

Optimized transgene sequence for ADORA2A/A2AR (SEQ ID NO. 58)
Generating engineered NK cells with gene knock-in
Human iPSC cells can be engineered by knocking in gene edits such as HLA-E, CD47, PDL2, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, and/or CD59. Such engineered iPSC cells can be differentiated into NK cells. Alternatively, human peripheral blood (PB) -NK cells can be engineered  with AAV system. Possible functional readouts to test the engineered NK cells for hypo-immunity include mixed lymphocyte reaction (MLR) , T cell activation assay, in vitro NK-cell-induced killing assay, and complement-dependent cytotoxicity.
Hypoimmunity via editing and differentiating iPSC
A testing scheme for generating NK cells or endothelial cells (EC) derived from iPSC cells for hypo-immunity is shown in FIG. 13. Briefly, iPSC can be edited with different knock-ins and knock-outs. Subsequently, these edited iPSC can be subjected for differentiation into iNK which can be further expanded into eNK. iNK cells can be used for T cell proliferation assay, and eNK can be used for NK cytotoxicity test and hypoimmunity test. Alternatively, edited iPSC cells can be differentiated into iEC cells (e.g., endothelial cells derived from iPSCs) which can be used for NK susceptibility assay.
Methods of generating engineered iPSC
Several methods can be used to engineer iPSCs for NK cells with hypo-immunity. Exemplary methods are shown in Table 10.
TABLE 10: Methods for generating engineered iPSC
Briefly, RNP method is a method of electroporating target cells with pre-mixed ribonucleoprotein (RNP) containing Cas9 and sgRNA. After delivery to the cells, the RNP edits the genome region paired to the sgRNA. Adenine base editor (ABE) method is a method where Cas proteins can be fused to an enzyme that can deaminate a DNA nucleoside.
Confirmation of Edit-1 clones
Several edit-1 clones, clones 05, 07, 08, 104, 111, 112, were derived by electroporating human iPSC with RNP targeting B2M. The Edit-1 clones were confirmed to be B2M knock-out by FACS analysis for MHC-I (see FIG. 14A) and by Sanger sequencing (FIG. 14B) . Briefly, clone 05 was sequenced to have an insertion of one nucleotide in both B2M alleles, and clone 07 was sequenced to have an insertion of one nucleotide in one B2M allele and a deletion of two nucleotides in the other allele.
Confirmation of Edit-2 clones
Similarly, several edit-2 clones, clones 03 and 06, were derived by electroporating human iPSC with RNP targeting CIITA, followed by FACS sorting for single cells. The edit-2 clones were confirmed to be CIITA KO by sanger sequencing. As shown in FIG. 14C, clone 03 was sequenced to have an insertion of one nucleotide in both CIITA alleles, and clone 06 was sequenced to have an insertion of one  nucleotide in one CIITA allele and a deletion of sixteen nucleotides in the other allele.
Confirmation of Edit-3 clones
Several edit-3 clones, clones 04, 20, 25, 48, and 16, were derived by electroporating hiPSC with two RNPs, targeting B2M and CIITA, respectively. The Edit-3 clones were confirmed to be B2M knock-out by FACS analysis for MHC-I (see FIG. 14D) and sanger sequencing was used to confirm the knock-out of B2M and CIITA at the genomic level (see FIG. 14E) .
Confirmation of Edit-4 clones
Several Edit-4 clones, clones 02 and 30, were derived by electroporating hiPSC with a construct overexpressing PD-L1, PD-L2, TGF-β, HLA-E, HLA-G, CD47, IL-10, CCL-21, CD46, CD55, CD59 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS sorting for B2M-; PDL1+; CD47+ single cells. The Edit-4 clones were confirmed to be B2M-by FACS analysis for MHC-I, and PDL1+, PD2+, CD47+, CD46+, CD55+ and CD59+ (see FIG. 14F) and sanger sequencing was used to confirm the knock-out of B2M and CIITA at the genomic level. For CIITA, only 1 allele was confirmed to be knock-out (see FIG. 14G) .
Confirmation of Edit-5 clones
Several Edit-5 clones, clones 01, 02, and 26, were derived by electroporating hiPSC with a construct overexpressing of PD-L1, HLA-E, CD47, IL-10, CCL-21 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS sorting for B2M-; PDL1+; CD47+ single cells. The Edit-5 clones were confirmed to be B2M-by FACS analysis for MHC-I, and PDL1+, CD47+ (see FIG. 14H) and sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level (see FIG. 14I) .
Confirmation of Edit-6 clones
Several Edit-6 clones, clones 08, 13, 15, and 31, were derived by electroporating hiPSC with a construtct overexpressing PD-L1, HLA-E, CD47, CD46, CD55, CD59 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS sorting for B2M-; PDL1+; CD47+ single cells. The Edit-6 clones were confirmed to be B2M-by FACS analysis for MHC-I, and PDL1+, CD47+, CD46+, CD55+, CD59+ (see FIG. 14J) and sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level (see FIG. 14K) .
Confirmation of Edit-7 clones
Several Edit-7 clones, clones 32, 33, 39, and 42, were derived by electroporating hiPSC with a construct overexpressing PD-L1, HLA-E, CD47, CCL-21, CD55 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS sorting for B2M-; PDL1+; CD47+ single cells. The Edit-7 clones were confirmed to be B2M-by FACS analysis for MHC-I, and PDL1+, CD47+, HLA-E+, CD55+ (see FIG. 14L) and sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level (see FIG. 14M) .
Confirmation of Edit-8 clones
Several Edit-8 clones, clones 15, 36, 40, and 42, were derived by electroporating hiPSC with a construct overexpressing of CD47 and two RNPs, targeting B2M and CIITA, respectively, followed by FACS  sorting for B2M-; CD47+ single cells. The Edit-8 clones were confirmed to be B2M-by FACS analysis for MHC-I, and CD47+ (see FIG. 14N) and Sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level (see FIG. 14O) .
Confirmation of Edit-9 clones
Several Edit-9 clones, clones 03, 10, 22, 27, 34, 36, 37, and 63, were derived by electroporating hiPSC with a construct overexpressing PD-L1, PD-L2, TGF-β, HLA-E, HLA-G, CD47, IL-10, CCL-21, CD46, CD55, CD59, two RNPs, targeting B2M and CIITA, and 1 Base Editor plasmid overexpressing sgRNA targeting MICA, MICB and ULBP1, followed by FACS sorting for B2M-; PDL1+; CD47+ single cells. The Edit-9 clones were confirmed to be B2M-by FACS analysis for MHC-I, and CD47+, B2M+, HLA-E+, PDL1+, CD55+ , CD46+ and CD59+ (see FIG. 14P) and sanger sequencing was used to confirm the KO of B2M and CIITA at the genomic level. The KO of MICA/MICB/ULBP1 were confirmed using next generation sequencing. The symbol√ represents the gene was knocked out successfully (see FIG. 14Q) .
Resistance to antibody-mediated complement cytotoxicity
As shown in FIG. 16A, different edited iPSC clones were prelabeled with SSEA-4 antibody, followed by co-incubating for 45 min with human complement at different concentration ranging from 0%～50%. The results demonstrated that iPSC with Edit-4 but not Edit-5 or Edit-8 were resistant to antibody-mediated complement cytotoxicity.
Without wishing to be bound by theory, the enhanced resistance to antibody-mediated complement cytotoxicity may be attributed to having enhanced or introduced expression of one or more of the following immune regulator polypeptides: PD-L2, TGF-beta, CD46, CD55, CD59, and HLA-G (e.g., at least one or more of PD-L2, TGF-beta, CD46, CD55, and CD59) . Thus, without wishing to be bound by theory, cells comprising Edit-9 (see FIG. 15) may also exhibit enhanced resistance to antibody-mediated complement cytotoxicity, as compared to cells with either Edit-5 or Edit-8.
Without withing to be bound by theory, iPSCs with Edit-4 or Edit-9 can exhibit enhance resistance to antibody-mediated complement cytotoxicity in vitro or in vivo, as compared to that of iPSCs with either Edit-5 or Edit-9. Without withing to be bound by theory, differentiated cells (e.g., endothelial cells, immune cells, etc. ) derived from iPSCs comprising Edit-4 or Edit-9 can exhibit enhance resistance to antibody-mediated complement cytotoxicity in vitro or in vivo, as compared to that of comparably differentiated cells derived from iPSCs with either Edit-5 or Edit-9. Without withing to be bound by theory, immune cells derived from iPSCs comprising Edit-4 or Edit-9 can exhibit enhance resistance to antibody-mediated complement cytotoxicity in vitro or in vivo, as compared to that of immune cells derived from iPSCs with either Edit-5 or Edit-9. Without withing to be bound by theory, NK cells derived from iPSCs comprising Edit-4 or Edit-9 can exhibit enhance resistance to antibody-mediated complement cytotoxicity in vitro or in vivo, as compared to that of NK cells derived from iPSCs with either Edit-5 or Edit-9.
CBNK cytotoxicity
Targeted cells iEC differentiated from corresponding iPSC were pre-labeled with CFSE, and then co-cultured for 4h with CBNK at an Effector: Target ratio of 5: 1. After 4h, PI was added for staining dead cells. FACS was used to detect the percentage of dead cell. iEC without co-cultured with CBNK was regarded as spontaneous target cell death. To calculate the percentage of specific NK cell-mediated target cell lysis, the following formula was used: NK-mediated specific lysis (%) = (%PI positive target cells in coculture -%PI positive target cells in spontaneous cell death) / (100-%PI positive target cells in spontaneous cell death) . The quantification is shown in FIG. 16B, which indicates iEC differentiated from corresponding iPSC with Edit-4 but not Edit-5 or Edit-8 were resistant to CBNK cytotoxicity
Efficiency of differentiation from iPSC
The engineered iPSC were subjected for iNK differentiation. 200ul of the culture containing cells was collected and used for CD56 and NKG2A staining. After staining, the cells were resuspended in 100ul FACS buffer (PBS+1%BSA) . 80ul of the cell suspension were collected for analysis. The yield of CD56+ cell number was calculated as followed: (Absolute CD56+ cells collected by FACS) *18ml/ (200ul*0.8) = (Absolute CD56+ cells collected by FACS) *112.5 (see FIG. 16C) ; the CD56+ and NKG2A+ percentage were shown in FIG. 16D and 16E, respectively. Suggested from the above figures, iPSC with different edits could all differentiate into iNK, with varied yields and efficiencies.
Functional property of NK cells with hypo-immunity
FIG. 16F shows single time killing against K562. Corresponding eNK was co-cultured with CFSE-prelabeled K562 cells at a ratio of E: T =3: 1 or 1: 1. After 4h, PI was added for staining dead cells. FACS was used to detect percentage of dead cell. K562 without co-cultured with eNK was regarded as spontaneous target cell death. To calculate the percentage of specific NK cell-mediated target cell lysis, the following formula was used: NK-mediated specific lysis (%) = (%PI positive target cells in coculture -%PI positive target cells in spontaneous cell death) / (100-%PI positive target cells in spontaneous cell death) .
FIG. 16G shows serial killing against K562. Corresponding eNK was co-cultured with K562-EGFP cells at a ratio of E: T=3: 1, the co-culture was performed in Incucyte. K562-EGFP cells were monitored by the imaging with a frequency of every 3h. K562 was added daily for 6 days. K562-GFP only sample was set as a control.
FIGs. 16F and 16G indicate that B2M/CIITA double Knock-Out eNK did not show any hyporesponsive phenotype against K562 cells, and most eNK with transgenes had comparable cytotoxicity to WT eNK, when tested against K562 cells. Without wishing to be bound by theory, the hyporesonsivitiy or cytotoxicity against other target cells can be different.
To test whether the iNK with different edits would stimulate the proliferation of CD8+ T cells in PBMC, corresponding eNK was co-cultured with CFSE-labeled PBMC. PBS was used a negative control while PHA as a positive control. After 5 days, the co-cultured cells were stained for CD3, CD4 and CD8.  CD3+CD8+CFSElow were regarded as proliferating CD8+ T cells. PBMC from different donors were tested in FIGs. 16H-16M. The data show that B2M/CIITA double Knock-Out NK with or without transgenes did not stimulate CD8+ T cell proliferation.
Similarly, corresponding eNK was co-cultured with CFSE-labeled PBMC. PBS was used a negative control while PHA as a positive control. After 5 days, the co-cultured cells were stained for CD3, CD4 and CD8. CD3+CD4+CFSElow were regarded as proliferating CD4+ T cells. PBMC from different donors were tested in FIGs. 16N-16S. The data show that B2M/CIITA double Knock-Out NK with or without transgenes did not stimulate CD4+ T cell proliferation.
Example 6: Engineered NK cells with enhanced function in tumor microenvironment (TME)
For improved function in tumor microenvironment (e.g., proliferation, persistence, hypo-immunity, anti-tumor activity, etc. ) , cells of interest can be engineered to carry (i) one or more enhanced or introduced genes (e.g., introduced transgenes) and/or (ii) one or more reduced expression level of endogenous genes (e.g., loss-of-function of genes of interest) . The reduced expression level of the endogenous genes can be induced by, e.g., CRISPP/Cas and one or more guide nucleic acid molecules, such as the non-limiting exemplary guide RNA sequences provided in TABLE 3. The cells of interest can be stem cells, such as isolated stem cells (e.g., embryonic stem cells) or induced stem cells (e.g., iPSCs) . The cells of interest can be immune cells (e.g., NK cells) . Such immune cells can be derived from the stem cells as disclosed herein. Alternatively, such immunes can be immune cell lines (e.g., NK cell lines) .
For example, for improved function in tumor microenvironment, NK cells can be engineered to carry certain transgenes and/or loss-of-function of genes of interest, as shown in TABLE 11.
TABLE 11: guide RNA sequences for enhanced function in tumor microenvironment




Exemplary optimized transgene sequences for KLRD1, CD96, CD244, CCR4, CCR9, CXCR6, CCR2, CXCR2, CX3CR1, KLRC2, TGFBR2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, MIR21, MIR181B1, MIR181A1, MIR144, and MIR150 are shown in SEQ ID NOs: 59-85, respectively.
Example 7. Anti-tumor activity of the engineered NK cells.
NOG mice were intravenously injected with 1x105 luciferase expressing Nalm6 cells at day -1 as shown in Figure 27A. On days 0, 3, and 6, mice were given either intravenous injections of 1x107 CB-NK cells or QN-019a (anti-CD19 CAR) NK cells. An additional negative control group was included that did not introduce any heterologous NK cells to the mice. From days 0-7, mice were given 1 μg per mouse (/mouse) of IL-15 once a day. From days 0-21, mice were given 100,000 IU/mouse of IL-2 every 2-3 days. Mice were imaged using In Vivo Imaging System (IVIS) weekly. As seen in Figure 27B, the quantification of IVIS imagine time course (day 7, 14, 18) showed that QN-019a NK cells effectively inhibited tumor growth in the mice. When a tumor burden curve was generated, as seen in Figure 27C, the QN-019a NK cell group showed lower tumor fluorescence values than the CB-NK and the Nalm6 (tumor) only groups.
Example 8: Treatment of autoimmune disease by using the engineered NK cell.
We evaluate the in vivo efficacy of Dsg3 CAAR-NK cells against AK23 target cells in a PV mouse model. A total number of 2x105 AK23 hybridoma cells will be injected intravenously into NSG mice (age 6-8 weeks) , after pre-treatment of mice daily for 2 days with 600 mg/kg intravenous immunoglobulin (IVIG, Privigen) to minimize FcγR-mediated toxicity against hybridoma cells. After 5 days, either Dsg3 CAAR-NK cells or control NK cells will be injected intravenously in a final volume of 250 μl. And the disease  burden will be serially and objectively quantitated by bioluminescence imaging.
Example 9: Treatment of cancer by using the engineered NK cell alone or in combination with antibodyNOG mice were intravenously injected with 1x106 luciferase expressing Raji cells at day 0. On days 0, 3, and 6, mice were given intravenous injections of 1x107 QN-019a (anti-CD19 CAR) NK cells. An additional negative control group was included that did not introduce any heterologous NK cells to the mice. Mice were either left untreated or treated with QN-019a alone or in combination with 300ug Rituximab on day 1. Mice were imaged using In Vivo Imaging System (IVIS) weekly. As seen in Figure 28A, the quantification of IVIS imagine time course (day 3, 6, 10, 14 and 18) showed that QN-019a NK cells effectively inhibited tumor growth in the mice. When a tumor burden curve was generated, as seen in Figure 28B, the QN-019a NK cell with Rituximab treatment showed lower tumor fluorescence values than the QN-019a NK cell only group.
It shall be understood that different aspects of the invention can be appreciated individually, collectively, or in combination with each other. Various aspects of the invention described herein may be applied to any of the particular applications disclosed herein. The compositions of matter including the engineered immune cells of the present disclosure may be utilized in the method section including methods of use and production disclosed herein, or vice versa.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
SEQUENCE LISTING















Claims (30)

  1. A cell or a population thereof, wherein
    a) the cell is (a) an induced pluripotent cell (iPSC) , a clonal iPSC, or an iPS cell line cell, or embryonic stem cell (ESC) ; (b) a derivative cell obtained from differentiating the cell of (a) ; or (c) an immune cell derived from an animal body,
    b) the cell comprises a component which comprising an ectodomain, a transmembrane domain, and an intracellular domain;
    wherein the ectodomain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, and a full length or at least a portion of CD64 or its variant; and
    wherein the transmembrane domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at least a portion of the native or modified transmembrane domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, CD25, CD122, CD132, CD127, CD218, CD360, and ICAM-1 polypeptide,
    wherein the intracellular domain is selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, a full length or at least a portion of CD89 or its variant, and a full length or at least a portion of the native or modified intracellular domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, ICAM-1, CD25, CD122, CD132, CD127, CD218, CD360, and CD3ζ, and wherein the cell has enhanced or acquired ADCC (antibody-dependent cell-mediated cytotoxicity) or ADCP (antibody-dependent cell-mediated phagocytosis) in comparison to a control cell or population thereof without the component.
  2. The cell or population thereof of claim 1, wherein the component further comprises a full length or at least a portion of the native or modified costimulatory domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, and NKp80 polypeptide.
  3. The cell or population thereof of claim 1 or 2, wherein the cell is a hemopoietic cell, an NK cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, or the derivative thereof, preferably the NK cell is an iPSC NK cell, a PBNK cell, a CBNK cell, or an NK92 cell.
  4. The cell or population thereof of any one of claims 1-3, wherein:
    when the ectodomain is a full length or at least a portion of CD16a or its variant, the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant; or
    when the ectodomain is a full length or at least a portion of CD32a or its variant, the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD16a or its variant, a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant; or when the ectodomain is a full length or at least a portion of CD64 or its variant, the transmembrane domain and the intracellular domain are selected from the group consisting of: a full length or at least a portion of CD32a or its variant, a full length or at least a portion of CD64 or its variant, and a full length or at least a portion of CD89 or its variant.
  5. The cell or population thereof of one of claims 1-4, wherein:
    when the ectodomain is CD16a with F176V or S197P, the transmembrane domain and the intracellular domain are selected from the group consisting of CD32a, CD64, and CD89; or
    when the ectodomain is CD32a or CD32a with H131 variant, the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89; or
    when the ectodomain is CD64, the transmembrane domain and the intracellular domain are selected from the group consisting of CD16a, CD32a, CD64, and CD89.
  6. The cell or population thereof of any one of claims 1-5, wherein the component comprises or consists of a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%identity to any one selected from the group consisting of SEQ ID NOs: 1-10.
  7. The cell or population thereof of one of claims 1-6, wherein the cell further comprises at least one feature selected from the group consisting of:
    (i) a chimeric antigen receptor (CAR) or a T cell receptor (TCR) , or the nucleotide coding sequence thereof;
    (ii) a persistence component, or the nucleotide coding sequence thereof;
    (iii) a hypo-immunity regulator, or the nucleotide coding sequence thereof;
    (iv) an immune activity component, or the nucleotide coding sequence thereof;
    (v) a safety switch component, or the nucleotide coding sequence thereof;
    (vi) a gene modification to enhance or reduce the expression of one or more endogenous or heterologous genes; and/or
    (vii) a tumor microenvironment component, or the nucleotide coding sequence thereof.
  8. The cell or population thereof of claim 7, wherein the CAR or the TCR specifically recognizes a tumor antigen selected from the group consisting of Adhesion G protein-coupled receptor E2 (ADGRE2) , Armadillo repeat-containing X-linked protein 3 (ARMCX3) , Carbonic Anhydrase IX (CA1X) , CCRI, CCR4, Carcinoembryonic Antigen (CEA) , CD3ζ, CD5, CD7, CD8, CD9, CD10, CD19, CD20, CD22, CD25, CD26, CD30, CD33, CD34, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD52, CD56, CD70, CD74, CD82, CD99, CD123, CD133, CD138, CD200, CD269 (BCMA) , CD S, CLEC12A, Collectin Liver 1 (CLL1) , an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen) , Decoy Receptor 2 (DCR2) , Density Enhanced Phosphatase 1 (DEP1) , Dipeptidyl peptidase-4 (DPP4) , Dopamine Receptor D2 (DRD2) , ERM-binding phosphoprotein-50 (EBP50) , epithelial glycoprotein2 (EGP 2) , epithelial glycoprotein-40 (EGP-40) , epithelial cell adhesion molecule (EpCAM) , EGFRvIII, receptor tyrosine-protein kinases erb-B2, 3, 4, EGFIR, EGFRVIII, ERBB folate-binding protein (FBP) , fetal acetylcholine receptor (AChR) , folate receptor-a, Ganglioside G2 (GD2) , Ganglioside G3 (GD3) , gp100, gp120, gp160, G-protein coupled receptor 56 (GPR56) , human Epidermal Growth Factor Receptor 2 (HER-2) , human telomerase reverse transcriptase (hTERT) , ICAM-1, Integrin B7, interleukin 6 receptor (IL6R) , Interleukin-13 receptor subunit alpha-2 (IL-13Rα2) , κ-light chain, kinase insert domain receptor (KDR) , Kappa, Lewis A (CA19.9) , LanC-like protein 1 (LANCL1) , Lewis Y (LeY) , L1 cell adhesion molecule (L1-CAM) , LILRB2, MART-1, melanoma antigen family A 1 (MAGE-A1) , MICA/B, Mucin 1 (Muc-1) , Mucin 16 (Muc-16) , Mesothelin (MSLN) , NKCSI, NKG2D ligand, c-Met, cancer-testis antigen NY-ESO-1, NY-ESO-2, oncofetal antigen (h5T4) , PRAIVIE, prostate stem cell antigen (PSCA) , PRAME, prostate-specific membrane antigen (PSMA) , Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) , Syntaxin 4 (STX4) , Tumor-Associated Glycoprotein 72 (TAG-72) , TIM-3, TRBCI, TRBC2, Trophoblast Cell-Surface Antigen 2 (Trop-2) , Urokinase Receptor (uPAR) , Vesicle Associated Membrane Protein 3 (VAMP3) , vascular endothelial growth factor R2 (VEGF-R2) , Wilms tumor protein (WT-1) , and pathogen antigen derived from a virus, bacteria, fungi, parasite and protozoa capable of causing diseases.
  9. The cell or population thereof of claim 8, wherein the pathogen antigen is derived from HIV, HBV, EBV, HPV, Lasse Virus, Influenza Virus, or Coronavirus.
  10. The cell or population thereof of any one of claims 7-9, wherein the CAR comprises at least one of the following domains:
    a) a full length or at least a portion of the native or modified transmembrane domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, DNAM1, NKG2D, CD8, CD8a, CD8b, NKp30, NKp44, NKp46, NKp80, NKG2C, and ICAM-1 polypeptide;
    b) a full length or at least a portion of the native or modified costimulatory domain selected from 2B4, 4-1BB, CD28, OX40, DAP10, DAP12, ICOS, CD27, and NKp80 polypeptide; and
    c) a full length or at least a portion of the native or modified signaling domain selected from CD3ζ, 2B4, 4-1BB, DAP10, DAP12, NKG2D, NKp30, NKp44, NKp46, DNAM1, and NKp80 polypeptide.
  11. The cell or population thereof of claim 10, wherein the CAR specifically binds to CD33 antigen and comprises at least one of a transmembrane domain of CD8, a costimulatory domain of 2B4, and a signaling domain of CD3ζ.
  12. The cell or population thereof of claim 10, wherein the transmembrane domain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 11-13, the costimulatory domain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 14-16, and/or the signaling domain has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to SEQ ID NO: 17.
  13. The cell or population thereof of any one of claims 7-12, wherein
    the CAR comprises or consists of (i) an scFv having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 18 and 20-21; or (ii) an scFv encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NO: 22.
  14. The cell or population thereof of any one of claims 7-13, wherein the CAR further comprises a hinge domain having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 23-24.
  15. The cell or population thereof of any one of claims 7-14, wherein the CAR comprises or consists of (i) a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 25-29; or (ii) an amino acid encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NO: 30.
  16. The cell or population thereof of any one of claims 7-15, wherein the persistence component is selected from the group consisting of:
    a) a full length or at least a portion of IL15, preferably in secretion form;
    b) a full length or at least a portion of IL15 fused with a full length or at least a portion of IL15Ra;
    c) a transgene for p-STAT5 enhancement;
    d) a modification to an endogenous gene for p-STAT5 enhancement, preferably a disruption of an  endogenous gene;
    e) a reduced expression of one or more genes selected from the group consisting of BCL3, CBLB, CDK8, FCER1G, IL17A, IL17F, SHIP1, SOCS1, SOCS2, SOCS3, STAT3, TET3, PTPN6, CD70; and
    f) an enhanced expression of one or more genes selected from the group consisting of CD25, CD122, and NKG2C.
  17. The cell or population thereof of any one of claims 7-16, wherein the persistence component comprises or consists of (i) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting SEQ ID NOs: 31-37; (ii) an amino acid sequence encoded by a nucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 38-39.
  18. The cell or population thereof of any one of claims 7-17, wherein the hypo-immunity regulator comprises a reduced expression of one or more genes selected from the group consisting of B2M, CIITA, TAP1, TAP2, tapasin, NLRC5, RFXANK, RFX5, RFXAP, CD80, CD86, ICOSL, CD40L, ICAM1, MICA, MICB, and a NKG2DL (e.g., ULBP1) , and/or an enhanced expression of one or more genes selected from the group consisting of HLA-E, CD47, CD113, PDL1, PDL2, A2AR, HLA-G, TGF-beta, CCL21, IL10, CD46, CD55, CD59, CD3, CD4, CD80, 41BBL, and CD131, preferably selected from the group consisting of PD-L2, TGF-beta, CD46, CD55, and CD59, preferably the hypo-immunity regulator comprises or consists of an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%identity to any one selected from the group consisting of SEQ ID NOs: 57-58.
  19. The cell or population thereof of any one of claims 7-18, wherein the immune activity component comprises a reduced expression of one or more genes selected from the group consisting of TGFb receptor, TIGIT, PD1, PDL1, SIGLEC9, CTLA-4, TIM-3, KIR2D, CD94, NKG2A, CD96, LAG3, and 2B4, and/or an enhanced expression of one or more genes selected from the group consisting of NCR, NKp30, NKp44, NKp46, NKG2D, NKp80, DNAM1, other NK activating receptors, chemokines or chemikine receptors.
  20. The cell or population thereof of any one of claims 7-19, wherein the safety switch component 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.
  21. The cell or population thereof of any one of claims 7-20, wherein the gene modification comprises a reduced or disrupted expression of FcRγ.
  22. The cell or population thereof of any one of claims 7-21, wherein the tumor microenvironment component comprises a reduced or an enhanced expression of one or more genes selected from the group consisting of KLRD1, CD96, CD244, CCR4, CCR9, CXCR6, CCR2, CXCR2, CX3CR1, KLRC2, TGFBR2, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, MIR21, MIR181B1, MIR181A1 MIR144, and MIR150.
  23. A method of preparing the cell or population thereof of any one of claims 1-22, comprising:
    a) providing a cell which is (a) an induced pluripotent cell (iPSC) , a clonal iPSC, or an iPS cell line cell, or embryonic stem cell (ESC) ; (b) a derivative cell obtained from differentiating the cell of (a) ; or (c) an immune cell derived from an animal body;
    b) introducing the component as defined in any one of claims 1-6 into the cell provided by step a) ; and
    c) optionally introducing the at least one of the feature as defined in any one of claims 7-22 into the cell provided by step b) .
  24. A composition or a kit comprising the cell or population thereof of any one of claims 1-22.
  25. The composition or the kit of claim 24, further comprising an antibody selected from the group consisting of: 20- (74) - (74) (milatuzumab; veltuzumab) , 20-2b-2b, 3F8, 74- (20) - (20) (milatuzumab; veltuzumab) , 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, zlintuzumab) , actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003/SIM-BD0801 (sevacizumab) , APX005M, arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also referred to as tocilizumab) , atorolimumab, Avelumab, B-701, bapineuzumab, basiliximab, bavituximab, BAY1129980, BAY1187982, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, Betalutin (177Lu-tetraxetan-tetulomab) , bevacizumab, BEVZ92 (bevacizumab biosimilar) , bezlotoxumab, BGB-A317, BHQ880, BI 836880, BI-505, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab) , CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch. 14.18, cidfusituzumab, cidtuzumab, citatuzumab bogatox, cixutumumab, clazakizumab,  clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, Cotara (iodine I-131 derlotuximab biotin) , cR6261, crenezumab, DA-3111 (trastuzumab biosimilar) , dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, Daratumumab Enhanze (daratumumab) , Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, Depatuxizumab, Depatuxizumab mafodotin, derlotuximab biotin, detumomab, DI-B4, dinutuximab, diridavumab, DKN-01, DMOT4039A, dorlimomab aritox, drozitumab, DS-1123, DS-8895, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, Etanercept, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, femzumab, fezakinumab, FF-21101, FGFR2 AntibodyDrug Conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, FPA144, fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, Gerilimzumab, gevokizumab, girentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hu14.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5B1, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (claudiximab) , imalumab, IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan) , IMMU-114, ImmuTune IMP701 Antagonist Antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, Ipafricept, IPH4102, ipilimumab, iratumumab, isatuximab, Istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2/L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L-DOS47, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (inebilizumab) , MEDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006/S80880, MGD007, MGD009, MGD011, milatuzumab, Milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, nolovizumab, NOV-10, numavizumab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab,  odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, OX002/MEN1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, PankoMabGEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pecfusituzumab, pectuzumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab) , PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, quilizumab, racotumomab, radretumab, rafivirumab, ralivizumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/SAR439684, reslivizumab, reslizumab, resyvizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, RO7009789, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-CD19A, SGN-CD19B, SGNCD33A, SGN-CD70A, SGN-LIV1A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, SYD985, SYM004 (futuximab and modotuximab) , Sym015, TAB08, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, Tanibirumab, taplitumomab paptox, tarextumab, TB-403, tefibazumab, Teleukin, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, TG-1303, TGN1412, Thorium-227-Epratuzumab Conjugate, ticilimumab, tigatuzumab, tildrakizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH 011, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tucusituzumab, tuvirumab, U3-1565, U3-1784, ublituximab, ulocuplumab, umavizumab, urelumab, urtoxazumab, ustekinumab, Vadastuximab Talirine, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB-101, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.
  26. A use of the cell or population thereof of any one of claims 1-22 or the composition or the kit of claim 24 or 25 in the manufacture of a medicament for treating a tumor, an infection disease, or an autoimmune disease.
  27. The use of claim 26, wherein the tumor is selected from the group consisting of: Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic  leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia,  Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, Tcell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis  and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms'tumor.
  28. The use of claim 26, wherein the infection disease is selected from the group consisting of: a viral infection, a bacterial infection, and a parasitic infection.
  29. The use of claim 26, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE) , myasthenia gravis (MG) , multiple sclerosis, scleroderma, Addison's Disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barré syndrome, Sjogren syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy of undetermined significance (MGUS) , Waldenstrom's macroglobulinemia (WM) , chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) , Hashimoto's Encephalopathy (HE) , Hashimoto's Thyroiditis, Graves’ Disease, Wegener’s Granulomatosis, and antibody-mediated transplantation rejection (e.g., for tissue transplants such as renaltransplant) .
  30. A use of the cell or population thereof of any one of claims 1-22 or the composition or the kit of claim 24 or 25 in the manufacture of a medicament for anti-aging.
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