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WO2025207798A1 - Genetically engineered cells having anti-cd123 chimeric antigen receptors, and uses thereof - Google Patents

Genetically engineered cells having anti-cd123 chimeric antigen receptors, and uses thereof

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Publication number
WO2025207798A1
WO2025207798A1 PCT/US2025/021593 US2025021593W WO2025207798A1 WO 2025207798 A1 WO2025207798 A1 WO 2025207798A1 US 2025021593 W US2025021593 W US 2025021593W WO 2025207798 A1 WO2025207798 A1 WO 2025207798A1
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Prior art keywords
cell
seq
ipsc
antigen
domain
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PCT/US2025/021593
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French (fr)
Inventor
Raisa UCHUROVA
John Wheeler
Michael Miller
Jill Marinari CARTON
Michael Francis NASO
Barry Morse
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Century Therapeutics Inc
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Century Therapeutics Inc
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Publication of WO2025207798A1 publication Critical patent/WO2025207798A1/en
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/50—Cellular immunotherapy characterised by the use of allogeneic cells
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    • 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
    • C12N5/0636—T lymphocytes
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    • 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
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    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/53—Liver
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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    • A61K2239/55—Lung
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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    • C12N2510/00—Genetically modified cells

Definitions

  • TECHNICAL FIELD This application provides genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof. Also provided are uses of the iPSCs or derivative cells thereof to express a chimeric antigen receptor for cell therapy. Also provided are related vectors, polynucleotides, and pharmaceutical compositions.
  • CARs Chimeric antigen receptors
  • AML acute myeloid leukemia
  • CD123- 1 sf-6638784 32429-20001.40 targeted CAR T cells can eliminate leukemic stem cells which mediate relapse, but on-target off-tumor toxicity has been a concern due to CD123 expression on normal hematopoietic progenitor cells.
  • Existing treatments have additional limitations. For example, autologous treatments need to be generated on a custom-made basis, which remains a limiting factor for large-scale clinical application due to the production costs and the risk of production failure.
  • CAR-T technology and its wider application is also limited due to a number of other key shortcomings, including, e.g., a) an inefficient anti-tumor response, b) limited penetration and susceptibility of adoptively transferred CAR T cells to an immunosuppressive tumor microenvironment (TME), c) poor persistence of CAR-T cells in vivo, d) serious adverse events in the patients including cytokine release syndrome (CRS) and graft-versus-host disease (GVHD) mediated by the CAR-T, and e) the time required for manufacturing.
  • TEE immunosuppressive tumor microenvironment
  • CRS cytokine release syndrome
  • GVHD graft-versus-host disease
  • CAR-T cells with dual-antigen targeting have shown potential for overcoming the antigen downregulation in myeloid and lymphocytic leukemias that is a common cause for treatment failure.
  • the allogeneic, tumor-targeting cell therapies of the present disclosure have the potential to overcome these challenges and satisfy the unmet need for therapeutically sufficient and functional antigen-specific immune cells for effective immunotherapy.
  • the present disclosure provides an induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising at least one exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), said one or more CARs comprising a first antigen-binding domain targeting a Cluster of Differentiation 123 (CD123) antigen.
  • iPSC induced pluripotent stem cell
  • CD123 Cluster of Differentiation 123
  • the iPSC or derivative cell thereof further comprises at least one of: (i) a deletion or reduced expression of one or more of Beta 2 Microglobulin (B2M), Transporter Associated with Antigen Processing (TAP) 1, TAP 2, Tapasin, Regulatory Factor X Associated Ankyrin Containing Protein (RFXANK), Class II Major Histocompatibility Complex Transactivator (CIITA), Regulatory Factor X5 (RFX5), and Regulatory Factor X Associated Protein (RFXAP) genes; (ii) an exogenous polynucleotide encoding a sf-6638784 2 32429-20001.40 human leukocyte antigen E (HLA-E) and/or a human leukocyte antigen G (HLA-G); (iii) an exogenous polynucleotide encoding a cluster of differentiation 16 (CD16; natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (Fc ⁇ R
  • the one or more CARs consists of a dual-targeting CAR comprising the first antigen-binding domain and a second antigen-binding domain.
  • the one or more CARs comprises a plurality of CARs including a first CAR and a second CAR, wherein the first CAR comprises the first antigen- binding domain, and wherein the second CAR comprises a second antigen-binding domain.
  • the first antigen-binding domain comprises an anti- CD123 VHH single domain antibody.
  • the dual-targeting CAR comprises: (i) optionally, a signal peptide; (ii) an extracellular domain comprising the first antigen-binding domain and the second antigen-binding domain; (vi) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and/or (vi) a co-stimulatory domain.
  • each of the first CAR and the second CAR comprises: (i) optionally, a signal peptide; (ii) an extracellular domain comprising the first antigen-binding domain or the second antigen-binding domain; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and/or (vi) a co-stimulatory domain.
  • the first antigen-binding domain comprises an anti-CD123 VHH single domain antibody.
  • the first antigen-binding domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 126-148.
  • the first antigen-binding domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 149-171.
  • the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ sf-6638784 3 32429-20001.40 ID NO: 1 or 103;
  • the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 126-148;
  • the hinge region comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21, 22, 61, 99, 100, or 102;
  • the transmembrane domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:
  • the signal peptide comprises amino acids having the sequence of SEQ ID NO: 1 or 103; the extracellular domain comprises amino acid having the sequence of one of SEQ ID NOs: 126-148; the hinge region comprises amino acids having the sequence of SEQ ID NO: 21, 22, 61, 99, 100, or 102; the transmembrane domain comprises amino acids having the sequence of SEQ ID NO: 23 or 24; and the intracellular signaling or co-stimulatory domains comprise amino acids having the sequence of one or more of SEQ ID NO: 6, 8-20, 243, and 244.
  • the cytokine comprises Interleukin-15 (IL-15).
  • iPSC or derivative cell thereof can further comprise an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope, wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide.
  • the IL-15 comprises (i) an IL-15 and an IL-15 receptor alpha (IL-15R ⁇ ) fusion polypeptide, or (ii) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 247.
  • the IL-15 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72.
  • the iPSC or derivative cell thereof comprises the deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.
  • the iPSC or the derivative cell thereof comprises the exogenous polynucleotide encoding the HLA-E and/or the HLA-G.
  • the CD16 protein is a CD16 variant protein.
  • the CD16 variant protein is a high affinity CD16 variant. In some embodiments, the CD16 variant protein is a non-cleavable CD16 variant. In some embodiments, the CD16 variant protein comprises wild-type sf-6638784 4 32429-20001.40 CD16 comprising amino acids having the sequence of SEQ ID NO: 186 and one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, and T220A. In some embodiments, the CD16 variant protein comprises amino acids having at least 90% sequence identity to SEQ ID NO: 187.
  • the iPSC or derivative cell thereof comprises the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by an autoprotease peptide.
  • the NKG2D protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a wildtype NKG2D protein.
  • the NKG2D protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 189.
  • the autoprotease peptide is selected from the group consisting of a porcine tesehovirus-1 2A (P2A) peptide, a foot-and-mouth disease virus 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide.
  • P2A porcine tesehovirus-1 2A
  • F2A foot-and-mouth disease virus 2A
  • E2A Equine Rhinitis A Virus
  • T2A cytoplasmic polyhedrosis virus 2A
  • BmCPV2A cytoplasmic polyhedrosis virus 2A
  • BmIFV2A Flacherie Virus 2A
  • the autoprotease peptide is a P2A peptide comprising amino acids having at least 90% sequence identity to SEQ ID NO: 73 or 191.
  • the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein comprises a nucleic acid having at least 90% sequence identity to SEQ ID NO: 184.
  • the exogenous polynucleotide(s) are integrated into the chromosome of the cell at one or more loci selected from the group consisting of AAVS1, B2M, CIITA, CD33, and CLYBL.
  • the iPSC or derivative cell thereof comprises a deletion or reduced expression of one or both of the B2M and the CIITA genes.
  • the iPSC or derivative cell thereof comprises a deletion or reduced expression of both of the B2M and the CIITA genes.
  • the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the iPSC is derived from a re- programmed T-cell.
  • the iPSC or the derivative cell thereof comprises an exogenous polynucleotide encoding a safety switch.
  • the safety switch comprises an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope.
  • the inactivated cell surface receptor is a truncated epithelial growth factor (tEGFR) variant.
  • the tEGFR variant consists of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71.
  • the safety switch comprises (i) an intracellular domain comprising a herpes simplex virus thymidine kinase (HSV-TK) or (ii) an inducible Caspase 9 (iCasp9).
  • the HSV-TK comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 248 or 249
  • the iCasp9 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 245 or 246.
  • the combined artificial cell death/reporter system polypeptide comprises the HSV-TK fused to a truncated variant PSMA polypeptide via the linker.
  • the at least one exogenous polynucleotide encoding one or more CARs comprises polynucleotides having at least one sequence selected from the group consisting of SEQ ID NOs: 174 and 175;
  • the exogenous polynucleotide encoding the HLA-E and/or the HLA-G comprises polynucleotides having the sequence of one or more of SEQ ID NOs: 67 and 70;
  • the exogenous polynucleotide encoding the CD16 protein and/or the NKG2D protein comprises polynucleotides having the sequence of one or more of SEQ ID NOs: 184, 188, and 190;
  • the at least one exogenous polynucleotide encoding the one or more CARs is integrated at a locus of the AAVS1 gene, the CD33 gene, or the CLYBL gene;
  • the exogenous polynucleotide encoding the HLA-E and/or the HLA-G is integrated at a locus of the B2M gene;
  • the exogenous polynucleotide encoding the CD16 protein and/or the NKG2D protein is integrated at a locus of the CD70 gene;
  • the exogeneous polynucleotide encoding the cytokine is integrated at the locus of the NKG2A gene;
  • the iPSC or the derivative cell thereof comprises a deletion or reduced expression of the CIITA gene; and, optionally (vi) the exogenous sf-6638784 8 32429-20001.40 polynucleotide encoding the safety switch or the
  • the dual-targeting CAR comprises a CD123 loop. In some embodiments, comprising the dual-targeting CAR, the dual-targeting CAR comprises one or more polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 126-148.
  • the dual- targeting CAR comprises one or more polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 149-171.
  • the iPSC or the derivative cell thereof comprises the exogenous polynucleotide encoding CXCR4 or the fragment or variant thereof.
  • the CXCR4 or the fragment or variant thereof is selected from the group consisting of a wild-type CXCR4 and a mutated variant of CXCR4.
  • the wild-type CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:192.
  • the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:200.
  • the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with a deletion of the C-terminal domain between 10 and 20 amino acid residues. In some embodiments, comprising the mutated variant of CXCR4, the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A.
  • the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 193-199.
  • the derivative cell is a NK cell or a T cell.
  • the derivative cell is a NK cell.
  • the derivative cell is a T cell.
  • the T cell is a gamma delta T cell.
  • the T cell is a gamma delta V ⁇ 9/V ⁇ 1 T cell.
  • the present disclosure provides a composition comprising the iPSC or the derivative cell thereof.
  • the composition further comprises or is used in combination with one or more therapeutic agents selected from the group consisting of a small-molecule therapeutic agent, a biologic, a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), a siRNA, an oligonucleotide, mononuclear blood cells, an antibody, a chemotherapeutic agent, a radioactive moiety, and an immunomodulatory drug (IMiD).
  • a small-molecule therapeutic agent e.g., a biologic, a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), a siRNA, an oligonucleotide, mononuclear blood cells, an antibody,
  • the present disclosure provides an induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising one or more exogenous polynucleotides encoding: (i) a dual-targeting chimeric antigen receptor (CAR) targeting a CD123 antigen integrated into a CD33 gene locus; (ii) a human leukocyte antigen E (HLA-E) and a human leukocyte antigen G (HLA-G) integrated into a B2M gene locus; (iii) a cluster of differentiation 16 (CD16; natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (Fc ⁇ RIII)) protein and/or an NKG2D protein integrated into a CD70 gene locus; (iv) an IL-15 and an IL-15 receptor alpha (IL-15R ⁇ ) fusion polypeptide integrated into a NKG2A gene locus;(v) a C-X-C chemokine receptor type 4 (CXCR4) or
  • the dual-targeting CAR comprises a CD123 loop.
  • the at least one exogenous polynucleotide encoding the dual-targeting CAR comprises (i) nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 149-171; or (ii) nucleotides encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 126-148.
  • the exogenous polynucleotide encoding the HLA-E and the HLA-G is integrated into exon 2 of the B2M gene locus.
  • the exogeneous polynucleotide encoding the IL-15 and the IL-15 receptor alpha (IL- 15R ⁇ ) fusion polypeptide is integrated into exon 3 of the NKG2A gene locus.
  • the CXCR4 or the fragment or variant thereof is selected from the group consisting of a wild-type CXCR4 and a mutated variant of CXCR4.
  • the wild-type CXCR4 comprises amino acids having at least 90%, sf-6638784 10 32429-20001.40 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 192.
  • the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 200.
  • the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with a deletion of the C-terminal domain between 10 and 20 amino acid residues. In some embodiments comprising the mutated variant of CXCR4, the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A.
  • the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 193-199.
  • the present disclosure provides a dual-targeting chimeric antigen receptor (CAR) polypeptide comprising a first antigen-binding domain targeting a CD123 antigen.
  • the first antigen-binding domain comprises an anti-CD123 VHH single domain antibody.
  • the dual-targeting CAR comprises: a signal peptide; an extracellular domain comprising the first antigen-binding domain and the second antigen-binding domain; a hinge region; a transmembrane domain; an intracellular signaling domain; and/or a co- stimulatory domain.
  • the first antigen-binding domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 126-148.
  • the second antigen-binding domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 149-171.
  • the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering the derivative cell thereof or the composition to a subject in need thereof.
  • the cancer is selected from the group consisting of leukemias, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), and chronic lymphocytic leukemia (CLL), lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma, multiple sf-6638784 11 32429-20001.40 myeloma, and follicular lymphoma.
  • leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), and chronic lymphocytic leukemia (CLL)
  • lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma, multiple sf-6638784 11 32429-20
  • the cancer is a B-cell malignancy, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, or follicular lymphoma.
  • the cancer is a myeloid cell malignancy.
  • the myeloid cell malignancy is acute myeloid leukemia (AML) or chronic myeloid leukemia (CML).
  • the present disclosure provides a method of manufacturing a derivative cell comprising differentiating the iPSC under conditions for cell differentiation to thereby obtain the derivative cell.
  • the iPSC is obtained by genomic engineering an unmodified iPSC, wherein the genomic engineering comprises targeted editing.
  • the targeted editing comprises deletion, insertion, or in/del carried out by CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these methods.
  • MOLM13_LUC cells were implanted subcutaneously (SQ) at 1x10 6 cells / mouse 3 days prior to CAR-T intravenous (IV) injection at 5x10 6 cells/mouse.
  • TGI Tumor growth inhibition
  • FIG. 2 shows results of anti-CD33 VHH-Fc fusion proteins that were examined by flow cytometry for CD33 cellular binding in CHO-CD33 and CHO- parental cell lines. Results are presented as a ratio of absolute Geomean signal of VHH-Fc binding to CHO-CD33 over CHO-parental. A total of 30 CD33 VHH-Fc sf-6638784 12 32429-20001.40 were tested for cell binding. 28/30 VHH-Fc demonstrated positive, specific binding to CHO-CD33 at 50nM and 5nM. No binding to CHO-Parental cells was observed. FIGs.
  • FIG. 3A-3B show results of anti-CD123 VHH-Fc fusion proteins that were examined by flow cytometry for binding to HL-60, Kasumi-3, MOLM-13 and Jurkat cell lines.
  • a total of 19 anti-CD123 VHH-Fc were tested for cell binding at (FIG. 3A) 50 nM and (FIG. 3B) 5 nM concentrations.
  • 12 anti-CD123 VHH-Fc samples demonstrated positive / specific binding to CHO-CD123 at 50nM (10 strong binders and 2 weak binders (PROT939, PROT937)).
  • 9 VHH-Fc samples also showed positive binding to Kasumi-3 cells at 50nM.
  • MOLM13_LUC cells were implanted subcutaneously (SQ) at 1x10 6 cells / mouse 3 days prior to CAR-T intravenous (IV) injection at 5x10 6 cells / mouse.
  • Tumor growth inhibition (TGI) was sf-6638784 13 32429-20001.40 observed with CD123 CAR-T constructs: 3 VHH-CAR showed approximately 99% TGI (P1407, P1412, P1414), and 4 CAR-T samples showed approximately 80-90% TGI (P1710, P1713).
  • Untransduced T cells (same donor) were used to supplement CAR-T cell suspensions so that the total number of T cells was equal for all groups.
  • FIGs. 9A-9D show schematics of (FIG.
  • FIG. 13 shows results of in vivo screening of dual-targeting -iNK cells using the SubQ MOLM-13 Fluc model in female NSG mice.
  • MOLM13_LUC cells were implanted subcutaneously (SQ) at 1x10 6 cells / mouse 3 days prior to CAR-iNK intravenous (IV) injection at 5x10 6 cells / mouse. Tumor growth inhibition (TGI) was observed with bispecific CAR-iNK cells.
  • FIG. 14 shows surface CXCR4 expression data in cells of the present disclosure that are engineered to express wild-type CXCR4, as assessed by FACS.
  • CXCR4-positive (+) iNK underwent engineering for expression of the human wild type CXCR4 protein via insertion into the CLYBL locus of the iPSC cell line through homology-directed repair. Following differentiation to iNK, this resulted in this particular bulk population of CXCR4+iNK with heterogenous expression of CXCR4 at 42.2%. Endogenous expression of CXCR4 in the CXCR4-negative (-) iNK cells (iPSC611) was 2.4%.
  • FIG. 15 shows surface CXCR4 expression data in cells of the present disclosure that are engineered to express wild-type CXCR4, as assessed by FACS.
  • Effector cells were co-cultured with target tumor cells at a 1:1 effector-to- target cell ratio.
  • Target cell growth was measured by Total Integrated Intensity readings from Incucyte Live-Cell Imaging system every 3 hours for 72 hours total.
  • Figure curves represent target cell growth curves when co-cultured with effector cells, normalized to target cell growth curves when target cells are cultured alone.
  • iPSC611 and iPSC1613 cells were equally potent in killing CD19-expressing target cells, indicating the CXCR4 transgene does not impact cytotoxicity in this assay.
  • FIG. 17 shows the CXCR4 expression in CAR iNKs differentiated to express a dual-targeting anti-CD33/anti-CD123 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984), following lentiviral transduction of CXCR4 at the hematopoietic progenitor stage of differentiation, and subsequent differentiation to iNK.
  • iPSC cells expressing a dual-targeting anti-CD33/anti-CD123 CAR (iPSC2984) sf-6638784 15 32429-20001.40 were transduced with a lentivirus-containing sequence encoding for WT CXCR4 upon integration into the cell line genome.
  • Hematopoietic progenitor cells derived from iPSC2984 were transduced with the WT CXCR4 lentivirus and then differentiated 14 days to iNK cells.
  • the lentivirus-transduced cells (CAR-iNK CXCR4+ ) were determined to be 54.5% CXCR4-positive versus 3.29% for non-transduced cells (CAR-iNK CXCR4- ).
  • Chemotactic Index was calculated as the number of cells in experimental wells (400 ng CXCL12) divided by the average of cells in the baseline wells (0 ng CXCL12). Shown are (FIG. 18A) raw cell counts at migration assay completion, and (FIG. 18B) calculated Chemotactic Index (ChI), which indicate pronounced migration of CXCR4-positive cells in the presence of CXCL12. FIGs.
  • FIG. 19A-19B show cytotoxicity of CAR iNK cells that are CXCR4- negative (open shape) and CXCR4-positive (filled shape), and also express a dual- targeting anti-CD33/anti-CD123 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984), towards NucLight Red expressing antigen-positive (diamond) and antigen-negative (triangle) tumor lines. Effector cells were co-cultured with target tumor cells at (FIG. 19A) 1:1 and (FIG. 19B) 2:1 effector-to-target cell ratios. Target cell growth was measured by Total Integrated Intensity readings from Incucyte Live-Cell Imaging system every 3 hours for 72 hours total.
  • Figure curves represent target cell growth curves when co-cultured with effector cells, normalized to target cell growth curves when target cells are cultured alone.
  • CXCR4-positive and CXCR4-negative CAR iNK cells were equally potent in killing antigen-expressing target cells, indicating the CXCR4 transgene does not impact cytotoxicity in this assay. No cytotoxicity against antigen-negative target cells was observed.
  • CXCR4-positive and CXCR4-negative CAR-iNK cells that express a dual-targeting anti-CD33/anti-CD123 sf-6638784 16 32429-20001.40 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984).
  • Luciferase-labeled tumor cells expressing CAR-specific antigen were intravenously (IV) injected into NOD SCID Gamma (NSG) mice on Day -4 (D-4). On Day 0 (D0), mice were intravenously injected with CXCR4-positive or -negative CAR-iNK. Mice underwent bioluminescent imaging to monitor tumor burden on Day -1 and twice weekly thereafter.
  • FIG. 22 shows bone marrow presence of CAR-iNK cells engineered to express a dual-targeting anti-CD33/anti-CD123 CAR of the present disclosure (iNKs differentiated from iPSC2984) in the presence and absence of tumor, assessed by FACS.
  • CXCR4-positive and CXCR4-negative CAR-iNK cells were intravenously injected into tumor-bearing and non-tumor bearing mice.
  • FIG. 23 shows positive immunohistochemical (IHC) staining for C-X-C motif chemokine 12 (CXCL12) in mouse bone marrow.
  • FIG. 24 shows immunohistochemical (IHC) staining for iNK cells in mouse tissues from mice dosed with CXCR4-positive and CXCR4-negative CAR-iNK cells engineered to express a dual-targeting anti-CD33/anti-CD123 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984).
  • Mice that received CXCR4- positive CAR-iNK had greater tissue iNK infiltration (bone, spleen, liver, and lung), indicating enhanced migration as a result of CXCR4 engineering of iNK.
  • any numerical values such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non- sf-6638784 18 32429-20001.40 exclusive or open-ended.
  • a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.
  • a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first.
  • a third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.”
  • subject means any animal, preferably a mammal, most preferably a human.
  • mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human. sf-6638784 19 32429-20001.40 It should also be understood that the terms “about,” “approximately,” “generally,” “substantially,” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension/characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art.
  • C-X-C CHEMOKINE RECEPTOR TYPE 4 and “CXCR4” refer to a G protein-coupled receptor that binds the chemokine CXCL12. It plays important roles in embryogenesis, hematopoiesis, organogenesis, vascularization, and tumorigenesis.
  • the wild type CXCR4 receptor contains 352 amino acids and has seven transmembrane domains, with an extracellular N-terminus and intracellular C- terminus.
  • CXCR4 Upon binding CXCL12, CXCR4 activates downstream signaling pathways such as Ras/MAPK and PI3K/Akt that regulate cell migration, proliferation, and survival.
  • engineered cell and “genetically modified cell” as used herein can be used interchangeably.
  • the terms mean containing and/or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny.
  • the terms refer to cells, preferentially T cells which are manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state.
  • T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface.
  • the sequences encoding the CAR may be delivered into cells using a retroviral or lentiviral vector.
  • the term “primary cell” refers to a cell isolated directly from a multicellular organism. Primary cells typically have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are cells that have been isolated and then used immediately. In other cases, primary cells cannot divide indefinitely and thus cannot be cultured for long periods of time in vitro.
  • a primary cell can comprise a primary immune cell (e.g., a primary T-cell)
  • a primary immune cell e.g., a primary T-cell
  • target refers to an antigen or epitope associated with a cell that should be recognized specifically by an antigen-binding domain, e.g., an antigen-binding domain of an antibody or of a CAR.
  • the antigen or epitope for antibody recognition can be bound to the cell surface but also be secreted, part of the extracellular membrane, or shed from the cell.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci.
  • nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • isolated means a biological component (such as a nucleic acid, peptide, protein, or cell) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues.
  • Nucleic acids, peptides, proteins, and cells that have been “isolated” thus include nucleic acids, peptides, proteins, and cells purified by standard purification methods and purification methods described herein.
  • nucleic acids, peptides, proteins, and cells can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • polynucleotide synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as sf-6638784 23 32429-20001.40 typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
  • Viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, Sendai virus vector, and the like.
  • integration it is meant that one or more nucleotides of a construct is stably inserted into the cellular genome, e.g., covalently linked to the nucleic acid sequence within the cell's chromosomal DNA.
  • targeted integration it is meant that the nucleotide(s) of a construct is inserted into the cell's chromosomal or mitochondrial DNA at a pre-selected site or “integration site”.
  • integration as used herein further refers to a process involving insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of an endogenous sequence or nucleotide at the integration site.
  • integration can further comprise replacement of the endogenous sequence or a nucleotide that is deleted with the one or more inserted nucleotides.
  • exogenous is intended to mean that the referenced molecule or the referenced activity is introduced into, or non-native to, the host cell.
  • the molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non- chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell.
  • the term “endogenous” refers to a referenced molecule or activity that is present in the host cell in its native form.
  • the term when used in reference to expression of an encoding nucleic sf-6638784 24 32429-20001.40 acid refers to expression of an encoding nucleic acid natively contained within the cell and not exogenously introduced.
  • a gene of interest may encode an miRNA, an shRNA, a native polypeptide (e.g., a polypeptide found in nature) or fragment thereof; a variant polypeptide (e.g., a mutant of the native polypeptide having less than 100% sequence identity with the native polypeptide) or fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like.
  • “Operably-linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably-linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (e.g., the coding sequence or functional RNA is under the transcriptional control of the promoter).
  • Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
  • expression refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications.
  • the expressed CAR can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane.
  • the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein.
  • the terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can comprise modified amino acids, and it can sf-6638784 25 32429-20001.40 be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • the peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C- terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.
  • the term “engineered immune cell” refers to an immune cell, also referred to as an immune effector cell, that has been genetically modified by the addition of exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • ISCs Induced Pluripotent Stem Cells
  • IPSCs Immune Effector Cells
  • Use of iPSCs enables cellular engineering to produce a controlled cell bank of modified cells that can be expanded and differentiated into desired immune effector cells, supplying large amounts of homogeneous allogeneic therapeutic products.
  • Provided herein are genetically engineered iPSCs and derivative cells thereof. The selected genomic modifications provided herein enhance the therapeutic properties of the derivative cells.
  • the derivative cells are functionally improved and suitable for allogenic off-the-shelf cell therapies following a combination of selective modalities being introduced to the cells at the level of iPSC through genomic engineering. This approach can help to reduce the side effects mediated by CRS/GVHD and prevent long-term autoimmunity while providing excellent efficacy.
  • the term "differentiation” is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell.
  • Specialized cells include, for example, a blood cell or a muscle cell.
  • a differentiated or differentiation- induced cell is one that has taken on a more specialized ("committed") position within the lineage of a cell.
  • sf-6638784 26 32429-20001.40 when applied to the process of differentiation, 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 refers to the ability of a cell to form all lineages of the body or soma or 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 is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), 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 terms "reprogramming” or “dedifferentiation” 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 (e.g., can differentiate into more cell types) compared to the same cell in the non-reprogrammed state.
  • hematopoietic stem and progenitor cells refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation.
  • Hematopoietic stem cells include, for example, multipotent hematopoietic stem cells (hematoblasts), myeloid progenitors, megakaryocyte progenitors, erythrocyte progenitors, and lymphoid progenitors.
  • Hematopoietic stem and progenitor cells 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 sf-6638784 27 32429-20001.40 cells).
  • myeloid monocytes and macrophages
  • neutrophils neutrophils
  • basophils basophils
  • eosinophils neutrophils
  • erythrocytes erythrocytes
  • megakaryocytes/platelets dendritic cells
  • dendritic cells lymphoid lineages
  • T cells B cells, NK sf-6638784 27 32429-20001.40 cells.
  • CD34+ hematopoietic progenitor cell refers to an HPC that expresses CD34 on its
  • immune cell refers to a cell that is involved in an immune response. Immune response includes, for example, the promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes.
  • T lymphocyte and “T cell” are used interchangeably and refer to a type of white blood cell that completes maturation in the thymus and that has various roles in the immune system. A T cell can have the roles including, e.g., the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells.
  • a T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal.
  • the T cell can be CD3+ cells.
  • CD4 are 55-kD glycoproteins originally defined as differentiation antigens on T-lymphocytes, but also found on other cells including monocytes/macrophages. CD4 antigens are members of the immunoglobulin supergene family and are implicated as associative recognition elements in MHC (major histocompatibility complex) class II-restricted immune responses. On T-lymphocytes they define the helper/inducer subset. sf-6638784 28 32429-20001.40 “CD8+ T cells” refers to a subset of T cells which express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells.
  • CD8 molecules are differentiation antigens found on thymocytes and on cytotoxic and suppressor T- lymphocytes. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions.
  • NK cell or “natural killer cell” refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 and CD45 and the absence of the T cell receptor (TCR chains).
  • the NK cell can also refer to a genetically engineered NK cell, such as a NK cell modified to express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the NK cell can also be differentiated from a stem cell or progenitor cell.
  • the term “genetic imprint” refers to genetic or epigenetic information that contributes to preferential therapeutic attributes in a source cell or an iPSC, and is retainable in the source cell derived iPSCs, and/or the iPSC-derived hematopoietic lineage cells.
  • a source cell is a non-pluripotent cell that may be used for generating iPSCs through reprogramming, and the source cell derived iPSCs may be further differentiated to specific cell types including any hematopoietic lineage cells.
  • the source cell derived iPSCs, and differentiated cells therefrom are sometimes collectively called “derived” or “derivative” cells depending on the context.
  • iPSC parental cell lines may be generated from peripheral blood mononuclear cells (PBMCs) or T-cells using any known method for introducing re-programming factors into non-pluripotent cells such as the episomal plasmid-based process as previously described in U.S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference.
  • PBMCs peripheral blood mononuclear cells
  • T-cells any known method for introducing re-programming factors into non-pluripotent cells such as the episomal plasmid-based process as previously described in U.S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference.
  • the reprogramming factors may be in a form of sf-6638784 29 32429-20001.40 polynucleotides, and thus are introduced to the non-pluripotent cells by vectors such as a retrovirus, a Sendai virus, an adenovirus, an episome, and a mini-circle.
  • the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector.
  • the one or more polynucleotides are introduced by a Sendai viral vector.
  • the iPSC’s are clonal iPSC’s or are obtained from a pool of iPSCs and the genome edits are introduced by making one or more targeted integration and/or in/del at one or more selected sites.
  • the iPSC’s are obtained from human T cells having antigen specificity and a reconstituted TCR gene (hereinafter, also refer to as "T-iPS” cells) as described in US Pat. Nos. 9,206,394 and 10,787,642 hereby incorporated by reference into the present application.
  • an iPSC cell or a derivative cell thereof comprises one or more first exogenous polynucleotides encoding a first and a second chimeric antigen receptor (CAR), such as a CAR targeting one or more tumor antigens.
  • CAR targets a CD123 antigen.
  • the CAR targets a CD123 antigen, and the targeting regions (e.g., the extracellular domains) of the CAR comprises an antibody fragment (e.g., a VHH domain).
  • the CAR can be a dual-targeting CAR targeting two or more antigens.
  • a therapeutic cell of the present disclosure can express a dual-targeting CAR targeting at least CD123.
  • the targeting domains of the CAR e.g., the antigen binding regions
  • the CAR can be a dual-targeting CAR in a loop configuration.
  • the extracellular binding domain comprises a loop format. sf-6638784 30 32429-20001.40
  • an iPSC cell or a derivative cell thereof comprises at least one exogenous polynucleotide encoding one of more CARs.
  • the one or more CARs comprise an antigen-binding domain targeting a CD123 antigen.
  • the antigen-binding domain can be derived from the same species or a different species than the species in which the CAR will be used in.
  • the term “dual-targeting” refers to a protein (e.g., a chimeric protein) capable of binding to two different antigens.
  • a dual-targeting protein of the present disclosure e.g., a CAR having two or more tumor or cancer antigen-binding domains
  • a dual-targeting protein of the present disclosure does not naturally occur and is produced by a genetic engineering method or other method.
  • an engineered iPSC or derivative cell of the present disclosure can comprise one or more exogenous polynucleotides encoding a CAR having a first antigen-binding domain that binds to an antigen of interest and a second antigen-binding domain that specifically binds CD123.
  • an engineered iPSC or derivative cell comprises one or more polynucleotides encoding a first CAR having a first antigen-binding domain that binds to an antigen of interest and a second CAR having a second antigen-binding domain that specifically binds CD123.
  • signal peptide refers to a leader sequence at the amino-terminus (N-terminus) of a nascent CAR protein, which co-translationally or post-translationally directs the nascent protein to the endoplasmic reticulum and subsequent surface expression.
  • extracellular antigen-binding domain refers to the part of a CAR that is located outside of the cell membrane and is capable of binding to an antigen, target or ligand.
  • the term “hinge region” or “hinge domain” refers to the part of a CAR that connects two adjacent domains of the CAR protein, e.g., the extracellular domain and the transmembrane domain of the CAR protein.
  • the term “transmembrane domain” refers to the portion of a CAR that extends across the cell membrane and anchors the CAR to cell membrane.
  • the term “intracellular signaling domain,” “cytoplasmic signaling domain,” or “intracellular signaling domain” refers to the part of a CAR that is located inside of the cell membrane and is capable of transducing an effector signal.
  • the term “stimulatory molecule” refers to a molecule expressed by an immune cell (e.g., NK cell or T cell) that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of receptors in a stimulatory way for at least some aspect of the immune cell signaling pathway.
  • Stimulatory molecules comprise two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation (referred to as “primary signaling domains”), and those that act in an antigen-independent manner to provide a secondary of co-stimulatory signal (referred to as “co-stimulatory signaling domains”).
  • the extracellular domain comprises an antigen- binding domain and/or an antigen-binding fragment.
  • the antigen-binding fragment can, for example, be an antibody or antigen-binding fragment thereof that specifically binds a tumor antigen.
  • the antigen-binding fragments of the application possess one or more desirable functional properties, including but not limited to high-affinity binding to a tumor antigen, high specificity to a tumor antigen, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and/or antibody-dependent cellular-mediated cytotoxicity (ADCC) against cells expressing a tumor antigen, and the ability to inhibit tumor growth in subjects in sf-6638784 32 32429-20001.40 need thereof and in animal models when administered alone or in combination with other anti-cancer therapies.
  • CDC complement-dependent cytotoxicity
  • ADPC antibody-dependent phagocytosis
  • ADCC antibody-dependent cellular-mediated cytotoxicity
  • antibody is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (e.g., IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub- classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.
  • antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (e.g., complementarity determining regions 1-3; CDR1, CDR2, and CDR3).
  • the light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.
  • an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the specific tumor antigen is substantially free of antibodies that do not bind to the tumor antigen).
  • an isolated antibody is substantially free of other cellular material and/or chemicals.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts.
  • the monoclonal antibodies of the application can be made by the hybridoma method, phage display sf-6638784 33 32429-20001.40 technology, single lymphocyte gene cloning technology, or by recombinant DNA methods.
  • the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene.
  • the term “antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdAb), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a minibody, a nanobody, a domain antibody, a bivalent domain antibody, a light chain variable domain (VL), a variable domain (V H H) of a camelid antibody, or any other antibody fragment that binds to an antigen-binding
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds.
  • the term “single-chain antibody” refers to a conventional single-chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide).
  • the term “single domain antibody” refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain.
  • a multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.
  • the term “bispecific antibody” refers to a multispecific antibody that binds no more than two epitopes or two antigens.
  • a bispecific antibody is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein).
  • the first and second epitopes overlap or substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • the term X/Y loop refers to an extracellular region in which one VH or VL region (of, for example, CD123) is nested in between the other VL and VH region.
  • X and Y may be the same antigen.
  • X and Y may be different antigens.
  • X and Y are tumor antigens.
  • binding and “specifically binds” or “specific for” with respect to an antigen-binding domain of a ligand like an antibody, of a fragment thereof or of a CAR refer to an antigen-binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample.
  • An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific.
  • An antigen- binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.).
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (e.g., Kd/Ka) and is expressed as a molar concentration (M).
  • KD values for antibodies can be determined using methods in the art in view of the present disclosure.
  • the KD of an antigen-binding domain or antigen-binding fragment can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system.
  • Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.
  • the antigen-binding fragment is an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single-domain antibody (sdAb) or nanobody, a dAb fragment, a Fd' fragment, a Fd fragment, a heavy chain variable region, an isolated complementarity determining region (CDR), a diabody, a triabody, or a decabody.
  • CDR complementarity determining region
  • the antigen-binding fragment is an scFv fragment. In some embodiments, the antigen-binding fragment is a VHH.
  • extracellular tag-binding domain is used interchangeably with “extracellular domain.” In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single-domain antibody or nanobody. In some embodiments, the extracellular domain or the antigen-binding domain comprises a single-domain antibody or nanobody. In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH.
  • the extracellular domain and the antigen-binding domain each comprise a VHH.
  • the extracellular tag-binding domain and the tag each comprise an scFv.
  • the extracellular domain comprises an scFv.
  • the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a scFv.
  • the extracellular domain and the antigen-binding domain each comprise a scFv.
  • Alternative scaffolds to immunoglobulin domains that exhibit similar functional characteristics, such as high-affinity and specific binding of target biomolecules, may also be used in the CARs of the present disclosure.
  • Non-limiting examples of alternative scaffolds that may be used in the CAR of the present disclosure include engineered, tenascin- derived, tenascin type III domain (e.g., CentyrinTM); engineered, gamma-B crystallin- derived scaffold or engineered, ubiquitin-derived scaffold (e.g., Affilins); engineered, fibronectin-derived, 10th fibronectin type III (10Fn3) domain (e.g., monobodies, AdNectinsTM, or AdNexinsTM);; engineered, ankyrin repeat motif containing polypeptide (e.g., DARPinsTM); engineered, low-density-lipoprotein-receptor-derived, A domain (LDLR-A) (e.g., AvimersTM); lipocalin (e.g., anticalins); engineered, protease inhibitor
  • LDLR-A low-density-lipoprotein-receptor-derived
  • the alternative scaffold is Affilin or Centyrin.
  • the first polypeptide of the CARs of the present disclosure comprises a leader sequence.
  • the leader sequence may be positioned at the N-terminus the extracellular tag-binding domain.
  • the leader sequence may be positioned at the N-terminus the extracellular domain.
  • the leader sequence may be optionally cleaved from the extracellular tag-binding domain during cellular processing and localization of the CAR to the cellular membrane.
  • the leader sequence may be optionally cleaved from the extracellular domain during cellular processing and localization of the CAR to the cellular membrane. Any of various leader sequences known to one of skill in the art may be used as the leader sequence.
  • Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), Fc ⁇ R, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8 ⁇ , or any of various other proteins secreted by T cells.
  • GMCSFR granulocyte-macrophage colony-stimulating factor receptor
  • Fc ⁇ R human immunoglobulin (IgG) heavy chain (HC) variable region
  • CD8 ⁇ human immunoglobulin secreted by T cells.
  • the leader sequence is compatible with the secretory pathway of a T cell.
  • the leader sequence is derived from human immunoglobulin heavy chain (HC).
  • the leader sequence is an IgK signal peptide variant comprising the amino acid sequence set forth in SEQ ID NO: 103, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, sf-6638784 39 32429-20001.40 at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 103.
  • the nucleic acid encoding the IgK signal peptide variant comprises the nucleotide sequence set forth in SEQ ID NO: 101.
  • the first polypeptide of the CARs of the present disclosure comprises a transmembrane domain, fused in frame between the extracellular tag-binding domain and the cytoplasmic domain. In some embodiments, the first polypeptide of the CARs of the present disclosure comprises a transmembrane domain, fused in frame between the extracellular domain and the cytoplasmic domain.
  • the transmembrane domain may be derived from the protein contributing to the extracellular tag-binding domain, the protein contributing the signaling or co- signaling domain, or by a totally different protein.
  • the transmembrane domain may be derived from the protein contributing to the extracellular domain, the protein contributing the signaling or co-signaling domain, or by a totally different protein.
  • the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex. In some instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to avoid binding of proteins naturally associated with the transmembrane domain. In certain embodiments, the transmembrane domain includes additional amino acids to allow for flexibility and/or optimal distance between the domains connected to the transmembrane domain.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • transmembrane domains of particular use in this disclosure may be derived from (e.g., comprise at least the transmembrane region(s) of) the ⁇ or ⁇ chain of the T-cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8 ⁇ , CD9, CD16, CD22, CD28, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154.
  • TCR T-cell receptor
  • CD28 CD3 epsilon
  • CD45 CD4
  • CD5 CD8 ⁇
  • CD9 CD16
  • CD22 CD22
  • CD28 CD33
  • CD37 CD40
  • CD64, CD80 CD86, CD134, CD137, or CD154
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and/or valine can be found at each end of a synthetic transmembrane domain.
  • sf-6638784 40 32429-20001.40 it will be desirable to utilize the transmembrane domain of the ⁇ , ⁇ or Fc ⁇ R1 ⁇ chains which contain a cysteine residue capable of disulfide bonding, so that the resulting chimeric protein will be able to form disulfide linked dimers with itself, or with unmodified versions of the ⁇ , ⁇ or Fc ⁇ R1 ⁇ chains or related proteins.
  • the transmembrane domain will be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • it will be desirable to employ the transmembrane domain of ⁇ , ⁇ or Fc ⁇ R1 ⁇ and - ⁇ , MB1 (Ig ⁇ .), B29 or CD3- ⁇ , ⁇ , or ⁇ , in order to retain physical association with other members of the receptor complex.
  • the transmembrane domain is derived from CD8 or CD28.
  • the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 23.
  • the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23.
  • the nucleic acid encoding the CD8 transmembrane domain comprises the polynucleotide sequence set forth in SEQ ID NO: 96.
  • the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 24, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 24.
  • the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 24.
  • the nucleic acid encoding the CD28 transmembrane domain comprises the polynucleotide sequence set forth in SEQ ID NO: 97.
  • the first polypeptide of the CAR of the present disclosure comprises a spacer region between the extracellular tag-binding domain and the transmembrane domain, wherein the extracellular tag-binding domain, linker, and the transmembrane domain are in frame with each other.
  • the first polypeptide of the CAR of the present disclosure comprises a spacer region between the extracellular domain and the transmembrane domain, wherein the sf-6638784 41 32429-20001.40 extracellular domain, linker, and the transmembrane domain are in frame with each other.
  • spacer region as used herein generally means any oligo- or polypeptide that functions to link the extracellular tag-binding domain to the transmembrane domain.
  • a spacer region can be used to provide more flexibility and accessibility for the extracellular tag-binding domain.
  • a spacer region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • a spacer region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the spacer region may be a synthetic sequence that corresponds to a naturally occurring spacer region sequence, or may be an entirely synthetic spacer region sequence.
  • Non-limiting examples of spacer regions which may be used in accordance to the disclosure include a part of human CD8 ⁇ chain, partial extracellular domain of CD28, Fc ⁇ RIIIa receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof.
  • additional linking amino acids are added to the spacer region to ensure that the antigen-binding domain is an optimal distance from the transmembrane domain.
  • the spacer when the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding.
  • the spacer region comprises a hinge domain.
  • the hinge domain may be derived from CD8, CD8 ⁇ , CD28, or an immunoglobulin (IgG).
  • the IgG hinge may be from IgG1, IgG2, IgG3, IgG4, IgG4 CH3, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.
  • the hinge domain comprises an immunoglobulin IgG hinge or functional fragment thereof.
  • the IgG hinge is from IgG1, IgG2, IgG3, IgG4, IgG4 CH3, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof.
  • the hinge domain comprises the CH1, CH2, CH3 and/or hinge region of the immunoglobulin.
  • the hinge domain comprises the core hinge region of the immunoglobulin.
  • core hinge can be used interchangeably with the term “short hinge” (a.k.a “SH”).
  • suitable hinge domains are the core immunoglobulin hinge regions include EPKSCDKTHTCPPCP (SEQ ID NO: 57) from IgG1, ERKCCVECPPCP (SEQ ID NO: 58) from IgG2, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP) 3 (SEQ ID NO: 59) from IgG3, sf-6638784 42 32429-20001.40 ESKYGPPCPSCP (SEQ ID NO: 60) from IgG4 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes), and ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA
  • the hinge domain is a fragment of the immunoglobulin hinge. In some embodiments, the hinge domain is a fragment of or derived from the immunoglobulin hinge. In one embodiment, the hinge domain is an immunoglobulin hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 61, or a variant thereof of having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with SEQ ID NO: 61.
  • the hinge domain comprises the sequence set forth in SEQ ID NO: 100, or a variant thereof of having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with SEQ ID NO: 100.
  • the hinge domain comprises the sequence set forth in SEQ ID NO: 100.
  • the hinge domain is derived from CD8 or CD28.
  • the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 22.
  • the nucleic acid sequence encoding the CD28 hinge domain comprises the sequence set forth in SEQ ID NO: 5.
  • the transmembrane domain and/or hinge domain is derived from CD8 or CD28. In some embodiments, both the transmembrane domain and hinge domain are derived from CD8.
  • ADCs include recombinant monoclonal antibodies covalently bound to biologically active drugs (“payload,” e.g., cytotoxic chemicals) by chemical linkers.
  • the ADC binds to the antigen (e.g., CD123) on the surface of target cells, such as tumor cells, and then is absorbed or internalized. After the ADC is internalized, the biologically active drug is released in the lysosomes and transported to cytosol to kill the target cells.
  • ADCs can also trigger antibody- dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular- mediated phagocytosis of target cells, e.g., tumor cells.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the inactivated cell surface receptor When expressed by iPSCs or derivative cells thereof, the inactivated cell surface receptor is signaling inactive or significantly impaired, but can still be specifically recognized by an antibody.
  • the specific binding of the antibody to the inactivated cell surface receptor enables the elimination of the iPSCs or derivative cells thereof by ADCC and/or ADCP mechanisms, as well as direct killing with antibody drug conjugates with toxins or radionuclides.
  • Epidermal growth factor receptor also known as EGFR, ErbB1 and HER1 is a cell-surface receptor for members of the epidermal growth factor family of extracellular ligands.
  • truncated EGFR “tEGFR,” “short EGFR” or “sEGFR” refers to an inactive EGFR variant that lacks the EGF-binding domains and the intracellular signaling domains of the EGFR.
  • An exemplary tEGFR variant contains residues 322-333 of domain 2, all of domains 3 and 4 and the transmembrane domain of the native EGFR sequence containing the cetuximab binding epitope.
  • tEGFR is inactive when expressed by iPSCs or derivative cell thereof.
  • An exemplary inactivated cell surface receptor of the application comprises a tEGFR variant.
  • expression of the inactivated cell surface receptor in an engineered immune cell expressing a chimeric antigen receptor (CAR) induces cell suicide of the engineered immune cell when the cell is contacted with an anti-EGFR antibody.
  • CAR chimeric antigen receptor
  • the tEGFR variant comprises or consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 71, preferably the amino acid sequence of SEQ ID NO: 71.
  • the inactivated cell surface receptor comprises one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin.
  • the monoclonal antibody-specific epitope comprises or consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82, preferably the amino acid sequence of SEQ ID NO: 82.
  • the inactivated cell surface receptor further comprises a cytokine, such as interleukin-15 or interleukin-2.
  • IL-15 refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof.
  • a “functional portion” (“biologically active portion”) of a cytokine refers to a portion of the cytokine that retains one or more functions of full length or mature cytokine.
  • Such functions for IL-15 include the promotion of NK cell survival, regulation of NK cell and T cell activation and proliferation as well as the support of NK cell development from hematopoietic stem cells.
  • the sequence of a variety of IL-15 molecules are known in the art.
  • the IL-15 is a wild-type IL-15.
  • the IL-15 is a human IL-15.
  • the IL-15 comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 72, preferably the amino acid sequence of SEQ ID NO: 72.
  • Interleukin-2 refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof.
  • the IL-2 is a wild-type IL-2.
  • the IL-2 is a human IL-2.
  • the autoprotease peptide examples include, but are not limited to, a peptide sequence selected from the group consisting of porcine teschovirus-12A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), and a combination thereof.
  • the autoprotease peptide is an autoprotease peptide of porcine tesehovirus-12A (P2A).
  • the autoprotease peptide comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 73, preferably the amino acid sequence of SEQ ID NO: 73.
  • an inactivated cell surface receptor comprises a truncated epithelial growth factor (tEGFR) variant operably linked to an interleukin- 15 (IL-15) or IL-2 by an autoprotease peptide sequence.
  • tEGFR truncated epithelial growth factor
  • the inactivated cell surface receptor comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 74, preferably the amino acid sequence of SEQ ID NO: 74.
  • an inactivated cell surface receptor further comprises a signal sequence.
  • AP1903 is an experimental drug and is considered biologically inert since it does not interact with wild-type FKBP12. However clinical experience with this agent is limited to a very small number of patients (Di Stasi, A. et al. (2011) N. Engl. J. Med. 365, 1673-1683; and luliucci, J. D. et al. (2001) J. Clin. Pharmacol. 41, 870-879). AP1903 is also a relatively large and polar molecule and unlikely to cross the blood-brain barrier.
  • the iCasp9 comprises the amino acid sequence set forth in SEQ ID NO: 245, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 245. In some embodiments, the iCasp9 comprises the sequence set forth in SEQ ID NO: 245.
  • caspase 9 is fused to a modified human FK-binding protein, which has been mutated to selectively bind a chemical inducer of dimerization (CID) (Di Stasi, A. et al. (2011) N. Engl. J. Med. 365, 1673-1683). Accordingly, presence of the CID results in homodimerization and activation. It enables conditional dimerization in the presence of a small molecule CID AP1903.
  • CID chemical inducer of dimerization
  • the HSV-TK comprises the sequence set forth in SEQ ID NO: 249.
  • Non-limiting exemplary artificial cell death polypeptide / inactivated cell surface receptor sequences are provided in Table 2. sf-6638784 78 32429-20001.40 Table 2. 79 s f-6638784 32429-20001.40 s f-6638784 80 32429-20001.40
  • the inactivated cell surface receptor comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 79, preferably the amino acid sequence of SEQ ID NO: 79.
  • the truncated variant PSMA polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 250. In one embodiment, the truncated variant PSMA polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the PSMA cell tracer of fragment thereof and safety switch are operatively linked by a linker. In some embodiments, the linker comprises an autoprotease peptide, such as any autoprotease peptide disclosed herein.
  • the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 251. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 251. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 252.
  • the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 252. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 253. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 253. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 254.
  • the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 254. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 255. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 255. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 256.
  • the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 256. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 257. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 257. IV.
  • a presently disclosed method comprises independently knocking out and/or knocking down two genes selected from the group consisting B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes in an iPSC cell, in particular, B2M and CIITA to achieve class I and II HLA disruption.
  • an iPSC or derivative cell thereof of the application can be further modified by introducing an exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).
  • an iPSC or derivative cell thereof of the application can be further modified by introducing a third exogenous polynucleotide sf-6638784 83 32429-20001.40 encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).
  • a third exogenous polynucleotide sf-6638784 83 32429-20001.40 encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G).
  • the iPSC or derivative cell thereof comprises a third exogenous polypeptide encoding at least one of a human leukocyte antigen E (HLA- E) and human leukocyte antigen G (HLA-G).
  • HLA-E comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 65, preferably the amino acid sequence of SEQ ID NO: 65.
  • the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 66.
  • the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-G via a linker.
  • the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 69. V.
  • the genomic editing at one or more selected sites may comprise insertions of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, persistence, and/or survival of the genome-engineered iPSCs or derivative cells thereof.
  • the exogenous polynucleotides for insertion are operatively linked to (1) one or more exogenous promoters comprising CMV, Efla, sf-6638784 84 32429-20001.40 PGK, CAG, UBC, or other constitutive, inducible, temporal-, tissue-, or cell type- specific promoters; or (2) one or more endogenous promoters comprised in the selected sites comprising AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other locus meeting the criteria of a genome safe harbor.
  • the genome-engineered iPSCs generated using the above method comprise one or more different exogenous polynucleotides encoding proteins comprising caspase, thymidine kinase, cytosine deaminase, B-cell CD20, ErbB2 or CD79b wherein when the genome-engineered iPSCs comprise two or more suicide genes, the suicide genes are integrated in different safe harbor locus comprising AAVSl, CCR5, ROSA26, collagen, HTRP, Hll, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1.
  • exogenous polynucleotides encoding proteins may include those encoding PET reporters, homeostatic cytokines, and inhibitory checkpoint inhibitory proteins such as PD1, PD-L1, and CTLA4 as well as proteins that target the CD47/signal regulatory protein alpha (SIRP ⁇ ) axis.
  • the genome-engineered iPSCs generated using the method provided herein comprise in/del at one or more endogenous genes associated with targeting modality, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins suppressing engraftment, trafficking, homing, viability, self-renewal, persistence, and/or survival of the iPSCs or derivative cells thereof.
  • the present disclosure describes genetically engineered iPSCs and cells derived therefrom that exogenously express recombinant CD16 and recombinant NKG2D.
  • such cells also express one or more CARs (e.g., a CAR comprising a CD123 antigen-binding domain.
  • CARs e.g., a CAR comprising a CD123 antigen-binding domain.
  • such cells may express a single CAR comprising one or more antigen- binding domains that bind CD123 and another antigen.
  • such cells may express two or more CARs, wherein each CAR comprises an antigen- binding domain that binds a tumor antigen, wherein one of the tumor antigens is CD123.
  • CD16 and NKG2D are expressed from in a single polynucleotide construct as it is advantageous to reduce the number of gene edits of a cell.
  • an iPSC or derivative cell thereof containing an exogenous or isolated polynucleotide construct encoding a CD16 protein and an NKG2D protein.
  • described herein is an iPSC or derivative cell thereof expressing recombinant CD16 proteins and recombinant NKG2D proteins.
  • the recombinant proteins are encoded by an exogenous or isolated polynucleotide construct.
  • the polynucleotide construct encoding the CD16 protein and the NKG2D protein also includes a polynucleotide sequence encoding an autoprotease peptide or self-cleaving peptide.
  • an exogenous polynucleotide construct encoding the CD16 protein, the NKG2D protein and the self-cleaving peptide is introduced into the iPSC or derivative cell thereof.
  • the exogenous or isolated polynucleotide construct can be introduced into a gene locus of the iPSC or derivative cell thereof.
  • the cell expressing recombinant CD16 proteins, recombinant NKG2D proteins and CARs also expresses recombinant IL-15 proteins.
  • the cell expresses recombinant CD16 proteins, recombinant NKG2D proteins, CARs, recombinant IL-15 proteins, and either recombinant HLA-E, HLA-G, or both.
  • the cell expressing recombinant CD16 proteins, recombinant NKG2D proteins and CARs also expresses recombinant fusion proteins containing IL-15 and IL-15Ra.
  • the cell expresses recombinant sf-6638784 86 32429-20001.40 CD16 proteins, recombinant NKG2D proteins, CARs, recombinant fusion proteins containing IL-15 and IL-15Ra, and either recombinant HLA-E, HLA-G, or both.
  • the cell expressing recombinant CD16 proteins and recombinant NKG2D proteins also expresses recombinant IL-15 proteins.
  • the cell expressing recombinant CD16 proteins and recombinant NKG2D proteins also expresses recombinant fusion proteins containing IL-15 and IL-15Ra.
  • the cell expressing recombinant CD16 proteins, recombinant NKG2D proteins, and recombinant IL-15 proteins also expresses CARs.
  • the cell expressing recombinant CD16 proteins, recombinant NKG2D proteins, and recombinant fusion proteins containing IL-15 and IL-15Ra also expresses CARs.
  • the polynucleotide sequence encoding a CD16 protein and the polynucleotide sequence encoding an NKG2D protein are operably linked by a polynucleotide sequence encoding an autoprotease peptide or self- cleaving peptide.
  • the polynucleotide construct includes from 5’ to 3’ end: a polynucleotide sequence encoding a CD16 protein, a polynucleotide sequence encoding an autoprotease peptide or self-cleaving peptide and a polynucleotide sequence encoding a NKG2D protein.
  • the polynucleotide construct includes from 5’ to 3’ end: a polynucleotide sequence encoding an NKG2D protein, a polynucleotide sequence encoding an autoprotease peptide or self-cleaving peptide and a polynucleotide sequence encoding a CD16 protein.
  • the exogenous polynucleotide construct comprises the nucleic acid sequence of SEQ ID NO: 184.
  • the exogenous polynucleotide construct encodes for the amino acid sequence of SEQ ID NO: 185.
  • the CD16 protein (which is also referred to as “low affinity immunoglobulin gamma Fc region receptor III-A” or “Fc gamma receptor IIIa”) is a wildtype CD16 protein.
  • the human wildtype CD16 protein has the amino acid sequence set forth in NCBI Ref. Seq. No. NP_000560.7 or UniProt No. P08637.
  • the coding sequence of human wildtype CD16 is set forth in NCBI Ref. No. NM_000569.8.
  • the CD16 protein is a CD16 variant protein.
  • the CD16 variant protein comprises amino acids having at least 90%, e.g., sf-6638784 87 32429-20001.40 at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to wildtype CD16 such as that of SEQ ID NO: 186.
  • the CD16 variant is a high affinity CD16 variant.
  • the CD16 variant is a non-cleavable CD16 variant.
  • the CD16 variant is a high affinity and non-cleavable CD16 variant.
  • the CD16 variant comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has an F158V substitution and one or more substitutions selected from F176V, S197P, D205A, S219A, T220A, and any combination thereof. In one embodiment, the CD16 variant has an F176V substitution and one or more substitutions selected from F158V, S197P, D205A, S219A, T220A, and any combination thereof.
  • the CD16 variant has an S197P, substitution and one or more substitutions selected from F158V, F176V, D205A, S219A, T220A, and any combination thereof. In various embodiments, the CD16 variant has a D205A substitution and one or more substitutions selected from F158V, F176V, S197P, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has a substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof.
  • the CD16 variant has an S219A substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, T220A, and any combination thereof. In some embodiments, the CD16 variant has a T220A substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the variant CD16 protein has the sequence of SEQ ID NO: 187. In some embodiments, the nucleic acid sequence encoding the variant CD16 protein has the sequence of SEQ ID NO: 188. In some embodiments, the wildtype CD16 protein has the sequence of SEQ ID NO: 186.
  • the NKG2D protein (which is also referred to as NKG2-D type II integral membrane protein, CD314, killer cell lectin-like receptor subfamily K1 member 1 or KLRK1) is a wildtype NKG2D protein.
  • the human wildtype NKG2D protein has the amino acid sequence set forth in NCBI Ref. Seq. Nos. NP_001186734.1 or NP_031386.2 or UniProt No. P26718.
  • the coding sequence of human wildtype NKG2D is set sf-6638784 88 32429-20001.40 forth in NCBI Ref. Nos. NM_001199805.1 or NM_007360.3.
  • the NKG2D protein is a NKG2D variant protein.
  • the NKG2D variant protein comprises amino acids having at least 90%, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to wildtype NKG2D such as that of SEQ ID NO: 189.
  • the NKG2D protein has the amino acid sequence of SEQ ID NO: 189.
  • the nucleic acid sequence encoding the NKG2D protein has sequence of SEQ ID NO: 190.
  • 2A peptides include, but are not limited to, a porcine tesehovirus-12A (P2A) peptide, a foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide.
  • P2A porcine tesehovirus-12A
  • FMDV foot-and-mouth disease virus
  • F2A foot-and-mouth disease virus
  • E2A Equine Rhinitis A Virus
  • T2A cytoplasmic polyhedrosis virus
  • BmCPV2A cytoplasmic polyhedrosis virus
  • BmIFV2A Flacherie Virus 2A
  • the wild type CXCR4 receptor contains 352 amino acids and has seven transmembrane domains, with an extracellular N-terminus and intracellular C- terminus.
  • CXCR4 Upon binding CXCL12, CXCR4 activates downstream signaling pathways such as Ras/MAPK and PI3K/Akt that regulate cell migration, proliferation, and survival.
  • downstream signaling pathways such as Ras/MAPK and PI3K/Akt that regulate cell migration, proliferation, and survival.
  • Ras/MAPK Ras/MAPK
  • PI3K/Akt PI3K/Akt
  • CXCR4-R334X Another noteworthy CXCR4 mutation is CXCR4-R334X, which causes truncation of the C-terminal tail of the receptor. This mutation impairs CXCL12-mediated chemotaxis and calcium flux, highlighting the importance of the C-tail in G protein coupling and downstream signaling. Truncation of the CXCR4 C-tail (CXCR4-T328) can reduce CXCL12 binding affinity but maintains signaling capacity.
  • CXCR4 is an important therapeutic target in both WHIM syndrome and various cancers.
  • CXCR4 is also known to be specific to the chemokine stromal-derived-factor- 1 (SDF-1). Interaction of CXCR4 with SDF-1 can be involved in chemotaxis of lymphocytes specifically important for hematopoietic stem cell homing to bone marrow. Enforced human CXCR4 expression in primary derived NK or T cells has been shown to improve homing of the cells to the bone marrow.
  • the present disclosure provides embodiments in which wild type human CXCR4 has been introduced into iPSC-derived cells expressing a CAR, demonstrating that the expression of CXCR4 in differentiated iPSC-derived effector cells (i) improves both homing of the effector cells to the bone marrow (e.g., in mice), and (ii) significantly improves tumor killing (e.g., in a Human leukemia xenograft mouse model).
  • a primary cell, an iPSC or a derivative cell thereof according to the present disclosure can comprise one or more exogenous polynucleotides encoding CXCR4, and the CXCR4 or a fragment or variant thereof is selected from the group consisting of wild-type CXCR4 and a mutated variant of sf-6638784 90 32429-20001.40 CXCR4.
  • the CXCR4 is wild-type CXCR4.
  • the CXCR4 is wild-type CXCR4 and comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 192.
  • the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 200.
  • the CXCR4 is a mutated variant of CXCR4.
  • the CXCR4 is a mutated variant of CXCR4 and the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with a deletion of the C- terminal domain between 10 and 20 amino acid residues.
  • a CXCR4 with the deletion in the C-terminal domain corresponds to the deletion of the 10, 11, 12, 13, 14, 15, 16, 17, 18, 16, 17, 18, 19, or 20 amino acid residues in the C-terminal domain of the CXCR4 polypeptide.
  • the CXCR4 is a mutated variant of CXCR4 and the mutated variant of CXCR4 comprises wild- type CXCR4 according to SEQ ID NO: 192 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A.
  • the CXCR4 is a mutated variant of CXCR4 and the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 192-199.
  • the CXCR4 mutation is a CXCR4 whim mutation.
  • CXCR4 whim mutation refers to the autosomal dominant mutation associated with the rare combined primary immunodeficiency Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) Syndrome (WS) which have been linked to inherited autosomal dominant gain-of-function mutations in CXCR4 (see, e.g., Kawai, T. & Malech, H. L. WHIM syndrome: congenital immune deficiency disease. Current Opinion in Hematology 16, 20-26 (2009); and Dotta, L., Tassone, L. & Badolato, R.
  • the different autosomal dominant gain-of-function mutations in CXCR4 associated with Whim syndrome include: (i) R334X (gene mutation 1000C>T) the most frequent mutation resulting in carboxy- terminal truncation of 19 residues (Hernandez, et al.); (ii) G336X, (gene mutation 1000C>T) resulting in carboxy-terminus truncation of 17 residues (Gulino et al., 2004); (iii) E343X (gene mutation 1027G>T) resulting in carboxy-terminus truncation of the receptor of 10 residues (Hernandez, et al.); (iv) S339fs342X, (gene mutation 1016-1017delCT) resulting in carboxy-terminus truncation of the receptor of 13 residues and addition of 3 additional amino acid residues (Hernandez
  • the different autosomal dominant gain-of-function mutations in CXCR4 associated with Whim syndrome are located in the C-terminal domain of the CXCR4 receptor (see Table 3), a domain responsible for regulation of the receptor (internalization / inactivation). Accordingly, the C-terminal domain of the CXCR4 receptor, which is deleted in several Whim mutation could also be directly deleted to obtain the same biological effect (gain-of- function of CXCR4 associated with the long term CD8 memory response).
  • Biological activity of CD8 cells according to the invention can be measured for example with chemokine receptor internalization assay or cell migration after adding a CXCR4 agonist. sf-6638784 92 32429-20001.40 Table 3.
  • Genome editing, or genomic editing, or genetic editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and/or replaced in the genome of a targeted cell.
  • Targeted genome editing (interchangeable with “targeted genomic editing” or “targeted genetic editing”) enables insertion, deletion, and/or substitution at pre-selected sites in the genome.
  • targeted genomic editing or “targeted genetic editing”
  • targeted editing can also be used to disrupt endogenous gene expression with precision.
  • targeted integration referring to a process involving insertion of one or more exogenous sequences at pre-selected sites in the genome, with or without deletion of an endogenous sequence at the insertion site.
  • Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach.
  • nuclease-independent targeted editing approach homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be inserted, through the enzymatic machinery of the host cell.
  • targeted editing could be achieved with higher frequency through specific introduction of double strand breaks (DSBs) by specific rare-cutting endonucleases.
  • DSBs double strand breaks
  • Such nuclease-dependent targeted editing utilizes DNA repair mechanisms including non-homologous end joining (NHEJ), which occurs in response to DSBs.
  • NHEJ non-homologous end joining
  • the NHEJ often leads to random insertions or deletions (in/dels) of a small number of endogenous nucleotides.
  • HDR homology directed repair
  • a donor vector containing exogenous genetic material flanked by a pair of homology arms the exogenous genetic material can be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in a “targeted integration.”
  • Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), RNA-guided CRISPR (Clustered Regular Interspaced 96 sf-6638784 32429-20001.40 Short Palindromic Repeats) systems.
  • ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain.
  • ZFBD zinc finger DNA binding domain
  • a zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers.
  • a “designed” zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
  • a “selected” zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. ZFNs are described in greater detail in U.S. Pat. No.
  • a TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain.
  • transcription activator-like effector DNA binding domain By “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” it is meant the polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection.
  • TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD).
  • RVD repeat variable-diresidues
  • TALENs are described in greater detail in U.S. Patent Application No. 2011/0145940, which is herein incorporated by reference.
  • the most recognized example of a TALEN sf-6638784 97 32429-20001.40 in the art is a fusion polypeptide of the Fokl nuclease to a TAL effector DNA binding domain.
  • a targeted nuclease that finds use in the subject methods is a targeted Spoll nuclease, a polypeptide comprising a Spol l polypeptide having nuclease activity fused to a DNA binding domain, e.g., a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc. that has specificity for a DNA sequence of interest.
  • a DNA binding domain e.g., a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc.
  • Additional examples of targeted nucleases suitable for the present application include, but not limited to Bxbl, phiC3 l, R4, PhiBTl, and Wp/SPBc/TP90l-l, whether used individually or in combination.
  • CRISPR/Cpf1 comprises two major components: (1) a CPf1 endonuclease and (2) a crRNA.
  • RNP ribonucleoprotein
  • the crRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cpf1 to target selected sequences. These two components can then be delivered to mammalian cells via transfection or transduction.
  • gRNA chimeric guide RNA
  • MAD7 is an engineered Cas12a variant originating from the bacterium Eubacterium rectale that has a preference for 5′-TTTN-3′ and 5′-CTTN-3′ PAM sites and does not require a tracrRNA. See, for example, PCT Publication No. 2018/236548, the disclosure of which is incorporated herein by reference.
  • DICE mediated insertion uses a pair of recombinases, for example, phiC31 and Bxbl, to provide unidirectional integration of an exogenous DNA that is tightly restricted to each enzyme’s own small attB and attP recognition sites. Because these sf-6638784 98 32429-20001.40 target att sites are not naturally present in mammalian genomes, they must be first introduced into the genome, at the desired integration site. See, for example, U.S. Application Publication No. 2015/0140665, the disclosure of which is incorporated herein by reference.
  • One aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genome integration.
  • the construct further comprises a pair of homologous arms specific to a desired integration site
  • the method of targeted integration comprises introducing the construct to cells to enable site specific homologous recombination by the cell host enzymatic machinery.
  • the pair of homologous arms e.g., left homology arm and right homology arm
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a ZFN-mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a TALEN-mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cpf1 expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cpf1-mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cas9 expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cas9-mediated insertion.
  • the method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases to a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing an expression cassette for DICE recombinases, to enable DICE- mediated targeted integration.
  • the guide sequence specific to a desired integration site is provided in Table 4.
  • the guide sequence is specific to an AAVS1 locus and set forth in SEQ ID NO: 225.
  • the guide sequence is specific to an AAVS1 locus and set forth in SEQ ID NO: 94.
  • the guide sequence is specific to a B2M locus and set forth in SEQ ID NO: 226 or SEQ ID NO: 227.
  • the guide sequence is specific to a B2M locus and set forth in SEQ ID NO: 238.
  • the guide sequence is specific to a B2M locus and set forth in SEQ ID NO: 93.
  • the guide sequence is specific to a CIITA locus and set forth in SEQ ID NO: 228. In one embodiment, the guide sequence is specific to a CIITA locus and set forth in SEQ ID NO: 229. In one embodiment, the guide sequence is specific to a CIITA locus and set forth in SEQ ID NO: 95. In one embodiment, the guide sequence is specific to a CD70 locus and set forth in SEQ ID NO: 230. In one embodiment, the guide sequence is specific to a CLYBL locus and set forth in SEQ ID NO: 231. In one embodiment, the guide sequence is specific to a NKG2A locus and set forth in SEQ ID NO: 232.
  • the guide sequence is specific to a TRAC locus and set forth in SEQ ID NO: 233. In one embodiment, the guide sequence is specific to a CD38 locus and set forth in SEQ ID NO: 234. In one embodiment, the guide sequence is specific to a CD33 locus and set forth in SEQ ID NO: 235 or SEQ ID NO: 236. In one embodiment, the guide sequence is specific to an IL2 locus and set forth in SEQ ID NO: 237.
  • Sites for targeted integration include, but are not limited to, genomic safe harbors, which are intragenic or extragenic regions of the human genome that, theoretically, are able to accommodate predictable expression of newly integrated DNA without adverse effects on the host cell or organism.
  • the genome safe harbor for the targeted integration is one or more loci of genes selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes.
  • the site for targeted integration is selected for deletion or reduced expression of an endogenous gene at the insertion site.
  • the term “deletion” with respect to expression of a gene refers to any genetic modification that abolishes the expression of the gene.
  • Examples of “deletion” of expression of a gene include, e.g., a removal or deletion of a DNA sequence of the gene, an insertion sf-6638784 100 32429-20001.40 of an exogenous polynucleotide sequence at a locus of the gene, and one or more substitutions within the gene, which abolishes the expression of the gene.
  • Genes for target deletion include, but are not limited to, genes of major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must be matched for histocompatibility in allogeneic recipients to avoid allogeneic rejection problems.
  • MHC major histocompatibility complex
  • MHC deficient including MHC- class I deficient, or MHC-class II deficient, or both, refers to cells that either lack, or no longer maintain, or have reduced level of surface expression of a complete MHC complex comprising a MHC class I protein heterodimer and/or a MHC class II heterodimer, such that the diminished or reduced level is less than the level naturally detectable by other cells or by synthetic methods.
  • MHC class I deficiency can be achieved by functional deletion of any region of the MHC class I locus (chromosome 6p2l), or deletion or reducing the expression level of one or more MHC class-I associated genes including, not being limited to, beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene and Tapasin genes.
  • B2M gene encodes a common subunit essential for cell surface expression of all MHC class I heterodimers.
  • B2M null cells are MHC-I deficient.
  • MHC class II deficiency can be achieved by functional deletion or reduction of MHC-II associated genes including, not being limited to, RFXANK, CIITA, RFX5 and RFXAP.
  • CIITA is a transcriptional coactivator, functioning through activation of the transcription factor RFX5 required for class II protein expression.
  • CIITA null cells are MHC-II deficient.
  • one or more of the exogenous polynucleotides are integrated at one or more loci of genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby delete or reduce the expression of the gene(s) with the integration.
  • the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell, preferably the one or more loci are of genes selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hl l, GAPDH, RUNX1, B2M, TAPI, 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, or TIGIT genes, provided at least one of the one or more loci is of a MHC gene, such as a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.
  • a MHC gene such as a gene selected from the
  • the one or more exogenous polynucleotides are integrated sf-6638784 101 32429-20001.40 at a locus of an MHC class-I associated gene, such as a beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene or Tapasin gene; and at a locus of an MHC-II associated gene, such as a RFXANK, CIITA, RFX5, RFXAP, or CIITA gene; and optionally further at a locus of a safe harbor gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes.
  • an MHC class-I associated gene such as a beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene or Tapasin gene
  • B2M beta-2 microglobulin
  • MHC-II associated gene such as a RFXANK, CIITA
  • the one or more of the exogenous polynucleotides are integrated at the loci of CIITA, AAVS1 and B2M genes.
  • the first exogenous polynucleotide is integrated at a locus of AAVS1 gene;
  • the second exogenous polypeptide is integrated at a locus of CIITA gene;
  • the third exogenous polypeptide is integrated at a locus of B2M gene; wherein integrations of the exogenous polynucleotides delete or reduce expression of CIITA and B2M genes.
  • the first exogenous polynucleotide comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or both of SEQ ID NOs: 174 and 175;
  • the second exogenous polynucleotide comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 75;
  • the third exogenous polynucleotide comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 67.
  • the first exogenous polynucleotide comprises polynucleotides having the sequence of one or both of SEQ ID NOs: 174 and 175;
  • the second exogenous polynucleotide comprises polynucleotides having the sequence of SEQ ID NO: 75; and
  • the third exogenous polynucleotide comprises polynucleotides having the sequence of SEQ ID NO: 67.
  • the invention relates to a cell derived from differentiation of an iPSC, a derivative cell. As described above, the genomic edits introduced into the iPSC cell are retained in the derivative cell.
  • the application provides a NK cell or a T cell comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a deletion or reduced expression of an MHC class I associated gene and an MHC class II associated gene, such as an MHC class-I associated gene selected from the group consisting of a B2M gene, TAP 1 gene, TAP 2 gene and Tapasin gene, and an MHC-II associated gene selected from the group consisting of a RFXANK gene, CIITA gene, RFX5 gene, RFXAP gene, and CIITA gene, preferably the B2M gene and CIITA gene; and optionally (iii) an exogenous polynucleotide encoding a chimeric IL-15RA and an IL-15, wherein the IL-15RA and IL-15 are operably linked.
  • CAR chimeric antigen receptor
  • the NK cell or T cell further comprises an exogenous polynucleotide encoding at least one of a human leukocyte antigen E (HLA-E) and a human leukocyte antigen G (HLA-G).
  • a NK cell or a T cell comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) having the amino acid sequence of one or both of SEQ ID NOs: 172 and 173; (ii) an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66; and optionally (iii) an exogenous polynucleotide encoding a chimeric IL-15RA and an interleukin-15 (IL-15), wherein the IL-15RA and IL-15 are operably linked, having the amino acid sequence of SEQ ID NO:247, and wherein the exogen
  • CAR
  • the first exogenous polynucleotide comprises polynucleotides having the sequence of one or both of SEQ ID NOs: 174 and 175; the second exogenous polynucleotide comprises polynucleotides having the sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises polynucleotides having the sequence of SEQ ID NO: 67.
  • HPC hematopoietic progenitor cell
  • iPSC induced pluripotent stem cell
  • a CD34+ hematopoietic progenitor cell derived from an induced pluripotent stem cell (iPSC) comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) an exogenous polynucleotide encoding an inactivated cell surface receptor that comprises a monoclonal antibody- specific epitope and an IL-15, wherein the inactivated cell surface receptor and IL-15 sf-6638784 103 32429-20001.40 are operably linked by an autoprotease peptide sequence; and (iii) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes.
  • the CD34+ HPC further comprises an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and/or human leukocyte antigen G (HLA-G).
  • the CAR comprises (i) optionally, a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to CD123; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a co-stimulatory domain, such as a co-stimulatory domain comprising a CD28 signaling domain. Also provided is a method of manufacturing the derivative cell.
  • the method comprises differentiating the iPSC under conditions for cell differentiation to thereby obtain the derivative cell.
  • An iPSC of the application can be differentiated by any method known in the art. Exemplary methods are described in US8846395, US8945922, US8318491, WO2010/099539, WO2012/109208, WO2017/070333, WO2017/179720, WO2016/010148, WO2018/048828 and WO2019/157597, each of which are herein incorporated by reference in its entirety.
  • the differentiation protocol may use feeder cells or may be feeder-free.
  • feeder cells are terms describing cells of one type that are co-cultured with cells of a second type to provide an environment in which the cells of the second type can grow, expand, or differentiate, as the feeder cells provide stimulation, growth factors and nutrients for the support of the second cell type.
  • the iPSC derivative cells of the invention are NK cells which are prepared by a method of differentiating an iPSC cell into an NK cell by subjecting the cells to a differentiation protocol including the addition of recombinant human IL-12p70 for the final 24 hours of culture.
  • the coding sequence of a CAR can be changed (e.g., having nucleotides substituted) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding CARs of the application can be altered without changing the amino acid sequences of the proteins.
  • the isolated nucleic acid encodes a CAR targeting CD123.
  • the isolated nucleic acid encoding the CAR comprises a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to one of SEQ ID NOs: 174 and 175.
  • the application provides a vector comprising a polynucleotide encoding a CAR useful for an invention according to embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector.
  • the application provides vectors for targeted integration of a CAR useful for an invention according to embodiments of the application.
  • the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding a CAR according to an embodiment of the application; and (c) a terminator/polyadenylation signal.
  • the promoter is a CAG promoter.
  • the CAG promoter is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 63.
  • Other promoters can also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta actin.
  • the terminator/ polyadenylation signal is a SV40 signal.
  • the SV40 signal is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 64.
  • Other terminator sequences can also be used, examples of which include, but are not limited to, BGH, hGH, and PGK.
  • the polynucleotide sequence encoding a CAR comprises polynucleotides having a sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to one or more of SEQ ID NOs: 149-171, 174, and 175.
  • the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.
  • left homology arm and right homology arm refers to a pair of nucleic acid sequences that flank an exogenous polynucleotide and facilitate the integration of the exogenous polynucleotide into a specified chromosomal locus. Sequences of the left and right arm homology arms can be designed based on the integration site of interest. In some embodiment, the left or right arm homology arm is homologous to the left or right side sequence of the integration site.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 90.
  • the left homology arm is a polynucleotide set forth in SEQ ID NO: 90.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 91.
  • the right homology arm is a polynucleotide set forth in SEQ ID NO: 91.
  • the left homology arm and right homology arms comprise any of the nucleotide sequences provided in Table 4. sf-6638784 122 32429-20001.40
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 201.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 201.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 213.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 213.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 202.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 202.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 214.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 214.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 203.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 203.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 215.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 215.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 204.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 204.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 216.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 216.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, sf-6638784 123 32429-20001.40 98%, 99% or 100%, identical to SEQ ID NO: 205.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 205.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 217.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 217.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 206.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 206.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 218.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 218.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 207.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 207.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 219.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 219.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 208.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 208.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 220.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 220.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 209.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 209.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 221.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 221.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 210.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 210.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 222.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 222.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 211.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 211.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 223.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 223.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 212.
  • the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 212.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 224.
  • the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 224.
  • the vector comprises polynucleotides having a sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 92, preferably the polynucleotide sequence of SEQ ID NO: 92.
  • the invention relates to an isolated nucleic acid encoding an inactivated cell surface receptor useful for an invention according to embodiments of the application.
  • an isolated nucleic acid encodes any inactivated cell surface receptor described herein, such as that comprises a monoclonal antibody- specific epitope, and a cytokine, such as an IL-15 or IL-2, wherein the monoclonal antibody-specific epitope and the cytokine are operably linked by an autoprotease peptide sequence.
  • the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by an antibody, such as abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab, infliximab, ipilimumab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, polatuzumab vedotin, ranibizumab, rituximab, tiuxetan
  • the isolated nucleic acid encodes an inactivated cell surface receptor having a truncated epithelial growth factor (tEGFR) variant.
  • the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, necitumumab or panitumumab, preferably cetuximab.
  • the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin.
  • the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD20, such as an epitope specifically recognized by rituximab. In certain embodiments, the isolated nucleic acid encodes an sf-6638784 126 32429-20001.40 inactivated cell surface receptor having one or more epitopes of Her 2 receptor, such as an epitope specifically recognized by trastuzumab. In certain embodiments, the autoprotease peptide sequence is porcine tesehovirus-12A (P2A).
  • the truncated epithelial growth factor (tEGFR) variant consists of amino acids having a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71.
  • the monoclonal antibody-specific epitope specifically recognized by polatuzumab vedotin consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78.
  • the monoclonal antibody-specific epitope specifically recognized by rituximab consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 80.
  • the monoclonal antibody-specific epitope specifically recognized by trastuzumab consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82.
  • the IL-15 comprises amino acids having a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72.
  • the autoprotease peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 73.
  • the inactivated cell surface receptor comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74.
  • the isolated nucleic acid encoding the inactivated cell surface receptor is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 75, preferably the polynucleotide sequence of SEQ ID NO: 75.
  • the application provides a vector comprising a polynucleotide sequence encoding an inactivated cell surface receptor useful for an invention according to embodiments of the application.
  • Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector.
  • the vector is a recombinant expression vector such as a plasmid.
  • the vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication.
  • the promoter can be a constitutive, inducible, or repressible promoter.
  • a number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of an inactivated cell surface receptor in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.
  • the application provides a vector for targeted integration of an inactivated cell surface receptor useful for an invention according to embodiments of the application.
  • the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an inactivated cell surface receptor, such as an inactivated cell surface receptor comprising a truncated epithelial growth factor (tEGFR) variant and an (IL-15, wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide sequence, such as porcine tesehovirus-12A (P2A), and (c) a terminator/polyadenylation signal.
  • the promoter is a CAG promoter.
  • the CAG promoter is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 63.
  • Other promoters can also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta actin.
  • the terminator/polyadenylation signal is a SV40 signal.
  • the SV40 signal is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 64.
  • Other terminator sf-6638784 128 32429-20001.40 sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK .
  • the polynucleotide sequence encoding an inactivated cell surface receptor is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 75.
  • the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.
  • the left homology arm and the light homology arm comprise the sequence set forth in Table 4 or sequences set forth in Section VII.C(1) above.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 84.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 85.
  • the vector is a polynucleotide having a sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 86, preferably the polynucleotide sequence of SEQ ID NO: 86.
  • Nucleic acids encoding an HLA construct In another general aspect, the invention relates to an isolated nucleic acid encoding an HLA construct useful for an invention according to embodiments of the application.
  • the coding sequence of an HLA construct can be changed (e.g., having nucleotides substituted) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding an HLA construct of the application can be altered without changing the amino acid sequences of the proteins.
  • the isolated nucleic acid encodes an HLA construct comprising a signal peptide, such as an HLA-G signal peptide, operably linked to an HLA coding sequence, such as a coding sequence of a mature B2M, and/or a mature HLA-E.
  • the HLA coding sequence encodes the HLA-G and sf-6638784 129 32429-20001.40 B2M, which are operably linked by a 4X GGGGS linker, and/or the B2M and HLA- E, which are operably linked by a 3X GGGGS linker.
  • the isolated nucleic acid encoding the HLA construct is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67.
  • the isolated nucleic acid encoding the HLA construct is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.
  • the application provides a vector comprising a polynucleotide sequence encoding a HLA construct useful for an invention according to embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector.
  • the vector is a recombinant expression vector such as a plasmid.
  • the vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication.
  • the promoter can be a constitutive, inducible, or repressible promoter.
  • a number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a HLA construct in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application.
  • the application provides vectors for targeted integration of a HLA construct useful for an invention according to embodiments of the application.
  • the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an HLA construct; and (c) a terminator/polyadenylation signal.
  • the promoter is a CAG promoter.
  • the CAG promoter is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 63.
  • Other promoters can also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta actin.
  • the terminator/ polyadenylation signal is a SV40 signal.
  • the SV40 signal is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 64.
  • Other terminator sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK .
  • a polynucleotide sequence encoding a HLA construct comprises a signal peptide, such as a HLA-G signal peptide, a mature B2M, and a mature HLA-E, wherein the HLA-G and B2M are operably linked by a 4X GGGGS linker (SEQ ID NO: 31) and the B2M transgene and HLA-E are operably linked by a 3X GGGGS linker (SEQ ID NO: 25).
  • the HLA construct is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67.
  • the HLA construct is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70.
  • the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide.
  • the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 87.
  • the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 88.
  • the vector comprises polynucleotides having a sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 89, preferably the polynucleotide sequence of SEQ ID NO: 89.
  • the application provides a host cell comprising a vector of the application and/or an isolated nucleic acid encoding a construct of the sf-6638784 131 32429-20001.40 application.
  • any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of exogenous polynucleotides of the application.
  • the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.
  • host cells include, for example, recombinant cells containing a vector or isolated nucleic acid of the application useful for the production of a vector or construct of interest; or an engineered iPSC or derivative cell thereof containing one or more isolated nucleic acids of the application, preferably integrated at one or more chromosomal loci.
  • a host cell of an isolated nucleic acid of the application can also be an immune effector cell, such as a T cell or NK cell, comprising the one or more isolated nucleic acids of the application.
  • the immune effector cell can be obtained by differentiation of an engineered iPSC of the application. Any suitable method in the art can be used for the differentiation in view of the present disclosure.
  • the immune effector cell can also be obtained transfecting an immune effector cell with one or more isolated nucleic acids of the application.
  • the application provides a composition comprising an isolated polynucleotide of the application, a host cell and/or an iPSC or derivative cell thereof of the application.
  • the composition further comprises one or more therapeutic agents 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), siRNA, oligonucleotide, mononuclear blood cells, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent, a radioactive moiety, or an immunomodulatory drug (iMiD).
  • a therapeutic agents 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), siRNA, oligonucleotide, mononuclear blood cells, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent, a radioactive moiety, or
  • the composition is a pharmaceutical composition comprising an isolated polynucleotide of the application, a host cell and/or an iPSC or derivative cell thereof of the application and a pharmaceutically acceptable carrier.
  • pharmaceutical composition means a product comprising an isolated polynucleotide of the application, an isolated polypeptide of the application, a host cell of the application, and/or an iPSC or derivative cell thereof of sf-6638784 132 32429-20001.40 the application together with a pharmaceutically acceptable carrier.
  • polypeptides, host cells, and/or iPSCs or derivative cells thereof of the application and compositions comprising them are also useful in the manufacture of a medicament for therapeutic applications mentioned herein.
  • carrier refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.
  • the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition described herein or the biological activity of a composition described herein.
  • any pharmaceutically acceptable carrier suitable for use in a polynucleotide, polypeptide, host cell, and/or iPSC or derivative cell thereof can be used.
  • the formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g., 21 st edition (2005), and any later editions).
  • Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers, and chelating agents.
  • One or more pharmaceutically acceptable carrier may be used in formulating the pharmaceutical compositions of the application. E. Methods of use
  • Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination.
  • the definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a “clinically detectable” tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation on physical examination, and/or which is detectable because of the sf-6638784 133 32429-20001.40 expression of one or more cancer-specific antigens in a sample obtainable from a patient.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • X-ray ultrasound or palpation on physical examination
  • Cancer conditions may be characterized by the abnormal proliferation of malignant cancer cells and may include leukemias, such as AML, CML, ALL and CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP).
  • leukemias such as AML, CML, ALL and CLL
  • lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma
  • Cancer cells within an individual may be immunologically distinct from normal somatic cells in the individual.
  • the cancerous tumor may be immunogenic, i.e., the cancer cells may be capable of eliciting a systemic immune response in the individual against one or more antigens expressed by the cancer cells.
  • the tumor antigens that elicit the immune response may be specific to cancer cells or may be shared by one or more normal cells in the individual.
  • the cancer cells of an individual suitable for treatment as described herein may express the antigen and/or may be of correct HLA type to bind the antigen receptor expressed by the ⁇ cells.
  • An individual suitable for treatment as described above may be a mammal. In preferred embodiments, the individual is a human.
  • non-human mammals especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, primate, porcine, canine, or rabbit animals) may be employed.
  • the individual may have minimal residual disease (MRD) after an initial cancer treatment.
  • the individual may have no minimal residual disease after one or more cancer treatments or repeated dosing.
  • An individual with cancer may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of cancer in accordance with clinical standards known in the art.
  • diagnosis of a cancer in an individual may include identification of a particular cell type (e.g., a cancer cell) in a sample of a body fluid or tissue obtained from the individual.
  • a particular cell type e.g., a cancer cell
  • An anti-tumor effect is a biological effect which can be manifested by a reduction in the rate of tumor growth, decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An "anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies, also ⁇ cells which may be obtained according to the methods of the present invention, as described herein in prevention of the occurrence of tumors in the first place.
  • Treatment may be any treatment and/or therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment.
  • Treatment may also be prophylactic (e.g., prophylaxis).
  • an individual susceptible to or at risk of the occurrence or re-occurrence of cancer may be treated as described herein.
  • Such treatment may prevent or delay the occurrence or re-occurrence of cancer in the individual.
  • treatment may include inhibiting cancer growth, including complete cancer remission, and/or inhibiting cancer metastasis.
  • Cancer growth generally refers to any one of a number of indices that indicate change within the cancer to a more developed form.
  • indices for measuring an inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumor growth, a destruction of tumor vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of ⁇ cells, and a decrease in levels of tumor-specific antigens.
  • CT computed tomographic
  • Administration of ⁇ cells modified as described herein may improve the capacity of sf-6638784 135 32429-20001.40 the individual to resist cancer growth, in particular growth of a cancer already present the subject and/or decrease the propensity for cancer growth in the individual.
  • This application provides a method of treating a disease or a condition in a subject in need thereof. The methods comprise administering to the subject in need thereof a therapeutically effective amount of cells of the application and/or a composition of the application.
  • the disease or condition is cancer.
  • the cancer can, for example, be a solid or a liquid cancer.
  • the cancer can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a liver cancer, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, an endometrial cancer, a prostate cancer, a thyroid cancer, a glioma, a glioblastoma, and other solid tumors, and a non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma/disease (HD), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors.
  • NHL non-Hodgkin’s lymph
  • the cancer is a non-Hodgkin’s lymphoma (NHL).
  • the composition comprises a therapeutically effective amount of an isolated polynucleotide, an isolated polypeptide, a host cell, and/or an iPSC or derivative cell thereof.
  • therapeutically effective amount refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject. A therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose.
  • a therapeutically effective amount means an amount of the cells and/or the pharmaceutical composition that modulates an immune response in a subject in need thereof.
  • a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be sf-6638784 136 32429-20001.40 treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the re
  • the therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.
  • the compositions described herein are formulated to be suitable for the intended route of administration to a subject.
  • the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.
  • the cells of the application and/or the pharmaceutical compositions of the application can be administered in any convenient manner known to those skilled in the art.
  • the cells of the application can be administered to the subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and/or transplantation.
  • the compositions comprising the cells of the application can be administered transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrapleurally, by intravenous (i.v.) injection, or intraperitoneally.
  • the cells of the application can be administered with or without lymphodepletion of the subject.
  • compositions comprising cells of the application can be provided in sterile liquid preparations, typically isotonic aqueous solutions with cell suspensions, or optionally as emulsions, dispersions, or the like, which are typically sf-6638784 137 32429-20001.40 buffered to a selected pH.
  • the compositions can comprise carriers, for example, water, saline, phosphate buffered saline, and the like, suitable for the integrity and viability of the cells, and for administration of a cell composition.
  • Sterile injectable solutions can be prepared by incorporating cells of the application in a suitable amount of the appropriate solvent with various other ingredients, as desired.
  • compositions can include a pharmaceutically acceptable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like, that are suitable for use with a cell composition and for administration to a subject, such as a human.
  • a pharmaceutically acceptable carrier such as sterile water, physiological saline, glucose, dextrose, or the like
  • Suitable buffers for providing a cell composition are well known in the art.
  • Any vehicle, diluent, or additive used is compatible with preserving the integrity and viability of the cells of the application.
  • the cells of the application and/or the pharmaceutical compositions of the application can be administered in any physiologically acceptable vehicle.
  • a cell population comprising cells of the application can comprise a purified population of cells. Those skilled in the art can readily determine the cells in a cell population using various well known methods.
  • the ranges in purity in cell populations comprising genetically modified cells of the application can be from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%. Dosages can be readily adjusted by those skilled in the art, for example, a decrease in purity could require an increase in dosage.
  • the cells of the application are generally administered as a dose based on the number cells per kilogram (cells/kg) of body weight of the subject to which the cells and/or pharmaceutical compositions comprising the cells are administered.
  • the cell doses are in the range of about 10 4 to about 10 10 cells/kg of body weight, for example, about 10 5 to about 10 9 , about 10 5 to about 10 8 , about 10 5 to about 10 7 , or about 10 5 to about 10 6 , depending on the mode and location of administration.
  • a higher dose is used than in regional administration, where the immune cells of the application are administered in the region of a tumor and/or cancer.
  • Exemplary dose ranges include, but are not limited to, 1 x 10 4 to 1 x 10 8 , 2 x 10 4 to 1 x 10 8 , 3 x 10 4 to 1 x 10 8 , 4 x 10 4 to 1 x 10 8 , 5 x 10 4 to 6 x 10 8 , 7 x 10 4 to 1 x 10 8 , 8 x 10 4 to 1 x 10 8 , 9 x 10 4 to 1 x 10 8 , 1 x 10 5 to 1 x 10 8 , 1 x 10 5 to 9 x 10 7 , 1 x 10 5 to 8 x 10 7 , 1 x 10 5 to 7 x 10 7 , 1 x 10 5 to 6 x 10 7 , 1 x sf-6638784 138 32429-20001.40 10 5 to 5 x 10 7 , 1 x 10 5 to 4 x 10 7 , 1 x 10 5 to 4 x 10 7 , 1 x 10 5 to 3 x 10 7 , 1 x 10
  • a fixed dosage of cells can be administered to a patient.
  • Exemplary fixed dosages include, but are not limited to about 10 5 , about 10 6 , about 10 7 , or about 10 8 cells.
  • the fixed or weigh- based dose can be adjusted to account for whether a single dose is being administered or whether multiple doses are being administered. The precise determination of what would be considered an effective dose can be based on factors individual to each subject.
  • the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer, which is not necessarily discernible in the subject, but can be discernible in the subject.
  • treat can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer.
  • “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition.
  • “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject.
  • the cells of the application and/or the pharmaceutical compositions of the application can be administered in combination with one or more additional 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), siRNA, sf-6638784 139 32429-20001.40 oligonucleotide, mononuclear blood cells, 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
  • CD33-specific CARs were constructed with binding domains on the extracellular side of the protein construct, singly or in multiples.
  • scFv proteins were tested as scFv-IgG1 Fc fusions for protein affinity and specific binding to CD33+ cells.
  • Epitope mapping was performed by binding competition and using CD33 protein variants with selected domains deleted from the full-length protein.
  • VH and VL fragments were cloned into lentiviral vectors in frame with a hinge domain (IgG4 Fc or CD8 hinge), a transmembrane domain (CD28tm), a co- stimulatory domain (41BB, CD28, or DAP10) and a primary signaling domain from CD3z.
  • Lentivirus was produced for the various constructs and transduced into primary T-cells to examine antigen-dependent activation of this T-cell line.
  • scFv-CAR transduced cells were cultured for two weeks, then injected into tumor-bearing female NSG mice (FIG. 1). Clones with tumor growth inhibition (TGI) of 90% and higher were selected for further functional assessment.
  • TGI tumor growth inhibition
  • Example 2 Development of cells expressing CD33-specific VHH-CARs CARs were constructed with VHH binding domains on the extracellular side of the protein construct, singly or in multiples. The VHH antibody binding fragments were isolated through phage display of VHH libraries and selection on the soluble CD33 protein.
  • VHH proteins were tested as VHH-IgG1 Fc fusions for protein affinity and specific binding to CD33+ cells.
  • VHH sequences were fused to the human IgG1 Fc domain in a mammalian expression construct and expressed as VHH-Fc fusion proteins in Expi293 cells and purified with Protein A (MabSelect SuRe) resin.
  • VHH-Fc fusion proteins were assessed for binding to CD33 expressed on the cell surface.
  • VHH-Fc proteins were serially diluted and added to CHO-CD33 and CHO-K1 cells and incubated at 4C for 30 minutes.
  • VHH-specific VHH-CARs CARs were constructed with VHH binding domains on the extracellular side of the protein construct, singly or in multiples.
  • the VHH antibody binding fragments were isolated through phage display of VHH libraries and selection on the soluble CD123 protein.
  • VHH proteins were tested as VHH-IgG1 Fc fusions for protein affinity and specific binding to CD33+ cells.
  • VHH sequences were fused to the human IgG1 Fc domain in a mammalian expression construct and expressed as VHH- Fc fusion proteins in Expi293 cells and purified with Protein A (MabSelect SuRe) resin.
  • VHH fragments were cloned into lentiviral vectors in frame with a hinge domain (IgG4 Fc or CD8 hinge), a transmembrane domain (CD28tm), a co- stimulatory domain (41BB, CD28, DAP10) and a primary signaling domain from CD3z.
  • Lentivirus was produced for the various constructs and transduced into primary T-cells to examine antigen-dependent activation of this T-cell line.
  • VHH-CARs were tested in primary T-cells for killing CD123+ tumor target cells. Lentivirus for each of the CARs described was transduced into primary donor T-cells and grown for 14 days. Expression of the CARs was assessed by flow cytometry.
  • CD123+ cells (FIGs. 6A-6F) were labeled with CellTrace Violet (ThermoFisher) and co-cultured with the CAR-T cells at various E:T ratios for 48 hours. Target cell viability was then assessed by flow cytometry (FIGs. 7A-7F).
  • Anti-CD123 VHH binders demonstrated CAR cytotoxic activity against CD123+ tumor cell lines. VHH-CAR transduced cells were cultured for two weeks, then injected into tumor-bearing female NSG mice (FIG. 8). Clones with TGI of 90% and higher were selected for further functional assessment.
  • Example 4 Cytotoxicity Assays Using Therapeutic Cells Expressing Mono- and Dual-targeting CARs against CD33 and/or CD123 Target Cells Dual-targeting CAR ectodomains were engineered with select CD33 binders in combination with selected CD123 binders and with various combinations of hinge, transmembrane and co-stimulatory endodomains (FIGs. 9A-9D, FIGs. 10A-10E, and FIG. 11). Selected CARs were expressed and tested for cytotoxicity activity in iNK cells. CAR expression was confirmed using a FACS assay (FIGs. 12A-12D), and CAR-iNK cells were subject to in vivo efficacy study.
  • CAR-iNK cells (IPSC3081, IPSC3082, IPSC3083 derived from the iPSC1631 parental line and IPSC3084, IPSC3085, and IPSC3086 derived from the parental iPSC1650 parental line) were injected to tumor bearing female NSG mice, and the resulting cytotoxic effect was assessed by chemiluminescence readout at days 6 and 8 (FIG. 13; Table 5).
  • iPSC cell line engineered to express a CAR against a tumor cell antigen was further engineered, through homology-directed-repair (HDR) into the CLYBL safe harbor site, to express the human wild type CXCR4 protein.
  • the iPSC cell lines were differentiated to iNK cells, and evaluated for CXCR4 expression through the process.
  • HPC stage following engineering
  • Day 14 (D14) of iNK differentiation Day 21 (D21) of iNK differentiation
  • cells were analyzed for CXCR4 levels by flow cytometry (FIG. 14).
  • CXCR4 PE mouse anti- human CD184 [CXCR4] mAb
  • FACS was conducted on the BD Symphony flow cytometer, and results depicted as % CXCR4 positive cells of live cells.
  • CXCR4 was found to be expressed in 42.4% of the engineered Day 21 iNK cells (IPSC1613) versus 2.4% of the iNK cells not engineered to express CXCR4 (iPSC611).
  • the IPSC lines were differentiated at larger scale for use in functional assays, and CXCR4 expression was evaluated as previously indicated. At scale, the CXCR4-engineered iNK cells maintained 47.7% expression of CXCR4 (FIG. 15).
  • iNK cells differentiated from iPSC611 CAR+/CXCR4-
  • iPSC1613 CAR+/CXCR4+
  • a CAR-CXCR4- iT cell line iT CAR-CXCR4-
  • 2x10 5 effector cells were co-cultured with 2x10 5 NucLight Red (NLR) labeled NALM-6 tumor cells in flatbottom 96-well tissue culture treated plates and placed in an Incucyte live cell imager. Whole well images were taken every 3 hours for 72 hours total collecting phase contrast and NLR images.
  • IPSC cells expressing a dual-targeting CAR to two tumor targets was transduced with a lentivirus containing sequence encoding for WT CXCR4 upon integration into the cell line genome.
  • Hematopoietic progenitor cells (HPCs) derived from IPSC2984 were transduced with the WT CXCR4 lentivirus and then differentiated 14 days to iNK cells.
  • CXCR4 expression was evaluated as previously described, and the lentivirus- transduced cells determined to be 54.5% CXCR4 positive (versus 3.29% for non-transduced cells) (FIG. 17).
  • CXCR4 positive cells CAR-iNK CXCR4+
  • CXCR4 negative cells CAR-iNK CXCR4-
  • Transwell plates were coated with rat tail collagen at a concentration of 50 ⁇ g/mL for 2 hours. After coating, rat tail collagen solution was removed, and plates were allowed to dry for 30 minutes. In the lower chamber of the assay plate, 1 mL of assay media with or without 400 ng/mL C-X-C motif chemokine 12 (CXCL12) was added. The insert of the assay plate was loaded with 200 ⁇ L of cells at a concentration of 1x10 6 cells/mL in assay media. Assay plates were transferred to a tissue culture incubator at 37°C and incubated for 4 hours.
  • CXCL12 C-X-C motif chemokine 12
  • CAR-iNK CXCR4+ and CAR-iNK CXCR4- were tested for functionality in a cytotoxicity assay against NucLight Red-labeled antigen-positive and antigen-knock- out tumor cell lines as shown in FIGS. 19A-19B.
  • 2x10 5 target cells were co-cultured with either 2x10 5 or 4x10 5 effector cells for effector to target ratios (E:T) of 1:1 (FIG. 19A) and 2:1 (FIG. 19B), respectively.
  • Target cell growth was measured by Total Integrated Intensity readings from Incucyte Live-Cell Imaging system every 3 hours for 72 hours total.
  • Figure curves represent target cell growth curves when co-cultured with effector cells, normalized to target cell growth curves when target cells are cultured alone.
  • CAR-iNK cells with (CAR-iNK CXCR4+ ) and without (CAR-iNK CXCR4- ) CXCR4 engineering were subject to in vivo functional studies. As shown in FIG. 20, CAR-iNK cells were injected to tumor bearing female NSG mice, and the resulting cytotoxic effect was assessed over 9 days.
  • MOLM13-Luc cells were implanted intravenously (IV) at 1x10 5 cells/mouse 4 days prior to intravenous injection of CAR- iNK at 1x10 7 cells/mouse.
  • FIG. 21 shows the efficacy results of in vivo efficacy screening of CAR-iNK cells with (CAR-iNK CXCR4+ ) and without (CAR-iNK CXCR4- ) CXCR4 engineering using the disseminated MOLM13-Luc xenograft model in NSG mice.
  • MOLM13-Luc cells were implanted intravenously sf-6638784 145 32429-20001.40 (IV) at 1x10 5 cells/mouse 4 days prior to intravenous injection of CAR-iNK at 1x10 7 cells/mouse. Tumor burden was measured using bioluminescent imaging (BLI).
  • mice were humanely euthanized and sampled for blood, bone marrow, and tissue analysis by FACS and histology. Blood was collected into lithium heparin coated tubes, and bone marrow aspirate was collected and kept in serum-free RPMI until FACS analysis. Briefly, samples were centrifuged to pellet cells and medium discarded. Samples underwent RBC lysis and were resuspended for FACS antibody staining.
  • FIG. 22 shows bone marrow presence of CAR-iNK in the presence and absence of tumor, assessed by FACS. Both tumor-bearing and non-tumor-bearing mice that received CAR-iNK CXCR4- did not have iNK present in the bone marrow.
  • FIG. 23 Immunohistochemistry of normal NSG mouse bone marrow stained using CXCL12 antibody (SDF1/CXCL12 [D8G6H] Rabbit mAb, 1:100 dilution) is shown in FIG. 23.
  • Femur, spleen, liver, and lung were stained with CD3 antibody (NCL-L-CD3-565 [LN10] Mouse mAb) to detect iNK.
  • CAR-iNK CXCR4- and CAR-iNK CXCR4+ tissue infiltration of femur, spleen, liver, and lung is shown in FIG. 24.

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Abstract

Provided are genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof expressing a chimeric antigen receptor (CAR) and methods of using the same. Also provided are compositions, polypeptides, vectors, and methods of manufacturing.

Description

32429-20001.40 GENETICALLY ENGINEERED CELLS HAVING ANTI-CD123 CHIMERIC ANTIGEN RECEPTORS, AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS The present application claims priority from U.S. provisional application No. 63/570,197, filed March 26, 2024, entitled “GENETICALLY ENGINEERED CELLS HAVING ANTI-CD123 CHIMERIC ANTIGEN RECEPTORS, AND USES THEREOF,” the contents of which is incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY This application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “324292000140SEQLIST_skips.xml”, created March 25, 2025, which is 335,740 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. TECHNICAL FIELD This application provides genetically engineered induced pluripotent stem cells (iPSCs) and derivative cells thereof. Also provided are uses of the iPSCs or derivative cells thereof to express a chimeric antigen receptor for cell therapy. Also provided are related vectors, polynucleotides, and pharmaceutical compositions. 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. BACKGROUND Chimeric antigen receptors (CARs) have shown remarkable activity in the treatment of leukemia by enhancing anti-tumor activity of immune effector cell, including for CD123-associated malignancies. While targeting either antigen alone with chimeric antigen receptor (CAR) T cell therapy has shown some efficacy in treating acute myeloid leukemia (AML), challenges remain. For example, CD123- 1 sf-6638784 32429-20001.40 targeted CAR T cells can eliminate leukemic stem cells which mediate relapse, but on-target off-tumor toxicity has been a concern due to CD123 expression on normal hematopoietic progenitor cells. Existing treatments have additional limitations. For example, autologous treatments need to be generated on a custom-made basis, which remains a limiting factor for large-scale clinical application due to the production costs and the risk of production failure. The development of CAR-T technology and its wider application is also limited due to a number of other key shortcomings, including, e.g., a) an inefficient anti-tumor response, b) limited penetration and susceptibility of adoptively transferred CAR T cells to an immunosuppressive tumor microenvironment (TME), c) poor persistence of CAR-T cells in vivo, d) serious adverse events in the patients including cytokine release syndrome (CRS) and graft-versus-host disease (GVHD) mediated by the CAR-T, and e) the time required for manufacturing. CAR-T cells with dual-antigen targeting (e.g., using two CARs by co-administration or co- transduction into an immune cell) have shown potential for overcoming the antigen downregulation in myeloid and lymphocytic leukemias that is a common cause for treatment failure. The allogeneic, tumor-targeting cell therapies of the present disclosure have the potential to overcome these challenges and satisfy the unmet need for therapeutically sufficient and functional antigen-specific immune cells for effective immunotherapy. BRIEF SUMMARY In some aspects, the present disclosure provides an induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising at least one exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), said one or more CARs comprising a first antigen-binding domain targeting a Cluster of Differentiation 123 (CD123) antigen. In some embodiments, the iPSC or derivative cell thereof further comprises at least one of: (i) a deletion or reduced expression of one or more of Beta 2 Microglobulin (B2M), Transporter Associated with Antigen Processing (TAP) 1, TAP 2, Tapasin, Regulatory Factor X Associated Ankyrin Containing Protein (RFXANK), Class II Major Histocompatibility Complex Transactivator (CIITA), Regulatory Factor X5 (RFX5), and Regulatory Factor X Associated Protein (RFXAP) genes; (ii) an exogenous polynucleotide encoding a sf-6638784 2 32429-20001.40 human leukocyte antigen E (HLA-E) and/or a human leukocyte antigen G (HLA-G); (iii) an exogenous polynucleotide encoding a cluster of differentiation 16 (CD16; natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII)) protein and/or a Natural Killer Group 2 Member D (NKG2D) protein; (iv) a deletion or reduced expression of one or both of Natural Killer Group 2 Member A (NKG2A) and Cluster of Differentiation 70 (CD70) genes; (v) an exogeneous polynucleotide encoding a cytokine; (vi) an exogenous polynucleotide encoding a safety switch; (vii) an exogenous polynucleotide encoding C-X-C chemokine receptor type 4 (CXCR4) or a fragment or variant thereof; and (viii) an exogeneous polynucleotide encoding a Prostate-Specific Membrane Antigen (PSMA) cell tracer. In some embodiments, the one or more CARs consists of a dual-targeting CAR comprising the first antigen-binding domain and a second antigen-binding domain. In some embodiments, the one or more CARs comprises a plurality of CARs including a first CAR and a second CAR, wherein the first CAR comprises the first antigen- binding domain, and wherein the second CAR comprises a second antigen-binding domain. In some embodiments, the first antigen-binding domain comprises an anti- CD123 VHH single domain antibody. In some embodiments, the dual-targeting CAR comprises: (i) optionally, a signal peptide; (ii) an extracellular domain comprising the first antigen-binding domain and the second antigen-binding domain; (vi) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and/or (vi) a co-stimulatory domain. In some embodiments comprising the plurality of CARs, each of the first CAR and the second CAR comprises: (i) optionally, a signal peptide; (ii) an extracellular domain comprising the first antigen-binding domain or the second antigen-binding domain; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and/or (vi) a co-stimulatory domain. In some embodiments, the first antigen-binding domain comprises an anti-CD123 VHH single domain antibody. In some embodiments, the first antigen-binding domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 126-148. In some embodiments, the first antigen-binding domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 149-171. In some embodiments, in the first CAR in the plurality of CARs: the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ sf-6638784 3 32429-20001.40 ID NO: 1 or 103; the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 126-148; the hinge region comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21, 22, 61, 99, 100, or 102; the transmembrane domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23 or 24; and the intracellular signaling or co-stimulatory domains comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 6, 8-20, 243, and 244. In some embodiments, in the first CAR in the plurality of CARs: the signal peptide comprises amino acids having the sequence of SEQ ID NO: 1 or 103; the extracellular domain comprises amino acid having the sequence of one of SEQ ID NOs: 126-148; the hinge region comprises amino acids having the sequence of SEQ ID NO: 21, 22, 61, 99, 100, or 102; the transmembrane domain comprises amino acids having the sequence of SEQ ID NO: 23 or 24; and the intracellular signaling or co-stimulatory domains comprise amino acids having the sequence of one or more of SEQ ID NO: 6, 8-20, 243, and 244. In some embodiments, the cytokine comprises Interleukin-15 (IL-15). In some embodiments, iPSC or derivative cell thereof can further comprise an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope, wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide. In some embodiments, the IL-15 comprises (i) an IL-15 and an IL-15 receptor alpha (IL-15Rα) fusion polypeptide, or (ii) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 247. In some embodiments, the IL-15 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72. In some embodiments, the iPSC or derivative cell thereof comprises the deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. In some embodiments, the iPSC or the derivative cell thereof comprises the exogenous polynucleotide encoding the HLA-E and/or the HLA-G. In some embodiments, the CD16 protein is a CD16 variant protein. In some embodiments, the CD16 variant protein is a high affinity CD16 variant. In some embodiments, the CD16 variant protein is a non-cleavable CD16 variant. In some embodiments, the CD16 variant protein comprises wild-type sf-6638784 4 32429-20001.40 CD16 comprising amino acids having the sequence of SEQ ID NO: 186 and one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, and T220A. In some embodiments, the CD16 variant protein comprises amino acids having at least 90% sequence identity to SEQ ID NO: 187. In some embodiments, the iPSC or derivative cell thereof comprises the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by an autoprotease peptide. In some embodiments, the NKG2D protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a wildtype NKG2D protein. In some embodiments, the NKG2D protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 189. In some embodiments, the autoprotease peptide is selected from the group consisting of a porcine tesehovirus-1 2A (P2A) peptide, a foot-and-mouth disease virus 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide. In some embodiments, the autoprotease peptide is a P2A peptide comprising amino acids having at least 90% sequence identity to SEQ ID NO: 73 or 191. In some embodiments, the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein comprises a nucleic acid having at least 90% sequence identity to SEQ ID NO: 184. In some embodiments, the exogenous polynucleotide(s) are integrated into the chromosome of the cell at one or more loci selected from the group consisting of Adeno-associated Virus Integration Site 1 (AAVS1), B2M, Cbl Proto-oncogene B (CBL-B), C-C Motif Chemokine Receptor 5 (CCR5), CD33, Cluster of Differentiation 38 (CD38), CD70, CIITA, CIS, Cytokine Inducible SH2 Containing Protein (CISH), Citramalyl-CoA Lyase (CLYBL), Collagen, Cytotoxic T- Lymphocyte Associated Protein 4 (CTLA4), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), HTRP, Lymphocyte Activating 3 (LAG3), NKG2A, NKG2D, NLR Family CARD Domain Containing 5 (NLRC5), Programmed Cell Death 1 (PD1), RFX5, RFXANK, RFXAP, Reverse Orientation Splice Acceptor 26 (ROSA26), RUNX Family Transcription Factor 1 (RUNX1), Suppressor Of Cytokine Signaling 2 (SOCS2), TAP 1, TAP 2, Tapasin, TAP Binding Protein (TAPBP), TCR a or b constant region, T Cell Immunoreceptor With Ig And ITIM Domains (TIGIT), T-cell immunoglobulin and mucin-domain containing-3 (TIM3), sf-6638784 5 32429-20001.40 and T Cell Receptor Alpha Constant (TRAC) genes, provided at least one of the exogenous polynucleotides is integrated at a locus of a gene selected from the group consisting of AAVS1, B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby result in a deletion or reduced expression of the gene. In some embodiments, the exogenous polynucleotide(s) are integrated into the chromosome of the cell at one or more loci selected from the group consisting of AAVS1, B2M, CIITA, CD33, and CLYBL. In some embodiments, the iPSC or derivative cell thereof comprises a deletion or reduced expression of one or both of the B2M and the CIITA genes. In some embodiments, the iPSC or derivative cell thereof comprises a deletion or reduced expression of both of the B2M and the CIITA genes. In some embodiments, the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs). In some embodiments, the iPSC is derived from a re- programmed T-cell. In some embodiments, the iPSC or the derivative cell thereof comprises an exogenous polynucleotide encoding a safety switch. In some embodiments, the safety switch comprises an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope. In some embodiments, the monoclonal antibody specific epitope is selected from the group of epitopes specifically recognized by abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab, infliximab, ipilimumab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, polatuzumab vedotin, ranibizumab, rituximab, tiuxetan, tocilizumab, tositumomab, trastuzumab, tremelimumab, ustekinumab, and vedolizumab. In some embodiments, the inactivated cell surface receptor is a truncated epithelial growth factor (tEGFR) variant. In some embodiments, the tEGFR variant consists of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71. In some embodiments, the safety switch comprises (i) an intracellular domain comprising a herpes simplex virus thymidine kinase (HSV-TK) or (ii) an inducible Caspase 9 (iCasp9). In some embodiments, the iPSC or the derivative cell thereof comprises the exogeneous polynucleotide encoding the PSMA cell tracer, wherein the safety switch further comprises an extracellular domain comprising a PSMA cell tracer or fragment thereof. In some embodiments, the iPSC or the derivative cell thereof comprises the HSV-TK domain, wherein the HSV-TK is operatively linked to sf-6638784 6 32429-20001.40 the extracellular domain comprising the PSMA cell tracer or fragment thereof by a linker to form a combined artificial cell death/reporter system polypeptide. In some embodiments, the HSV-TK comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 248 or 249, or the iCasp9 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 245 or 246. In some embodiments, the combined artificial cell death/reporter system polypeptide comprises the HSV-TK fused to a truncated variant PSMA polypeptide via the linker. In some embodiments, the truncated variant PSMA polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 250. In some embodiments, the linker comprises an autoprotease peptide selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and a F2A peptide. In some embodiments, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 251. In some embodiments, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 252-254. In some embodiments, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 255-257. In some embodiments, the HLA-E comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 66, and/or the HLA-G comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 69. In some embodiments, (i) the at least one exogenous polynucleotide encoding one or more CARs comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 174 and 175; (ii) the exogenous polynucleotide encoding the HLA-E and/or the HLA-G comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 67 and 70; (iii) the exogenous polynucleotide encoding the CD16 protein and/or the NKG2D protein sf-6638784 7 32429-20001.40 comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 184, 188, and 190; (iv) the exogeneous polynucleotide encoding the cytokine comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 258; (v) the exogenous polynucleotide encoding the safety switch comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 255-257; and/or (vi) the exogeneous polynucleotide encoding the PSMA cell tracer comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 250. In some embodiments,(i) the at least one exogenous polynucleotide encoding one or more CARs comprises polynucleotides having at least one sequence selected from the group consisting of SEQ ID NOs: 174 and 175; (ii) the exogenous polynucleotide encoding the HLA-E and/or the HLA-G comprises polynucleotides having the sequence of one or more of SEQ ID NOs: 67 and 70; (iii) the exogenous polynucleotide encoding the CD16 protein and/or the NKG2D protein comprises polynucleotides having the sequence of one or more of SEQ ID NOs: 184, 188, and 190; (iv) the exogeneous polynucleotide encoding the cytokine comprises polynucleotides having the sequence of SEQ ID NO: 258; and/or (v) the exogenous polynucleotide encoding the safety switch comprises polynucleotides having the sequence of one of SEQ ID NOs: 255-257. In some embodiments, each of the exogenous polynucleotides are independently integrated into a gene locus selected from the group consisting of AAVS1, B2M, CBL-B, CCR5, CD33, CD38, CD70, CIITA, CIS, CISH, CLYBL, Collagen, CTLA4 , GAPDH, HTRP, LAG3, NKG2A, NKG2D, NLRC5, PD1, RFX5, RFXANK, RFXAP, ROSA26, RUNX1, SOCS2, TAP 1, TAP 2, Tapasin, TAPBP, TCR a or b constant region, TIGIT, TIM3, and TRAC. In some embodiments, (i) the at least one exogenous polynucleotide encoding the one or more CARs is integrated at a locus of the AAVS1 gene, the CD33 gene, or the CLYBL gene; (ii) the exogenous polynucleotide encoding the HLA-E and/or the HLA-G is integrated at a locus of the B2M gene; (iii) the exogenous polynucleotide encoding the CD16 protein and/or the NKG2D protein is integrated at a locus of the CD70 gene; (iv) the exogeneous polynucleotide encoding the cytokine is integrated at the locus of the NKG2A gene;(v) the iPSC or the derivative cell thereof comprises a deletion or reduced expression of the CIITA gene; and, optionally (vi) the exogenous sf-6638784 8 32429-20001.40 polynucleotide encoding the safety switch or the PSMA cell tracer is integrated at the locus of the CIITA gene. In some embodiments comprising the dual-targeting CAR, the dual-targeting CAR comprises a CD123 loop. In some embodiments, comprising the dual-targeting CAR, the dual-targeting CAR comprises one or more polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 126-148. In some embodiments, comprising the dual-targeting CAR, the dual- targeting CAR comprises one or more polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 149-171. In some embodiments, the iPSC or the derivative cell thereof comprises the exogenous polynucleotide encoding CXCR4 or the fragment or variant thereof. In some embodiments, the CXCR4 or the fragment or variant thereof is selected from the group consisting of a wild-type CXCR4 and a mutated variant of CXCR4. In some embodiments comprising the wild-type CXCR4, the wild-type CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:192. In some embodiments comprising the wild-type CXCR4, the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:200. In some embodiments comprising the mutated variant of CXCR4, the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with a deletion of the C-terminal domain between 10 and 20 amino acid residues. In some embodiments, comprising the mutated variant of CXCR4, the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A. In some embodiments, comprising the mutated variant of CXCR4, the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 193-199. In some embodiments, the derivative cell is a NK cell or a T cell. In some embodiments, the derivative cell is a NK cell. In some embodiments, the derivative cell is a T cell. In some embodiments, the T cell is a gamma delta T cell. In some embodiments, the T cell is a gamma delta Vγ9/Vδ1 T cell. sf-6638784 9 32429-20001.40 In some aspects, the present disclosure provides a composition comprising the iPSC or the derivative cell thereof. In some embodiments, the composition further comprises or is used in combination with one or more therapeutic agents selected from the group consisting of a small-molecule therapeutic agent, a biologic, a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), a siRNA, an oligonucleotide, mononuclear blood cells, an antibody, a chemotherapeutic agent, a radioactive moiety, and an immunomodulatory drug (IMiD). In some aspects, the present disclosure provides an induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising one or more exogenous polynucleotides encoding: (i) a dual-targeting chimeric antigen receptor (CAR) targeting a CD123 antigen integrated into a CD33 gene locus; (ii) a human leukocyte antigen E (HLA-E) and a human leukocyte antigen G (HLA-G) integrated into a B2M gene locus; (iii) a cluster of differentiation 16 (CD16; natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII)) protein and/or an NKG2D protein integrated into a CD70 gene locus; (iv) an IL-15 and an IL-15 receptor alpha (IL-15Rα) fusion polypeptide integrated into a NKG2A gene locus;(v) a C-X-C chemokine receptor type 4 (CXCR4) or a fragment or variant thereof integrated into a CLYBL gene locus; and a deletion or reduced expression of CD33, B2M, CD70, NKG2A, CLYBL, and CIITA genes. In some embodiments, the dual-targeting CAR comprises a CD123 loop. In some embodiments, the at least one exogenous polynucleotide encoding the dual-targeting CAR comprises (i) nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 149-171; or (ii) nucleotides encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 126-148. In some embodiments, the exogenous polynucleotide encoding the HLA-E and the HLA-G is integrated into exon 2 of the B2M gene locus. In some embodiments, the exogeneous polynucleotide encoding the IL-15 and the IL-15 receptor alpha (IL- 15Rα) fusion polypeptide is integrated into exon 3 of the NKG2A gene locus. In some embodiments, the CXCR4 or the fragment or variant thereof is selected from the group consisting of a wild-type CXCR4 and a mutated variant of CXCR4. In some embodiments, the wild-type CXCR4 comprises amino acids having at least 90%, sf-6638784 10 32429-20001.40 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 192. In some embodiments, the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 200. In some embodiments comprising the mutated variant of CXCR4, the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with a deletion of the C-terminal domain between 10 and 20 amino acid residues. In some embodiments comprising the mutated variant of CXCR4, the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A. In some embodiments comprising the mutated variant of CXCR4, the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 193-199. In some aspects, the present disclosure provides a dual-targeting chimeric antigen receptor (CAR) polypeptide comprising a first antigen-binding domain targeting a CD123 antigen. In some embodiments, the first antigen-binding domain comprises an anti-CD123 VHH single domain antibody. In some embodiments, the dual-targeting CAR comprises: a signal peptide; an extracellular domain comprising the first antigen-binding domain and the second antigen-binding domain; a hinge region; a transmembrane domain; an intracellular signaling domain; and/or a co- stimulatory domain. In some embodiments, the first antigen-binding domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 126-148. In some embodiments, the second antigen-binding domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 149-171. In some aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering the derivative cell thereof or the composition to a subject in need thereof. In some embodiments, the cancer is selected from the group consisting of leukemias, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), and chronic lymphocytic leukemia (CLL), lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma, multiple sf-6638784 11 32429-20001.40 myeloma, and follicular lymphoma. In some embodiments, the cancer is a B-cell malignancy, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, or follicular lymphoma. In some embodiments, the cancer is a myeloid cell malignancy. In some embodiments, the myeloid cell malignancy is acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). In some aspects, the present disclosure provides a method of manufacturing a derivative cell comprising differentiating the iPSC under conditions for cell differentiation to thereby obtain the derivative cell. In some embodiments, the iPSC is obtained by genomic engineering an unmodified iPSC, wherein the genomic engineering comprises targeted editing. In some embodiments, the targeted editing comprises deletion, insertion, or in/del carried out by CRISPR, ZFN, TALEN, homing nuclease, homology recombination, or any other functional variation of these methods. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings. FIG. 1 shows results of in vivo screening of anti-CD33 CAR-T using the SubQ MOLM-13 Fluc model in female NSG mice. MOLM13_LUC cells were implanted subcutaneously (SQ) at 1x106 cells / mouse 3 days prior to CAR-T intravenous (IV) injection at 5x106 cells/mouse. Tumor growth inhibition (TGI) was observed with CD33 CAR-T constructs, and 4 CAR-T samples (P3228 (ADI- 57195_HL); P3229 (ADI-53986_LH); P3232 (ADI-53983_LH); and P3235 (ADI- 59362_LH)) showed approximately 99-100% TGI. Untransduced T cells (same donor) were used to supplement CAR-T cell suspensions so that total number of T cells is equal for all groups. FIG. 2 shows results of anti-CD33 VHH-Fc fusion proteins that were examined by flow cytometry for CD33 cellular binding in CHO-CD33 and CHO- parental cell lines. Results are presented as a ratio of absolute Geomean signal of VHH-Fc binding to CHO-CD33 over CHO-parental. A total of 30 CD33 VHH-Fc sf-6638784 12 32429-20001.40 were tested for cell binding. 28/30 VHH-Fc demonstrated positive, specific binding to CHO-CD33 at 50nM and 5nM. No binding to CHO-Parental cells was observed. FIGs. 3A-3B show results of anti-CD123 VHH-Fc fusion proteins that were examined by flow cytometry for binding to HL-60, Kasumi-3, MOLM-13 and Jurkat cell lines. A total of 19 anti-CD123 VHH-Fc were tested for cell binding at (FIG. 3A) 50 nM and (FIG. 3B) 5 nM concentrations. 12 anti-CD123 VHH-Fc samples demonstrated positive / specific binding to CHO-CD123 at 50nM (10 strong binders and 2 weak binders (PROT939, PROT937)). 9 VHH-Fc samples also showed positive binding to Kasumi-3 cells at 50nM. FIG. 4 shows results of anti-CD123 VHH-Fc fusion proteins that were examined by flow cytometry for binding to CHO-CD123 and CHO-parental cell lines. Results are presented as a ratio of absolute Geomean signal of VHH-Fc binding to CHO-CD123 cells over CHO-parental cells. 10 anti-CD123 VHH-Fc samples showed a ratio of absolute Geomean signal over 1.5. FIGs. 5A-5B show results of anti-CD123 VHH-Fc fusion proteins that were examined by flow cytometry for binding to Jeko-1, Kasumi-3, MOLM-13, NALM6, Jurkat cell lines. A total of 9 anti-CD123 VHH-Fc were tested for cell binding at (FIG. 5A) 50 nM and (FIG. 5B) 5 nM concentrations. 4 anti-CD123 VHH-Fc also showed positive binding to Kasumi-3 at 50nM, and 2 showed positive binding to MOLM-13 at 50nM. FIGs. 6A-6F show CD123 antigen expression profiles assessed by flow cytometry in (FIG. 6A) Kasumi-3, (FIG. 6B) MOLM-13, (FIG. 6C) NALM6, (FIG. 6D) KG-1, (FIG. 6E) HL-60, (FIG. 6F) K-562 cell lines. FIGs. 7A-7F show cytotoxicity of anti-CD123 VHH-CAR expressing cells towards tumor and control cell lines, including (FIG. 7A) Kasumi-3, (FIG. 7B) MOLM-13, (FIG. 7C) NALM6, (FIG. 7D) KG-1, (FIG. 7E) HL-60, and (FIG. 7F) K-562 cells. An anti-CD123 VHH-CAR or scFv-CAR control was transduced into primary T-cells using lentivirus. Cells were grown for 14 days, then co-cultured with CD123+ and CD123- cells at effector to target (E:T) ratios of 2:1, 1:1, and 0.5:1 for 48 hours. Target cell killing was assessed by flow cytometry. FIG. 8 shows results of in vivo screening of anti-CD123 CAR-T cells using the SubQ MOLM-13 Fluc model in female NSG mice. MOLM13_LUC cells were implanted subcutaneously (SQ) at 1x106 cells / mouse 3 days prior to CAR-T intravenous (IV) injection at 5x106 cells / mouse. Tumor growth inhibition (TGI) was sf-6638784 13 32429-20001.40 observed with CD123 CAR-T constructs: 3 VHH-CAR showed approximately 99% TGI (P1407, P1412, P1414), and 4 CAR-T samples showed approximately 80-90% TGI (P1710, P1713). Untransduced T cells (same donor) were used to supplement CAR-T cell suspensions so that the total number of T cells was equal for all groups. FIGs. 9A-9D show schematics of (FIG. 9A) a mono-specific anti-CD33 VH/VL, (FIG. 9B) a mono-specific anti-CD123 VHH-CAR, and (FIG. 9C- FIG. 9D) dual-targeting CARs which are used in various embodiments of the present disclosure. CAR ectodomains were engineered with select CD33 and/or CD123 binders of the present disclosure. Selected CARs were expressed and tested for cytotoxicity activity in T-cells and in in vivo studies. FIGs. 10A-10E show schematics of hinge, transmembrane and endodomain combinations which are used in various CAR embodiments of the present disclosure, including the (FIG. 10A) CD28 CAR format, (FIG. 10B) DAP10 CAR format, (FIG. 10C) 41BB CAR format, (FIG. 10D) CD8/CD28 CAR format, and (FIG. 10E) 41BB_L CAR format. FIG. 11 shows a diagram of CAR-iPSC engineering. Parental iPSC line can be engineered to express 1) an anti-CD123 CAR; 2) an anti-CD33 CAR; 3) an anti- CD123 CAR and an anti-CD33 CAR (i.e., dual CARs); or 4) a dual-targeting targeting CD33 and CD123. FIGs. 12A-12D show CAR expression data in cells of the present disclosure that are engineered to express an anti-CD123 and anti-CD33 dual-targeting CAR, as assessed by FACS. iPSCs were genetically modified to express a transgene for the CD33/CD123 bispecific CAR. iPSCs were then differentiated into NK cells (iNK cells) and CAR expression was evaluated by staining with either anti-VHH antibody that binds to the CD33/CD123 recognition domain of the CAR (top row) or anti-huFc antibody that binds to the hinge region of the CAR (bottom row). Staining of cells was evaluated using flow cytometry. These results demonstrate that the CD33/CD123 bispecific CAR-iNK cells express a CAR molecule that has the potential to bind to either CD33 antigen or CD123 antigen on target cells. (FIG. 12A and FIG. 12B) show data for cells of the present disclosure (IPSC3081, IPSC3082, and IPSC3083) derived from the iPSC1631 parental line, and (FIG. 12C-FIG. 12D) show data for cells of the present disclosure (IPSC3084, IPSC3085, and IPSC3086) derived from the iPSC1650 parental line. sf-6638784 14 32429-20001.40 FIG. 13 shows results of in vivo screening of dual-targeting -iNK cells using the SubQ MOLM-13 Fluc model in female NSG mice. MOLM13_LUC cells were implanted subcutaneously (SQ) at 1x106 cells / mouse 3 days prior to CAR-iNK intravenous (IV) injection at 5x106 cells / mouse. Tumor growth inhibition (TGI) was observed with bispecific CAR-iNK cells. FIG. 14 shows surface CXCR4 expression data in cells of the present disclosure that are engineered to express wild-type CXCR4, as assessed by FACS. CXCR4-positive (+) iNK (iPSC1613) underwent engineering for expression of the human wild type CXCR4 protein via insertion into the CLYBL locus of the iPSC cell line through homology-directed repair. Following differentiation to iNK, this resulted in this particular bulk population of CXCR4+iNK with heterogenous expression of CXCR4 at 42.2%. Endogenous expression of CXCR4 in the CXCR4-negative (-) iNK cells (iPSC611) was 2.4%. FIG. 15 shows surface CXCR4 expression data in cells of the present disclosure that are engineered to express wild-type CXCR4, as assessed by FACS. iPSC611 cells were genetically modified to express a transgene for wild-type CXCR4 (iPSC1613). Subsequent differentiation yielded a bulk population of iPSC1613 with heterogenous expression of CXCR4 at 47.7%. The endogenous expression of CXCR4 of iPSC611 was 4.74%. FIG. 16 shows cytotoxicity of CD19 CAR iNK cells iPSC611 (CXCR4- negative, circle) and iPSC1613 (CXCR4-positive, square), and CAR-negative CXCR4-negative effector cells (triangle) towards NucLight Red expressing NALM6 tumor line. Effector cells were co-cultured with target tumor cells at a 1:1 effector-to- target cell ratio. Target cell growth was measured by Total Integrated Intensity readings from Incucyte Live-Cell Imaging system every 3 hours for 72 hours total. Figure curves represent target cell growth curves when co-cultured with effector cells, normalized to target cell growth curves when target cells are cultured alone. iPSC611 and iPSC1613 cells were equally potent in killing CD19-expressing target cells, indicating the CXCR4 transgene does not impact cytotoxicity in this assay. FIG. 17 shows the CXCR4 expression in CAR iNKs differentiated to express a dual-targeting anti-CD33/anti-CD123 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984), following lentiviral transduction of CXCR4 at the hematopoietic progenitor stage of differentiation, and subsequent differentiation to iNK. iPSC cells expressing a dual-targeting anti-CD33/anti-CD123 CAR (iPSC2984) sf-6638784 15 32429-20001.40 were transduced with a lentivirus-containing sequence encoding for WT CXCR4 upon integration into the cell line genome. Hematopoietic progenitor cells (HPCs) derived from iPSC2984 were transduced with the WT CXCR4 lentivirus and then differentiated 14 days to iNK cells. The lentivirus-transduced cells (CAR-iNKCXCR4+) were determined to be 54.5% CXCR4-positive versus 3.29% for non-transduced cells (CAR-iNKCXCR4-). FIGs. 18A-18B show migration of CXCR4-positive cells (iNKCXCR4+ and Jurkat) and CXCR4-negative cells (iNKCXCR4- and MOLM13) expressing a dual- targeting anti-CD33/anti-CD123 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984) in a transwell assay format. Transwell plates were coated with rat tail collagen, and test cells added to the upper chamber of the plates. Assay medium containing C-X-C motif chemokine 12 (CXCL12) was added to the lower chamber of the plates. Plates were incubated for 4 hours before being assessed by FACS for cell quantification. Chemotactic Index (ChI) was calculated as the number of cells in experimental wells (400 ng CXCL12) divided by the average of cells in the baseline wells (0 ng CXCL12). Shown are (FIG. 18A) raw cell counts at migration assay completion, and (FIG. 18B) calculated Chemotactic Index (ChI), which indicate pronounced migration of CXCR4-positive cells in the presence of CXCL12. FIGs. 19A-19B show cytotoxicity of CAR iNK cells that are CXCR4- negative (open shape) and CXCR4-positive (filled shape), and also express a dual- targeting anti-CD33/anti-CD123 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984), towards NucLight Red expressing antigen-positive (diamond) and antigen-negative (triangle) tumor lines. Effector cells were co-cultured with target tumor cells at (FIG. 19A) 1:1 and (FIG. 19B) 2:1 effector-to-target cell ratios. Target cell growth was measured by Total Integrated Intensity readings from Incucyte Live-Cell Imaging system every 3 hours for 72 hours total. Figure curves represent target cell growth curves when co-cultured with effector cells, normalized to target cell growth curves when target cells are cultured alone. CXCR4-positive and CXCR4-negative CAR iNK cells were equally potent in killing antigen-expressing target cells, indicating the CXCR4 transgene does not impact cytotoxicity in this assay. No cytotoxicity against antigen-negative target cells was observed. FIG. 20 depicts the study design for in vivo testing of CXCR4-positive and CXCR4-negative CAR-iNK cells that express a dual-targeting anti-CD33/anti-CD123 sf-6638784 16 32429-20001.40 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984). Luciferase-labeled tumor cells expressing CAR-specific antigen were intravenously (IV) injected into NOD SCID Gamma (NSG) mice on Day -4 (D-4). On Day 0 (D0), mice were intravenously injected with CXCR4-positive or -negative CAR-iNK. Mice underwent bioluminescent imaging to monitor tumor burden on Day -1 and twice weekly thereafter. On Day 9 (D9), tumor growth inhibition (TGI) was calculated, and mice were humanely euthanized for blood FACS analysis and histology sampling. FIG. 21 shows in vivo tumor burden by bioluminescent imaging (BLI). Tumor-bearing mice were either untreated controls (open circle), or intravenously injected with CXCR4-positive (filled diamond) and CXCR4-negative (open diamond) CAR-iNK cells engineered to express a dual-targeting anti-CD33/anti-CD123 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984) on Day 0, and tumor burden monitored twice weekly. On the final study day, percent tumor growth inhibition was calculated for each treatment as (1-[treated BLI/control BLI]) x 100. CXCR4-positive CAR-iNK elicited significant efficacy compared to CXCR4 negative CAR-iNK (p=0.0363) and untreated controls (p=0.0061). FIG. 22 shows bone marrow presence of CAR-iNK cells engineered to express a dual-targeting anti-CD33/anti-CD123 CAR of the present disclosure (iNKs differentiated from iPSC2984) in the presence and absence of tumor, assessed by FACS. CXCR4-positive and CXCR4-negative CAR-iNK cells were intravenously injected into tumor-bearing and non-tumor bearing mice. Nine days later, mice were sampled for bone marrow aspirates. Both tumor-bearing and non-tumor-bearing mice that received CXCR4-negative CAR-iNK did not have iNK present in the bone marrow. Significant numbers of iNK were present in the bone marrow of both tumor- bearing (p<0.0001) and non-tumor-bearing mice (p=0.0018) that received CXCR4- positive CAR-iNK. FIG. 23 shows positive immunohistochemical (IHC) staining for C-X-C motif chemokine 12 (CXCL12) in mouse bone marrow. FIG. 24 shows immunohistochemical (IHC) staining for iNK cells in mouse tissues from mice dosed with CXCR4-positive and CXCR4-negative CAR-iNK cells engineered to express a dual-targeting anti-CD33/anti-CD123 CAR of the present disclosure (p4573; iNKs differentiated from iPSC2984). Mice that received CXCR4- positive CAR-iNK had greater tissue iNK infiltration (bone, spleen, liver, and lung), indicating enhanced migration as a result of CXCR4 engineering of iNK. sf-6638784 17 32429-20001.40 DETAILED DESCRIPTION Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this application pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ± 10% of the recited value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise. Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the application. As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non- sf-6638784 18 32429-20001.40 exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). As used herein, the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and/or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.” As used herein, the term “consists of,” or variations such as “consist of” or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition. As used herein, the term “consists essentially of,” or variations such as “consist essentially of” or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure or composition. See M.P.E.P. § 2111.03. As used herein, “subject” means any animal, preferably a mammal, most preferably a human. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably a human. sf-6638784 19 32429-20001.40 It should also be understood that the terms “about,” “approximately,” “generally,” “substantially,” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension/characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit. The terms “C-X-C CHEMOKINE RECEPTOR TYPE 4” and “CXCR4” refer to a G protein-coupled receptor that binds the chemokine CXCL12. It plays important roles in embryogenesis, hematopoiesis, organogenesis, vascularization, and tumorigenesis. The wild type CXCR4 receptor contains 352 amino acids and has seven transmembrane domains, with an extracellular N-terminus and intracellular C- terminus. Upon binding CXCL12, CXCR4 activates downstream signaling pathways such as Ras/MAPK and PI3K/Akt that regulate cell migration, proliferation, and survival. The terms “engineered cell” and “genetically modified cell” as used herein can be used interchangeably. The terms mean containing and/or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny. Especially, the terms refer to cells, preferentially T cells which are manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are not expressed in these cells in the natural state. For example, T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface. For example, the sequences encoding the CAR may be delivered into cells using a retroviral or lentiviral vector. The term “primary cell” refers to a cell isolated directly from a multicellular organism. Primary cells typically have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are cells that have been isolated and then used immediately. In other cases, primary cells cannot divide indefinitely and thus cannot be cultured for long periods of time in vitro. In certain embodiments sf-6638784 20 32429-20001.40 of the present disclosure, a primary cell can comprise a primary immune cell (e.g., a primary T-cell) The term “target” as used herein refers to an antigen or epitope associated with a cell that should be recognized specifically by an antigen-binding domain, e.g., an antigen-binding domain of an antibody or of a CAR. The antigen or epitope for antibody recognition can be bound to the cell surface but also be secreted, part of the extracellular membrane, or shed from the cell. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences (e.g., CAR polypeptides and the CAR polynucleotides that encode them), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. sf-6638784 21 32429-20001.40 (1997) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N= -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. sf-6638784 22 32429-20001.40 A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. As used herein, the term “isolated” means a biological component (such as a nucleic acid, peptide, protein, or cell) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, e.g., other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and tissues. Nucleic acids, peptides, proteins, and cells that have been “isolated” thus include nucleic acids, peptides, proteins, and cells purified by standard purification methods and purification methods described herein. “Isolated” nucleic acids, peptides, proteins, and cells can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, protein, or cell. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. As used herein, the term “polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as sf-6638784 23 32429-20001.40 typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides. A “construct” refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo. A “vector,” as used herein refers to any nucleic acid construct capable of directing the delivery or transfer of a foreign genetic material to target cells, where it can be replicated and/or expressed. The term “vector” as used herein comprises the construct to be delivered. A vector can be a linear or a circular molecule. A vector can be integrating or non- integrating. The major types of vectors include, but are not limited to, plasmids, episomal vector, viral vectors, cosmids, and artificial chromosomes. Viral vectors include, but are not limited to, adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, Sendai virus vector, and the like. By “integration” it is meant that one or more nucleotides of a construct is stably inserted into the cellular genome, e.g., covalently linked to the nucleic acid sequence within the cell's chromosomal DNA. By “targeted integration” it is meant that the nucleotide(s) of a construct is inserted into the cell's chromosomal or mitochondrial DNA at a pre-selected site or “integration site”. The term “integration” as used herein further refers to a process involving insertion of one or more exogenous sequences or nucleotides of the construct, with or without deletion of an endogenous sequence or nucleotide at the integration site. In the case, where there is a deletion at the insertion site, “integration” can further comprise replacement of the endogenous sequence or a nucleotide that is deleted with the one or more inserted nucleotides. As used herein, the term “exogenous” is intended to mean that the referenced molecule or the referenced activity is introduced into, or non-native to, the host cell. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non- chromosomal genetic material such as a plasmid. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. The term “endogenous” refers to a referenced molecule or activity that is present in the host cell in its native form. Similarly, the term when used in reference to expression of an encoding nucleic sf-6638784 24 32429-20001.40 acid refers to expression of an encoding nucleic acid natively contained within the cell and not exogenously introduced. As used herein, a “gene of interest” or “a polynucleotide sequence of interest” is a DNA sequence that is transcribed into RNA and in some instances translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. A gene or polynucleotide of interest can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. For example, a gene of interest may encode an miRNA, an shRNA, a native polypeptide (e.g., a polypeptide found in nature) or fragment thereof; a variant polypeptide (e.g., a mutant of the native polypeptide having less than 100% sequence identity with the native polypeptide) or fragment thereof; an engineered polypeptide or peptide fragment, a therapeutic peptide or polypeptide, an imaging marker, a selectable marker, and the like. “Operably-linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably-linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (e.g., the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The term “expression” as used herein, refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed CAR can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane. As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can sf-6638784 25 32429-20001.40 be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. The peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C- terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated. As used herein, the term “engineered immune cell” refers to an immune cell, also referred to as an immune effector cell, that has been genetically modified by the addition of exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell. A. Induced Pluripotent Stem Cells (IPSCs) And Immune Effector Cells IPSCs have unlimited self-renewing capacity. Use of iPSCs enables cellular engineering to produce a controlled cell bank of modified cells that can be expanded and differentiated into desired immune effector cells, supplying large amounts of homogeneous allogeneic therapeutic products. Provided herein are genetically engineered iPSCs and derivative cells thereof. The selected genomic modifications provided herein enhance the therapeutic properties of the derivative cells. The derivative cells are functionally improved and suitable for allogenic off-the-shelf cell therapies following a combination of selective modalities being introduced to the cells at the level of iPSC through genomic engineering. This approach can help to reduce the side effects mediated by CRS/GVHD and prevent long-term autoimmunity while providing excellent efficacy. As used herein, the term "differentiation" is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell. Specialized cells include, for example, a blood cell or a muscle 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", sf-6638784 26 32429-20001.40 when applied to the process of differentiation, 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. As used herein, the term "pluripotent" refers to the ability of a cell to form all lineages of the body or soma or 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 is a continuum of developmental potencies ranging from the incompletely or partially pluripotent cell (e.g., an epiblast stem cell or EpiSC), 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). As used herein, the terms "reprogramming" or "dedifferentiation" 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 (e.g., 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. As used herein, the term "induced pluripotent stem cells" or, iPSCs, means that the stem cells are produced from differentiated adult, neonatal or fetal cells that have been induced or changed or 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. The term “hematopoietic stem and progenitor cells,” “hematopoietic stem cells,” “hematopoietic progenitor cells,” or “hematopoietic precursor cells” or “HPCs” refers to cells which are committed to a hematopoietic lineage but are capable of further hematopoietic differentiation. Hematopoietic stem cells include, for example, 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 sf-6638784 27 32429-20001.40 cells). As used herein, “CD34+ hematopoietic progenitor cell” refers to an HPC that expresses CD34 on its surface. As used herein, the term “immune cell” or “immune effector cell” refers to a cell that is involved in an immune response. Immune response includes, for example, the promotion of an immune effector response. Examples of immune cells include T cells, B cells, natural killer (NK) cells, mast cells, and myeloid-derived phagocytes. As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to a type of white blood cell that completes maturation in the thymus and that has various roles in the immune system. A T cell can have the roles including, e.g., the identification of specific foreign antigens in the body and the activation and deactivation of other immune cells. A T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. The T cell can be CD3+ cells. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+/CD8+ double positive T cells, CD4+ helper T cells (e.g., Thl and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes (PBLs), tumor infiltrating lymphocytes (TILs), memory T cells, naive T cells, regulator T cells, gamma delta T cells (gd T cells), and the like. Additional types of helper T cells include cells such as Th3 (Treg), Thl7, Th9, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tern cells and TEMRA cells). The T cell can also refer to a genetically engineered T cell, such as a T cell modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). The T cell can also be differentiated from a stem cell or progenitor cell. “CD4+ T cells” refers to a subset of T cells that express CD4 on their surface and are associated with cell-mediated immune response. They are characterized by the secretion profiles following stimulation, which may include secretion of cytokines such as IFN-gamma, TNF-alpha, IL2, IL4 and IL10. “CD4” are 55-kD glycoproteins originally defined as differentiation antigens on T-lymphocytes, but also found on other cells including monocytes/macrophages. CD4 antigens are members of the immunoglobulin supergene family and are implicated as associative recognition elements in MHC (major histocompatibility complex) class II-restricted immune responses. On T-lymphocytes they define the helper/inducer subset. sf-6638784 28 32429-20001.40 “CD8+ T cells” refers to a subset of T cells which express CD8 on their surface, are MHC class I-restricted, and function as cytotoxic T cells. “CD8” molecules are differentiation antigens found on thymocytes and on cytotoxic and suppressor T- lymphocytes. CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions. As used herein, the term “NK cell” or “natural killer cell” refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 and CD45 and the absence of the T cell receptor (TCR chains). The NK cell can also refer to a genetically engineered NK cell, such as a NK cell modified to express a chimeric antigen receptor (CAR). The NK cell can also be differentiated from a stem cell or progenitor cell. As used herein, the term “genetic imprint” refers to genetic or epigenetic information that contributes to preferential therapeutic attributes in a source cell or an iPSC, and is retainable in the source cell derived iPSCs, and/or the iPSC-derived hematopoietic lineage cells. As used herein, “a source cell” is a non-pluripotent cell that may be used for generating iPSCs through reprogramming, and the source cell derived iPSCs may be further differentiated to specific cell types including any hematopoietic lineage cells. The source cell derived iPSCs, and differentiated cells therefrom are sometimes collectively called “derived” or “derivative” cells depending on the context. For example, derivative effector cells, or derivative NK or “iNK” cells or derivative T or “iT” cells, as used throughout this application are cells differentiated from an iPSC, as compared to their primary counterpart obtained from natural/native sources such as peripheral blood, umbilical cord blood, or other donor tissues. As used herein, the genetic imprint(s) conferring a preferential therapeutic attribute is incorporated into the iPSCs either through reprogramming a selected source cell that is donor-, disease-, or treatment response- specific, or through introducing genetically modified modalities to iPSC using genomic editing. The induced pluripotent stem cell (iPSC) parental cell lines may be generated from peripheral blood mononuclear cells (PBMCs) or T-cells using any known method for introducing re-programming factors into non-pluripotent cells such as the episomal plasmid-based process as previously described in U.S. Pat. Nos. 8,546,140; 9,644,184; 9,328,332; and 8,765,470, the complete disclosures of which are incorporated herein by reference. The reprogramming factors may be in a form of sf-6638784 29 32429-20001.40 polynucleotides, and thus are introduced to the non-pluripotent cells by vectors such as a retrovirus, a Sendai virus, an adenovirus, an episome, and a mini-circle. In particular embodiments, the one or more polynucleotides encoding at least one reprogramming factor are introduced by a lentiviral vector. In some embodiments, the one or more polynucleotides introduced by an episomal vector. In various other embodiments, the one or more polynucleotides are introduced by a Sendai viral vector. In some embodiments, the iPSC’s are clonal iPSC’s or are obtained from a pool of iPSCs and the genome edits are introduced by making one or more targeted integration and/or in/del at one or more selected sites. In another embodiment, the iPSC’s are obtained from human T cells having antigen specificity and a reconstituted TCR gene (hereinafter, also refer to as "T-iPS” cells) as described in US Pat. Nos. 9,206,394 and 10,787,642 hereby incorporated by reference into the present application. According to a particular aspect, the application relates to an induced pluripotent stem cell (iPSC) cell or a derivative cell thereof comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a deletion or reduced expression of B2M and CIITA genes; and optionally (iii) an exogenous polynucleotide encoding a chimeric IL-15RA and an interleukin-15 (IL-15), wherein the IL-15RA and IL-15 are operably linked. I. Chimeric Antigen Receptor (CAR) Expression According to embodiments of the application, an iPSC cell or a derivative cell thereof comprises one or more first exogenous polynucleotides encoding a first and a second chimeric antigen receptor (CAR), such as a CAR targeting one or more tumor antigens. In one embodiment, the CAR targets a CD123 antigen. In another embodiment, the CAR targets a CD123 antigen, and the targeting regions (e.g., the extracellular domains) of the CAR comprises an antibody fragment (e.g., a VHH domain). In other embodiments, the CAR can be a dual-targeting CAR targeting two or more antigens. In one example, a therapeutic cell of the present disclosure can express a dual-targeting CAR targeting at least CD123. The targeting domains of the CAR (e.g., the antigen binding regions) can comprise VHH and/or scFv domains. In some embodiments, the CAR can be a dual-targeting CAR in a loop configuration. In some embodiments, the extracellular binding domain comprises a loop format. sf-6638784 30 32429-20001.40 In some embodiments of the application, an iPSC cell or a derivative cell thereof comprises at least one exogenous polynucleotide encoding one of more CARs. In some embodiments, the one or more CARs comprise an antigen-binding domain targeting a CD123 antigen. In some embodiments, the one or more CARs is a CD123 monospecific CAR. In some embodiments, the CAR targets a CD123 antigen and the targeting region (e.g., the extracellular domain) of the CAR comprises an antibody fragment (e.g., a VHH domain or scFv domain). As used herein, the term “chimeric antigen receptor” (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that binds specifically to an antigen or a target, a transmembrane domain and an intracellular signaling domain. Engagement of the extracellular domain of the CAR with the target antigen on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell. CARs redirect the specificity of immune effector cells and trigger proliferation, cytokine production, phagocytosis and/or production of molecules that can mediate cell death of the target antigen- expressing cell in a major histocompatibility (MHC)-independent manner. The targeting regions may comprise full length heavy chain, Fab fragments, scFvs, divalent single chain antibodies or diabodies, each of which are specific to the target antigen (e.g., CD123). The antigen-binding domain can be derived from the same species or a different species than the species in which the CAR will be used in. As used herein, the term “dual-targeting” refers to a protein (e.g., a chimeric protein) capable of binding to two different antigens. Specifically, a dual-targeting protein of the present disclosure (e.g., a CAR having two or more tumor or cancer antigen-binding domains) does not naturally occur and is produced by a genetic engineering method or other method. In one embodiment, an engineered iPSC or derivative cell of the present disclosure can comprise one or more exogenous polynucleotides encoding a CAR having a first antigen-binding domain that binds to an antigen of interest and a second antigen-binding domain that specifically binds CD123. This is in contrast with other examples of the present disclosure wherein an engineered iPSC or derivative cell comprises one or more polynucleotides encoding a first CAR having a first antigen-binding domain that binds to an antigen of interest and a second CAR having a second antigen-binding domain that specifically binds CD123. sf-6638784 31 32429-20001.40 As used herein, the term “signal peptide” refers to a leader sequence at the amino-terminus (N-terminus) of a nascent CAR protein, which co-translationally or post-translationally directs the nascent protein to the endoplasmic reticulum and subsequent surface expression. As used herein, the term “extracellular antigen-binding domain,” “extracellular domain,” or “extracellular ligand binding domain” refers to the part of a CAR that is located outside of the cell membrane and is capable of binding to an antigen, target or ligand. As used herein, the term “hinge region” or “hinge domain” refers to the part of a CAR that connects two adjacent domains of the CAR protein, e.g., the extracellular domain and the transmembrane domain of the CAR protein. As used herein, the term “transmembrane domain” refers to the portion of a CAR that extends across the cell membrane and anchors the CAR to cell membrane. As used herein, the term “intracellular signaling domain,” “cytoplasmic signaling domain,” or “intracellular signaling domain” refers to the part of a CAR that is located inside of the cell membrane and is capable of transducing an effector signal. As used herein, the term “stimulatory molecule” refers to a molecule expressed by an immune cell (e.g., NK cell or T cell) that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of receptors in a stimulatory way for at least some aspect of the immune cell signaling pathway. Stimulatory molecules comprise two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation (referred to as “primary signaling domains”), and those that act in an antigen-independent manner to provide a secondary of co-stimulatory signal (referred to as “co-stimulatory signaling domains”). In certain embodiments, the extracellular domain comprises an antigen- binding domain and/or an antigen-binding fragment. The antigen-binding fragment can, for example, be an antibody or antigen-binding fragment thereof that specifically binds a tumor antigen. The antigen-binding fragments of the application possess one or more desirable functional properties, including but not limited to high-affinity binding to a tumor antigen, high specificity to a tumor antigen, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent phagocytosis (ADPC), and/or antibody-dependent cellular-mediated cytotoxicity (ADCC) against cells expressing a tumor antigen, and the ability to inhibit tumor growth in subjects in sf-6638784 32 32429-20001.40 need thereof and in animal models when administered alone or in combination with other anti-cancer therapies. As used herein, the term “antibody” is used in a broad sense and includes immunoglobulin or antibody molecules including human, humanized, composite and chimeric antibodies and antibody fragments that are monoclonal or polyclonal. In general, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structures are well known. Immunoglobulins can be assigned to five major classes (e.g., IgA, IgD, IgE, IgG and IgM), depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub- classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Accordingly, the antibodies of the application can be of any of the five major classes or corresponding sub-classes. Preferably, the antibodies of the application are IgG1, IgG2, IgG3 or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Accordingly, the antibodies of the application can contain a kappa or lambda light chain constant domain. According to particular embodiments, the antibodies of the application include heavy and/or light chain constant regions from rat or human antibodies. In addition to the heavy and light constant domains, antibodies contain an antigen-binding region that is made up of a light chain variable region and a heavy chain variable region, each of which contains three domains (e.g., complementarity determining regions 1-3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3. As used herein, the term an “isolated antibody” refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the specific tumor antigen is substantially free of antibodies that do not bind to the tumor antigen). In addition, an isolated antibody is substantially free of other cellular material and/or chemicals. As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. The monoclonal antibodies of the application can be made by the hybridoma method, phage display sf-6638784 33 32429-20001.40 technology, single lymphocyte gene cloning technology, or by recombinant DNA methods. For example, the monoclonal antibodies can be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, such as a transgenic mouse or rat, having a genome comprising a human heavy chain transgene and a light chain transgene. As used herein, the term “antigen-binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdAb), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a minibody, a nanobody, a domain antibody, a bivalent domain antibody, a light chain variable domain (VL), a variable domain (VHH) of a camelid antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds. As used herein, the term “single-chain antibody” refers to a conventional single-chain antibody in the field, which comprises a heavy chain variable region and a light chain variable region connected by a short peptide of about 15 to about 20 amino acids (e.g., a linker peptide). As used herein, the term “single domain antibody” refers to a conventional single domain antibody in the field, which comprises a heavy chain variable region and a heavy chain constant region or which comprises only a heavy chain variable region. As used herein, the term “human antibody” refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide. As used herein, the term “humanized antibody” refers to a non-human antibody that is modified to increase the sequence homology to that of a human antibody, such that the antigen-binding properties of the antibody are retained, but its antigenicity in the human body is reduced. sf-6638784 34 32429-20001.40 As used herein, the term “chimeric antibody” refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. The variable region of both the light and heavy chains often correspond to the variable region of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capability, while the constant regions correspond to the sequences of an antibody derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species. As used herein, the term “multispecific antibody” refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes do not overlap or do not substantially overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment, a multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In an embodiment, a multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule. As used herein, the term “bispecific antibody” refers to a multispecific antibody that binds no more than two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment, the first and second epitopes overlap or substantially overlap. In an embodiment, the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein). In an embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light sf-6638784 35 32429-20001.40 chain variable domain sequence which have binding specificity for a second epitope. In an embodiment, a bispecific antibody comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment, a bispecific antibody comprises a VHH having binding specificity for a first epitope, and a VHH having binding specificity for a second epitope. In an embodiment, the term X/Y loop (wherein ‘X’ and ‘Y’ are antigens such as CD123 and any antigen of interest) refers to an extracellular region in which one VH or VL region (of, for example, CD123) is nested in between the other VL and VH region. In some embodiments, X and Y may be the same antigen. In some embodiments, X and Y may be different antigens. In some embodiments, X and Y are tumor antigens. The terms “binder” and “specifically binds” or “specific for” with respect to an antigen-binding domain of a ligand like an antibody, of a fragment thereof or of a CAR refer to an antigen-binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific. An antigen- binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific. As used herein, an antigen-binding domain or antigen-binding fragment that “specifically binds to a tumor antigen” refers to an antigen-binding domain or antigen-binding fragment that binds a tumor antigen, with a KD of 1×10−7 M or less, preferably 1×10−8 M or less, more preferably 5×10−9 M or less, 1×10−9 M or less, 5×10−10 M or less, or 1×10−10 M or less. The term “KD” refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (e.g., Kd/Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods in the art in view of the present disclosure. For example, the KD of an antigen-binding domain or antigen-binding fragment can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system. The smaller the value of the KD of an antigen-binding domain or antigen- sf-6638784 36 32429-20001.40 binding fragment, the higher affinity that the antigen-binding domain or antigen- binding fragment binds to a target antigen. In various embodiments, antibodies or antibody fragments suitable for use in the CAR of the present disclosure include, but are not limited to, monoclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, polypeptide-Fc fusions, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv), masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceuticals ("SMIPsTM"), intrabodies, minibodies, single domain antibody variable domains, nanobodies, VHHs, diabodies, tandem diabodies (TandAb®), anti-idiotypic (anti-Id) antibodies (including, e.g., anti- Id antibodies to antigen-specific TCR), and epitope-binding fragments of any of the above. Antibodies and/or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains. In some embodiments, the antigen-binding fragment is an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an scFv fragment, an Fv fragment, a dsFv diabody, a VHH, a VNAR, a single-domain antibody (sdAb) or nanobody, a dAb fragment, a Fd' fragment, a Fd fragment, a heavy chain variable region, an isolated complementarity determining region (CDR), a diabody, a triabody, or a decabody. In some embodiments, the antigen-binding fragment is an scFv fragment. In some embodiments, the antigen-binding fragment is a VHH. As used herein, the term “extracellular tag-binding domain” is used interchangeably with “extracellular domain.” In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a single-domain antibody or nanobody. In some embodiments, the extracellular domain or the antigen-binding domain comprises a single-domain antibody or nanobody. In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises a VHH. In some embodiments, at least one of the extracellular domain or the antigen-binding domain comprises a VHH. In some embodiments, the extracellular tag-binding domain and the tag each comprise a VHH. In some embodiments, the extracellular domain each comprise a VHH. sf-6638784 37 32429-20001.40 In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a VHH. In some embodiments, the extracellular domain and the antigen-binding domain each comprise a VHH. In some embodiments, at least one of the extracellular tag-binding domain, the antigen-binding domain, or the tag comprises an scFv. In some embodiments, at least one of the extracellular domain and the antigen-binding domain each comprise a VHH. In some embodiments, the extracellular tag-binding domain and the tag each comprise an scFv. In some embodiments, the extracellular domain comprises an scFv. In some embodiments, the extracellular tag-binding domain, the tag, and the antigen-binding domain each comprise a scFv. In some embodiments, the extracellular domain and the antigen-binding domain each comprise a scFv. Alternative scaffolds to immunoglobulin domains that exhibit similar functional characteristics, such as high-affinity and specific binding of target biomolecules, may also be used in the CARs of the present disclosure. Such scaffolds have been shown to yield molecules with improved characteristics, such as greater stability or reduced immunogenicity. Non-limiting examples of alternative scaffolds that may be used in the CAR of the present disclosure include engineered, tenascin- derived, tenascin type III domain (e.g., Centyrin™); engineered, gamma-B crystallin- derived scaffold or engineered, ubiquitin-derived scaffold (e.g., Affilins); engineered, fibronectin-derived, 10th fibronectin type III (10Fn3) domain (e.g., monobodies, AdNectins™, or AdNexins™);; engineered, ankyrin repeat motif containing polypeptide (e.g., DARPins™); engineered, low-density-lipoprotein-receptor-derived, A domain (LDLR-A) (e.g., Avimers™); lipocalin (e.g., anticalins); engineered, protease inhibitor-derived, Kunitz domain (e.g., EETI-II/AGRP, BPTI/LACI-D1/ITI- D2); engineered, Protein-A-derived, Z domain (Affibodies™); Sac7d-derived polypeptides (e.g., Nanoffitins® or affitins); engineered, Fyn-derived, SH2 domain (e.g., Fynomers®); CTLD3 (e.g., Tetranectin); thioredoxin (e.g., peptide aptamer); KALBITOR®; the β-sandwich (e.g., iMab); miniproteins; C-type lectin-like domain scaffolds; engineered antibody mimics; and any genetically manipulated counterparts of the foregoing that retains its binding functionality (Wörn A, Pluckthun A, J Mol Biol 305: 989-1010 (2001); Xu L et al., Chem Biol 9: 933-42 (2002); Wikman M et al., Protein Eng Des Sel 17: 455-62 (2004); Binz H et al., Nat Biolechnol 23: 1257-68 (2005); Hey T et al., Trends Biotechnol 23:514-522 (2005); Holliger P, Hudson P, sf-6638784 38 32429-20001.40 Nat Biotechnol 23: 1126-36 (2005); Gill D, Damle N, Curr Opin Biotech 17: 653-8 (2006); Koide A, Koide S, Methods Mol Biol 352: 95-109 (2007); Skerra, Current Opin. in Biotech., 200718: 295-304; Byla P et al., J Biol Chem 285: 12096 (2010); Zoller F et al., Molecules 16: 2467-85 (2011), each of which is incorporated by reference in its entirety). In some embodiments, the alternative scaffold is Affilin or Centyrin. In some embodiments, the first polypeptide of the CARs of the present disclosure comprises a leader sequence. The leader sequence may be positioned at the N-terminus the extracellular tag-binding domain. The leader sequence may be positioned at the N-terminus the extracellular domain. The leader sequence may be optionally cleaved from the extracellular tag-binding domain during cellular processing and localization of the CAR to the cellular membrane. The leader sequence may be optionally cleaved from the extracellular domain during cellular processing and localization of the CAR to the cellular membrane. Any of various leader sequences known to one of skill in the art may be used as the leader sequence. Non-limiting examples of peptides from which the leader sequence may be derived include granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), FcεR, human immunoglobulin (IgG) heavy chain (HC) variable region, CD8α, or any of various other proteins secreted by T cells. In various embodiments, the leader sequence is compatible with the secretory pathway of a T cell. In certain embodiments, the leader sequence is derived from human immunoglobulin heavy chain (HC). In some embodiments, the leader sequence is derived from GMCSFR. In one embodiment, the GMCSFR leader sequence comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 1. In one embodiment, the nucleic acid encoding the GMCSFR leader sequence comprises the nucleotide sequence set forth in SEQ ID NO: 2. In some embodiments, the leader sequence is derived from IgK. In one embodiment, the leader sequence is an IgK signal peptide variant comprising the amino acid sequence set forth in SEQ ID NO: 103, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, sf-6638784 39 32429-20001.40 at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 103. In one embodiment, the nucleic acid encoding the IgK signal peptide variant comprises the nucleotide sequence set forth in SEQ ID NO: 101. In some embodiments, the first polypeptide of the CARs of the present disclosure comprises a transmembrane domain, fused in frame between the extracellular tag-binding domain and the cytoplasmic domain. In some embodiments, the first polypeptide of the CARs of the present disclosure comprises a transmembrane domain, fused in frame between the extracellular domain and the cytoplasmic domain. The transmembrane domain may be derived from the protein contributing to the extracellular tag-binding domain, the protein contributing the signaling or co- signaling domain, or by a totally different protein. The transmembrane domain may be derived from the protein contributing to the extracellular domain, the protein contributing the signaling or co-signaling domain, or by a totally different protein. In some instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex. In some instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to avoid binding of proteins naturally associated with the transmembrane domain. In certain embodiments, the transmembrane domain includes additional amino acids to allow for flexibility and/or optimal distance between the domains connected to the transmembrane domain. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains of particular use in this disclosure may be derived from (e.g., comprise at least the transmembrane region(s) of) the α or β chain of the T-cell receptor (TCR), CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan and/or valine can be found at each end of a synthetic transmembrane domain. sf-6638784 40 32429-20001.40 In some embodiments, it will be desirable to utilize the transmembrane domain of the ζ, η or FcεR1γ chains which contain a cysteine residue capable of disulfide bonding, so that the resulting chimeric protein will be able to form disulfide linked dimers with itself, or with unmodified versions of the ζ, η or FcεR1γ chains or related proteins. In some instances, the transmembrane domain will be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In other cases, it will be desirable to employ the transmembrane domain of ζ, η or FcεR1γ and -β, MB1 (Igα.), B29 or CD3- γ, ζ, or η, in order to retain physical association with other members of the receptor complex. In some embodiments, the transmembrane domain is derived from CD8 or CD28. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 23. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23. In one embodiment, the nucleic acid encoding the CD8 transmembrane domain comprises the polynucleotide sequence set forth in SEQ ID NO: 96. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 24, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 24. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 24. In one embodiment, the nucleic acid encoding the CD28 transmembrane domain comprises the polynucleotide sequence set forth in SEQ ID NO: 97. In some embodiments, the first polypeptide of the CAR of the present disclosure comprises a spacer region between the extracellular tag-binding domain and the transmembrane domain, wherein the extracellular tag-binding domain, linker, and the transmembrane domain are in frame with each other. In some embodiments, the first polypeptide of the CAR of the present disclosure comprises a spacer region between the extracellular domain and the transmembrane domain, wherein the sf-6638784 41 32429-20001.40 extracellular domain, linker, and the transmembrane domain are in frame with each other. The term “spacer region” as used herein generally means any oligo- or polypeptide that functions to link the extracellular tag-binding domain to the transmembrane domain. A spacer region can be used to provide more flexibility and accessibility for the extracellular tag-binding domain. A spacer region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. A spacer region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the spacer region may be a synthetic sequence that corresponds to a naturally occurring spacer region sequence, or may be an entirely synthetic spacer region sequence. Non-limiting examples of spacer regions which may be used in accordance to the disclosure include a part of human CD8α chain, partial extracellular domain of CD28, FcγRIIIa receptor, IgG, IgM, IgA, IgD, IgE, an Ig hinge, or functional fragment thereof. In some embodiments, additional linking amino acids are added to the spacer region to ensure that the antigen-binding domain is an optimal distance from the transmembrane domain. In some embodiments, when the spacer is derived from an Ig, the spacer may be mutated to prevent Fc receptor binding. In some embodiments, the spacer region comprises a hinge domain. The hinge domain may be derived from CD8, CD8α, CD28, or an immunoglobulin (IgG). For example, the IgG hinge may be from IgG1, IgG2, IgG3, IgG4, IgG4 CH3, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof. In certain embodiments, the hinge domain comprises an immunoglobulin IgG hinge or functional fragment thereof. In certain embodiments, the IgG hinge is from IgG1, IgG2, IgG3, IgG4, IgG4 CH3, IgM1, IgM2, IgA1, IgA2, IgD, IgE, or a chimera thereof. In certain embodiments, the hinge domain comprises the CH1, CH2, CH3 and/or hinge region of the immunoglobulin. In certain embodiments, the hinge domain comprises the core hinge region of the immunoglobulin. The term “core hinge” can be used interchangeably with the term “short hinge” (a.k.a “SH”). Non- limiting examples of suitable hinge domains are the core immunoglobulin hinge regions include EPKSCDKTHTCPPCP (SEQ ID NO: 57) from IgG1, ERKCCVECPPCP (SEQ ID NO: 58) from IgG2, ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: 59) from IgG3, sf-6638784 42 32429-20001.40 ESKYGPPCPSCP (SEQ ID NO: 60) from IgG4 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes), and ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK (SEQ ID NO: 102), or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity. In certain embodiments, the hinge domain is a fragment of the immunoglobulin hinge. In some embodiments, the hinge domain is a fragment of or derived from the immunoglobulin hinge. In one embodiment, the hinge domain is an immunoglobulin hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 61, or a variant thereof of having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with SEQ ID NO: 61. In one embodiment, the hinge domain is an immunoglobulin hinge domain comprising the amino acid sequence set forth in SEQ ID NO: 61. In one embodiment, the nucleic acid encoding the immunoglobulin hinge domain comprises the sequence set forth in SEQ ID NO: 62. In one embodiment, the hinge domain comprises the sequence set forth in SEQ ID NO: 99, or a variant thereof of having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with SEQ ID NO: 99. In one embodiment, the hinge domain comprises the sequence set forth in SEQ ID NO: 99. In one embodiment, the hinge domain comprises the sequence set forth in SEQ ID NO: 100, or a variant thereof of having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with SEQ ID NO: 100. In one embodiment, the hinge domain comprises the sequence set forth in SEQ ID NO: 100. In some embodiments, the hinge domain is derived from CD8 or CD28. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50%, at least 55%, at least 60%, sf-6638784 43 32429-20001.40 at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 21. In one embodiment, the nucleic acid sequence encoding the CD8 hinge domain comprises the sequence set forth in SEQ ID NO: 4. In one embodiment, the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 22, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 22. In one embodiment, the nucleic acid sequence encoding the CD28 hinge domain comprises the sequence set forth in SEQ ID NO: 5. In some embodiments, the transmembrane domain and/or hinge domain is derived from CD8 or CD28. In some embodiments, both the transmembrane domain and hinge domain are derived from CD8. In some embodiments, both the transmembrane domain and hinge domain are derived from CD28. In certain aspects, the first polypeptide of CARs of the present disclosure comprises a cytoplasmic domain, which comprises at least one intracellular signaling domain. In some embodiments, cytoplasmic domain also comprises one or more co- stimulatory signaling domains. The cytoplasmic domain is responsible for activation of at least one of the normal effector functions of the host cell (e.g., T cell) in which the CAR has been placed in. The term “effector function” refers to a specialized function of a cell. Effector function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire signaling domain is present, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the signaling domain sufficient to transduce the effector function signal. Non-limiting examples of signaling domains which can be used in the CARs of the present disclosure include, e.g., signaling domains derived from DAP10, DAP12, Fc epsilon receptor I γ chain (FCER1G), FcR β, CD3δ, CD3ε, CD3γ, CD3ζ, CD5, CD22, CD226, CD66d, CD79A, CD79B, IL18R1, and IL18RAP. In some sf-6638784 44 32429-20001.40 embodiments, the signaling domain of the CAR can comprise the TIR domain sequence from IL18R1 or IL18RAP. In some embodiments, the cytoplasmic domain comprises a CD3ζ signaling domain. In one embodiment, the CD3ζ signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 6, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 6. In one embodiment, the CD3ζ signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 6. In one embodiment, the nucleic acid sequence encoding the CD3ζ signaling domain comprises the sequence set forth in SEQ ID NO: 7. In some embodiments, the cytoplasmic domain further comprises one or more co-stimulatory signaling domains. In some embodiments, the one or more co- stimulatory signaling domains are derived from CD28, 41BB, IL2Rb, IL18R1, IL18RAP, CD40, OX40 (CD134), CD80, CD86, CD27, ICOS, NKG2D, DAP10, DAP12, 2B4 (CD244), BTLA, CD30, GITR, CD226, CD79A, and HVEM. In some embodiments, the co-stimulatory signaling domain of the CAR can comprise the TIR domain sequence from IL18R1 or IL18RAP. In one embodiment, the co-stimulatory signaling domain is derived from CD28. In one embodiment, the CD28 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 20, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with SEQ ID NO: 20. In one embodiment, the CD28 co- stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 20. In one embodiment, the co-stimulatory signaling domain is derived from 41BB. In one embodiment, the 41BB co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 8, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 8. In one embodiment, the 41BB co- stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 8. sf-6638784 45 32429-20001.40 In one embodiment, the co-stimulatory signaling domain is derived from IL2Rb. In one embodiment, the IL2Rb co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 9. In one embodiment, the IL2Rb co- stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 9. In one embodiment, the co-stimulatory signaling domain is derived from CD40. In one embodiment, the CD40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 10. In one embodiment, the CD40 co- stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 10. In one embodiment, the co-stimulatory signaling domain is derived from OX40. In one embodiment, the OX40 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 11, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 11. In one embodiment, the OX40 co- stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 11. In one embodiment, the co-stimulatory signaling domain is derived from CD80. In one embodiment, the CD80 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 12, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 12. In one embodiment, the CD80 co- stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 12. In one embodiment, the co-stimulatory signaling domain is derived from CD86. In one embodiment, the CD86 co-stimulatory signaling domain comprises the sf-6638784 46 32429-20001.40 amino acid sequence set forth in SEQ ID NO: 13, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 13. In one embodiment, the CD86 co- stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 13. In one embodiment, the co-stimulatory signaling domain is derived from CD27. In one embodiment, the CD27 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 14, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 14. In one embodiment, the CD27 co- stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 14. In one embodiment, the co-stimulatory signaling domain is derived from ICOS. In one embodiment, the ICOS co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 15. In one embodiment, the ICOS co- stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 15. In one embodiment, the co-stimulatory signaling domain is derived from NKG2D. In one embodiment, the NKG2D co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 16, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 16. In one embodiment, the NKG2D co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 16. In one embodiment, the co-stimulatory signaling domain is derived from DAP10. In one embodiment, the DAP10 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 17, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least sf-6638784 47 32429-20001.40 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 17. In one embodiment, the DAP10 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 17. In one embodiment, the co-stimulatory signaling domain is derived from DAP12. In one embodiment, the DAP12 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 18. In one embodiment, the DAP12 co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 18. In one embodiment, the co-stimulatory signaling domain is derived from IL18R1 or IL18RAP. In one embodiment, the IL18R1 or IL18RAP co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 243 or 244, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 243 or 244. In one embodiment, the co-stimulatory signaling domain is derived from 2B4 (CD244). In one embodiment, the 2B4 (CD244) co-stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 19. In some embodiments, the CAR of the present disclosure comprises one co- stimulatory signaling domains. In some embodiments, the CAR of the present disclosure comprises two or more co-stimulatory signaling domains. In certain embodiments, the CAR of the present disclosure comprises two, three, four, five, six or more co-stimulatory signaling domains. In some embodiments, the signaling domain(s) and co-stimulatory signaling domain(s) can be placed in any order. In some embodiments, the signaling domain is upstream of the co-stimulatory signaling domains. In some embodiments, the signaling domain is downstream from the co-stimulatory signaling domains. In the sf-6638784 48 32429-20001.40 cases where two or more co-stimulatory domains are included, the order of the co- stimulatory signaling domains could be switched. Non-limiting exemplary CAR regions and sequences are provided in Table 1, including amino acid and nucleic acid sequences for various CAR constructs shown in FIGS. 9A-9D and 10A-10D. sf-6638784 49 32429-20001.40 Table 1. 50 sf-6638784 32429-20001.40 sf-6638784 51 32429-20001.40 sf-6638784 52 32429-20001.40 sf-6638784 53 32429-20001.40 sf-6638784 54 32429-20001.40 sf-6638784 55 32429-20001.40 sf-6638784 56 32429-20001.40 sf-6638784 57 32429-20001.40 sf-6638784 58 32429-20001.40 sf-6638784 59 32429-20001.40 sf-6638784 60 32429-20001.40 sf-6638784 61 32429-20001.40 sf-6638784 62 32429-20001.40 sf-6638784 63 32429-20001.40 sf-6638784 64 32429-20001.40 sf-6638784 65 32429-20001.40 sf-6638784 66 32429-20001.40 In some embodiments, the antigen-binding domain of the second polypeptide binds to an antigen. The antigen-binding domain of the second polypeptide may bind to more than one antigen or more than one epitope in an antigen. For example, the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six, seven, eight or more antigens. As another example, the antigen-binding domain of the second polypeptide may bind to two, three, four, five, six, seven, eight or more epitopes in the same antigen. The choice of antigen-binding domain may depend upon the type and number of antigens that define the surface of a target cell. For example, the antigen-binding sf-6638784 67 32429-20001.40 domain may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In certain embodiments, the CARs of the present disclosure can be genetically modified to target a tumor antigen of interest by way of engineering a desired antigen-binding domain that specifically binds to an antigen (e.g., on a tumor cell). Non-limiting examples of cell surface markers that may act as targets for the antigen-binding domain in the CAR of the disclosure include those associated with tumor cells or autoimmune diseases. In some embodiments, the antigen-binding domain binds to at least one tumor antigen or autoimmune antigen. In some embodiments, the antigen-binding domain binds to at least one tumor antigen. In some embodiments, the antigen-binding domain binds to two or more tumor antigens. In some embodiments, the two or more tumor antigens are associated with the same tumor. In some embodiments, the two or more tumor antigens are associated with different tumors. In some embodiments, the antigen-binding domain binds to at least one autoimmune antigen. In some embodiments, the antigen-binding domain binds to two or more autoimmune antigens. In some embodiments, the two or more autoimmune antigens are associated with the same autoimmune disease. In some embodiments, the two or more autoimmune antigens are associated with different autoimmune diseases. In some embodiments, the tumor antigen is associated with AML, CML, ALL and CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer such as glioblastoma, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, ovarian cancer, pancreatic cancer, renal cell carcinoma, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP). Non-limiting examples of tumor antigens that may be targeted by the antigen-binding domain include alpha- fetoprotein, A3, antigen specific for A33 antibody, AFP, B7H4, Ba 733, BCMA, BrE3-antigen, carbonic anhydrase EX, CA125, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD38, CD45, CD70, CD74, CD79, CD79a, CD80, CD123, CD133, CD138, c-MET, Claudin 18.2, CLL1, colon- specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, sf-6638784 68 32429-20001.40 EGFRvIII, EGP-I, EGP-2, Ep-CAM, EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FGFR1, FGFR3, Fit-I, Flt-3, folate receptor such as FOLR1, FSHR, GD2, GPC3, GPRC5D, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, HER2, hypoxia inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, IL13Rα2, insulin growth factor-1 (IGF- I), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, Mesothelin, MET, MUC1, MUC2, MUC3, MUC4, MUC16, NCA66, NCA95, NCA90, Nectin-4, antigen specific for PAM-4 antibody, PDGFRα, placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA, ROBO1, RS5, S100, SLAM F7, SLITRK6, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-1A-antigen, an angiogenesis marker, an oncogene marker or an oncogene product. In one embodiment, the antigen targeted by the antigen-binding domain is CD123. In one embodiment, the antigen-binding domain comprises an anti-CD123 VH and VL region. In another embodiment, the antigen-binding domain comprises an anti-CD123 VHH. In one embodiment, the anti-CD123 antigen-binding domain comprises amino acids having the sequence set forth in one of SEQ ID NOs: 126-148, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with one of SEQ ID NOs: 126-148. In one embodiment, the anti-CD123 antigen-binding domain is encoded by a polynucleotide having the sequence set forth in one of SEQ ID NOs: 149-171, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with one of SEQ ID NOs: 149-171. In some embodiments, the antigen is associated with an autoimmune disease or disorder. Such antigens may be derived from cell receptors and cells which produce “self”-directed antibodies. In some embodiments, the antigen is associated with an autoimmune disease or disorder such as rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren’s syndrome, systemic lupus erythematosus, sarcoidosis, Type 1 diabetes mellitus, insulin dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener’s granulomatosis, myasthenia gravis, sf-6638784 69 32429-20001.40 Hashimoto’s thyroiditis, Graves’ disease, chronic inflammatory demyelinating polyneuropathy, Guillain-Barre syndrome, Crohn’s disease or ulcerative colitis. In some embodiments, autoimmune antigens that may be targeted by the CAR disclosed herein include but are not limited to platelet antigens, myelin protein antigen, Sm antigens in snRNPs, islet cell antigen, rheumatoid factor, and anticitrullinated protein. Citrullinated proteins and peptides such as CCP-1, CCP-2 (cyclical citrullinated peptides), fibrinogen, fibrin, vimentin, filaggrin, collagen I and II peptides, alpha-enolase, translation initiation factor 4G1, perinuclear factor, keratin, Sa (cytoskeletal protein vimentin), components of articular cartilage such as collagen II, IX, and XI, circulating serum proteins such as RFs (IgG, IgM), fibrinogen, plasminogen, ferritin, nuclear components such as RA33/hnRNP A2, Sm, eukaryotic translation elongation factor 1 alpha 1, stress proteins such as HSP-65, -70, -90, BiP, inflammatory/immune factors such as B7-H1, IL-1 alpha, and IL-8, enzymes such as calpastatin, alpha-enolase, aldolase-A, dipeptidyl peptidase, osteopontin, glucose-6- phosphate isomerase, receptors such as lipocortin 1, neutrophil nuclear proteins such as lactoferrin and 25-35 kD nuclear protein, granular proteins such as bactericidal permeability increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, proteinase 3, platelet antigens, myelin protein antigen, islet cell antigen, rheumatoid factor, histones, ribosomal P proteins, cardiolipin, vimentin, nucleic acids such as dsDNA, ssDNA, and RNA, ribonuclear particles and proteins such as Sm antigens (including but not limited to SmD’s and SmB′/B), U1RNP, A2/B1 hnRNP, Ro (SSA), and La (SSB) antigens. In various embodiments, the extracellular domain used in the CAR of the present disclosure may include a linker between the VH and VL domains. The linker can be a peptide linker and may include any naturally occurring amino acid. Exemplary amino acids that may be included into the linker are Gly, Ser Pro, Thr, Glu, Lys, Arg, Ile, Leu, His and The. The linker should have a length that is adequate to link the VH and the VL in such a way that they form the correct conformation relative to one another so that they retain the desired activity, such as binding to an antigen. The linker may be about 5-50 amino acids long. In some embodiments, the linker is about 10-40 amino acids long. In some embodiments, the linker is about 10-35 amino acids long. In some embodiments, the linker is about 10-30 amino acids long. In some embodiments, the linker is about 10-25 amino acids long. In some embodiments, the linker is about 10- 20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. sf-6638784 70 32429-20001.40 Exemplary linkers that may be used are Gly rich linkers, Gly and Ser containing linkers, Gly and Ala containing linkers, Ala and Ser containing linkers, and other flexible linkers. In one embodiment, the linker is a Whitlow linker. In one embodiment, the Whitlow linker comprises the amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 3. In one embodiment, the nucleic acid encoding the Whitlow linker comprises the sequence set forth in SEQ ID NO: 98. In another embodiment, the linker is a (G4S)3 linker. In one embodiment, the (G4S)3 linker comprises the amino acid sequence set forth in SEQ ID NO: 25, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 25. Other linker sequences may include portions of immunoglobulin hinge area, CL or CH1 derived from any immunoglobulin heavy or light chain isotype. Exemplary linkers that may be used include any of SEQ ID NOs: 26-56 in Table 1. Exemplary linkers that may be used include SEQ ID NO: 259 or SEQ ID NO: 260 in Table 1. Additional linkers are described for example in Int. Pat. Publ. No. WO2019/060695, incorporated by reference herein in its entirety. In some embodiments, provided herein is a full-length antibody that binds to CD123. In some embodiments, the antibody comprises a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the antibody or antigen-binding fragment is recombinant. In some embodiments, the antigen binding fragment is or contains a single chain fragment. In some embodiments, the antigen- binding fragment is or contains a single chain Fv (scFv). In some embodiments, the antigen-binding fragment contains a VH and a VL region. In some embodiments, the VH and VL regions are joined by a flexible linker. In some embodiments, the antibody or antigen fragment binds a CD123 antigen. In some embodiments, the antibody or antigen fragment is monospecific. In some embodiments, the antibody or antigen fragment is bispecific. sf-6638784 71 32429-20001.40 In some embodiments, provided herein are antibody-drug conjugates (ADCs). In some embodiments, the antibody-drug conjugate is a CD123-directed antibody-drug conjugate. ADCs include recombinant monoclonal antibodies covalently bound to biologically active drugs (“payload,” e.g., cytotoxic chemicals) by chemical linkers. In some embodiments, the ADC, binds to the antigen (e.g., CD123) on the surface of target cells, such as tumor cells, and then is absorbed or internalized. After the ADC is internalized, the biologically active drug is released in the lysosomes and transported to cytosol to kill the target cells. In some embodiments, ADCs can also trigger antibody- dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular- mediated phagocytosis of target cells, e.g., tumor cells. In some embodiments, the ADC comprises an antibody or an antigen-binding fragment thereof that binds to CD123. In some embodiments, the ADC comprises a monospecific antibody. In some embodiments, the ADC comprises a bispecific antibody. II. Artificial Cell Death Polypeptide According to embodiments of the application, an iPSC cell or a derivative cell thereof comprises an exogenous polynucleotide encoding an artificial cell death polypeptide. As used herein, the term “artificial cell death polypeptide” refers to an engineered protein designed to prevent potential toxicity or otherwise adverse effects of a cell therapy. The artificial cell death polypeptide could 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 instance, the artificial cell death polypeptide is activated by an exogenous molecule, e.g., an antibody, that when activated, triggers apoptosis and/or cell death of a therapeutic cell. In certain embodiments, an artificial cell death polypeptide comprises an inactivated cell surface receptor that comprises an epitope specifically recognized by an antibody, particularly a monoclonal antibody, which is also referred to herein as a monoclonal antibody-specific epitope. When expressed by iPSCs or derivative cells thereof, the inactivated cell surface receptor is signaling inactive or significantly impaired, but can still be specifically recognized by an antibody. The specific binding of the antibody to the inactivated cell surface receptor enables the elimination of the iPSCs or derivative cells thereof by ADCC and/or ADCP mechanisms, as well as direct killing with antibody drug conjugates with toxins or radionuclides. sf-6638784 72 32429-20001.40 In certain embodiments, the inactivated cell surface receptor comprises an epitope that is selected from epitopes specifically recognized by an antibody including, but not limited to, abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab, infliximab, ipilimumab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, polatuzumab vedotin, ranibizumab, rituximab, tiuxetan, tocilizumab, tositumomab, trastuzumab, tremelimumab, ustekinumab, and vedolizumab. Epidermal growth factor receptor, also known as EGFR, ErbB1 and HER1, is a cell-surface receptor for members of the epidermal growth factor family of extracellular ligands. As used herein, “truncated EGFR,” “tEGFR,” “short EGFR” or “sEGFR” refers to an inactive EGFR variant that lacks the EGF-binding domains and the intracellular signaling domains of the EGFR. An exemplary tEGFR variant contains residues 322-333 of domain 2, all of domains 3 and 4 and the transmembrane domain of the native EGFR sequence containing the cetuximab binding epitope. Expression of the tEGFR variant on the cell surface enables cell elimination by an antibody that specifically binds to the tEGFR, such as cetuximab (Erbitux®), as needed. Due to the absence of the EGF-binding domains and intracellular signaling domains, tEGFR is inactive when expressed by iPSCs or derivative cell thereof. An exemplary inactivated cell surface receptor of the application comprises a tEGFR variant. In certain embodiments, expression of the inactivated cell surface receptor in an engineered immune cell expressing a chimeric antigen receptor (CAR) induces cell suicide of the engineered immune cell when the cell is contacted with an anti-EGFR antibody. Methods of using inactivated cell surface receptors are described in WO2019/070856, WO2019/023396, and WO2018/058002, the disclosure of each of which is incorporated herein by reference. For example, a subject who has previously received an engineered immune cell of the present disclosure that comprises a heterologous polynucleotide encoding an inactivated cell surface receptor comprising a tEGFR variant can be administered an anti-EGFR antibody in an amount effective to ablate in the subject the previously administered engineered immune cell. In certain embodiments, the anti-EGFR antibody is cetuximab, matuzumab, necitumumab or panitumumab, preferably the anti-EGFR antibody is cetuximab. sf-6638784 73 32429-20001.40 In certain embodiments, the tEGFR variant comprises or consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 71, preferably the amino acid sequence of SEQ ID NO: 71. In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin. In certain embodiments, the CD79b epitope comprises or consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78, preferably the amino acid sequence of SEQ ID NO: 78. In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of CD20, such as an epitope specifically recognized by rituximab. In certain embodiments, the CD20 epitope comprises or consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 80, preferably the amino acid sequence of SEQ ID NO: 80. In some embodiments, the inactivated cell surface receptor comprises one or more epitopes of Her 2 receptor or ErbB, such as an epitope specifically recognized by trastuzumab. In certain embodiments, the monoclonal antibody-specific epitope comprises or consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82, preferably the amino acid sequence of SEQ ID NO: 82. III. Cytokine Expression In some embodiments the inactivated cell surface receptor further comprises a cytokine, such as interleukin-15 or interleukin-2. As used herein “Interleukin-15” or “IL-15” refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof. A “functional portion” (“biologically active portion”) of a cytokine refers to a portion of the cytokine that retains one or more functions of full length or mature cytokine. Such functions for IL-15 include the promotion of NK cell survival, regulation of NK cell and T cell activation and proliferation as well as the support of NK cell development from hematopoietic stem cells. As will be appreciated by those of skill in the art, the sequence of a variety of IL-15 molecules are known in the art. In certain sf-6638784 74 32429-20001.40 embodiments, the IL-15 is a wild-type IL-15. In certain embodiments, the IL-15 is a human IL-15. In certain embodiments, the IL-15 comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 72, preferably the amino acid sequence of SEQ ID NO: 72. As used herein “Interleukin-2” refers to a cytokine that regulates T and NK cell activation and proliferation, or a functional portion thereof. In certain embodiments, the IL-2 is a wild-type IL-2. In certain embodiments, the IL-2 is a human IL-2. In certain embodiments, the IL-2 comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 76, preferably the amino acid sequence of SEQ ID NO: 76. In certain embodiments, an inactivated cell surface receptor comprises a monoclonal antibody-specific epitope operably linked to a cytokine, preferably by an autoprotease peptide sequence. Examples of the autoprotease peptide include, but are not limited to, a peptide sequence selected from the group consisting of porcine teschovirus-12A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), and a combination thereof. In one embodiment, the autoprotease peptide is an autoprotease peptide of porcine tesehovirus-12A (P2A). In certain embodiments, the autoprotease peptide comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 73, preferably the amino acid sequence of SEQ ID NO: 73. In certain embodiments, an inactivated cell surface receptor comprises a truncated epithelial growth factor (tEGFR) variant operably linked to an interleukin- 15 (IL-15) or IL-2 by an autoprotease peptide sequence. In a particular embodiment, the inactivated cell surface receptor comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 74, preferably the amino acid sequence of SEQ ID NO: 74. In some embodiments, an inactivated cell surface receptor further comprises a signal sequence. In certain embodiments, the signal sequence comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, sf-6638784 75 32429-20001.40 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 77, preferably the amino acid sequence of SEQ ID NO: 77. In some embodiments, an inactivated cell surface receptor further comprises a hinge domain. In some embodiments, the hinge domain is derived from CD8. In one embodiment, the CD8 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 21. In one embodiment, the nucleic acid sequence encoding the CD8 hinge domain comprises the sequence set forth in SEQ ID NO: 4. In certain embodiments, an inactivated cell surface receptor further comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from CD8. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 23. In certain embodiment, an inactivated cell surface receptor comprises one or more epitopes specifically recognized by an antibody in its extracellular domain, a transmembrane region and a cytoplasmic domain. In some embodiments, the inactivated cell surface receptor further comprises a hinge region between the epitope(s) and the transmembrane region. In some embodiments, the inactivated cell surface receptor comprises more than one epitope specifically recognized by an antibody, the epitopes can have the same or different amino acid sequences, and the epitopes can be linked together via a peptide linker, such as a flexible peptide linker have the sequence of (GGGGS)n, wherein n is an integer of 1-8 (SEQ ID NO: 25). In some embodiments, the inactivated cell surface receptor further comprises a cytokine, such as an IL-15 or IL-2. In certain embodiments, the cytokine is in the cytoplasmic domain of the inactivated cell surface receptor. Preferably, the cytokine is operably linked to the epitope(s) specifically recognized by an antibody, directly or indirectly, via an autoprotease peptide sequence, such as those described herein. In some embodiments, the cytokine is indirectly linked to the epitope(s) by connecting to the transmembrane region via the autoprotease peptide sequence. sf-6638784 76 32429-20001.40 In some embodiments, an exogenous polynucleotide encoding the cytokine comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 258. In some embodiments, the exogenous polynucleotide encoding the cytokine comprises the sequence set forth in SEQ ID NO: 258. In some embodiments, the artificial cell death polypeptide can comprise a safety switch. In some embodiments, the safety switch is an inducible Caspase 9 sequence (iCasp9) or a herpes simplex virus thymidine kinase (HSV-TK). In some embodiments, the artificial cell death polypeptide can comprise an inducible Caspase 9 sequence (iCasp9). Caspase 9 homodimerizes to become activated. The homodimer undergoes a conformational change and the proteolytic domain of one of a pair of dimers becomes active. Physiologically, this occurs by binding of the CARD domain of Caspase 9 to APAF-1. In iCasp9, the APAF-1 domain is replaced with a modified FKBP12 which has been mutated to selectively bind a chemical inducer of dimerization (CID). Presence of the CID results in homodimerization and activation. iCasp9 is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) (Straathof et al. (2005) Blood 105:4247-4254). It enables conditional dimerization in the presence of a small molecule CID, known as AP1903. AP1903 is an experimental drug and is considered biologically inert since it does not interact with wild-type FKBP12. However clinical experience with this agent is limited to a very small number of patients (Di Stasi, A. et al. (2011) N. Engl. J. Med. 365, 1673-1683; and luliucci, J. D. et al. (2001) J. Clin. Pharmacol. 41, 870-879). AP1903 is also a relatively large and polar molecule and unlikely to cross the blood-brain barrier. In some embodiments, the iCasp9 comprises the amino acid sequence set forth in SEQ ID NO: 245, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 245. In some embodiments, the iCasp9 comprises the sequence set forth in SEQ ID NO: 245. In some embodiments, the iCasp9 comprises the amino acid sequence set forth in SEQ ID NO: 256, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 256. In some embodiments, the iCasp9 comprises the sequence set forth in SEQ ID NO: 256. sf-6638784 77 32429-20001.40 In some embodiments, the artificial cell death polypeptide can comprise a herpes simplex virus thymidine kinase (HSV-TK). In HSV-TK, caspase 9 is fused to a modified human FK-binding protein, which has been mutated to selectively bind a chemical inducer of dimerization (CID) (Di Stasi, A. et al. (2011) N. Engl. J. Med. 365, 1673-1683). Accordingly, presence of the CID results in homodimerization and activation. It enables conditional dimerization in the presence of a small molecule CID AP1903. In some embodiments, the HSV-TK comprises the amino acid sequence set forth in SEQ ID NO: 248, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 248. In some embodiments, the HSV-TK comprises the sequence set forth in SEQ ID NO: 248. In some embodiments, the HSV-TK comprises the amino acid sequence set forth in SEQ ID NO: 249, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, sequence identity with SEQ ID NO: 249. In some embodiments, the HSV-TK comprises the sequence set forth in SEQ ID NO: 249. Non-limiting exemplary artificial cell death polypeptide / inactivated cell surface receptor sequences are provided in Table 2. sf-6638784 78 32429-20001.40 Table 2. 79 sf-6638784 32429-20001.40 sf-6638784 80 32429-20001.40 In a particular embodiment, the inactivated cell surface receptor comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 79, preferably the amino acid sequence of SEQ ID NO: 79. In a particular embodiment, the inactivated cell surface receptor comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 81, preferably the amino acid sequence of SEQ ID NO: 81. In a particular embodiment, the inactivated cell surface receptor comprises amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 83, preferably the amino acid sequence of SEQ ID NO: 83. In some embodiments, the artificial cell death polypeptide further comprises an extracellular domain comprising a PSMA cell tracer of fragment thereof. In some embodiments, the safety switch (e.g., HSV-TK or iCas9) comprised in the artificial cell death polypeptide is operatively linked to the extracellular domain comprising a PSMA cell tracer of fragment thereof to form a combined artificial cell death/reporter system polypeptide. In some embodiments, the safety switch is HSV-TK. In some embodiments, the PSMA cell tracer or fragment thereof is a truncated variant PSMA polypeptide. In one embodiment, the truncated variant PSMA polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 250. In one embodiment, the truncated variant PSMA polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the PSMA cell tracer of fragment thereof and safety switch are operatively linked by a linker. In some embodiments, the linker comprises an autoprotease peptide, such as any autoprotease peptide disclosed herein. sf-6638784 81 32429-20001.40 In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 251. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 251. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 252. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 252. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 253. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 253. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 254. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 254. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 255. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 255. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 256. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 256. In one embodiment, the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 257. In one embodiment, the artificial cell death/reporter system polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 257. IV. HLA Expression sf-6638784 82 32429-20001.40 In one aspect, MHC I and/or MHC II knock-out and/or knock down can be incorporated in the cells for use in “allogeneic” cell therapies, in which cells are harvested from a subject, modified to knock-out or knock-down, e.g., disrupt, B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP gene expression, and then returned to a different subject. Knocking out or knocking down the B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes as described herein can: (1) prevent Graft versus Host response; (2) prevent Host versus Graft response; and/or (3) improve cell safety and efficacy. Accordingly, certain embodiments of the present disclosure comprise independently knocking out and/or knocking down one or more genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes in an iPSC cell. In certain embodiments, a presently disclosed method comprises independently knocking out and/or knocking down two genes selected from the group consisting B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes in an iPSC cell, in particular, B2M and CIITA to achieve class I and II HLA disruption. In certain embodiments, an iPSC or derivative cell thereof of the application can be further modified by introducing an exogenous polynucleotide encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G). In particular, disruption of the B2M gene eliminates surface expression of all MHC class I molecules, leaving cells vulnerable to lysis by NK cells through the “missing self” response. Exogenous HLA-E expression can lead to resistance to NK-mediated lysis (Gornalusse et al., Nat Biotechnol. 2017; 35(8): 765–772). Incorporating MHC I and/or MHC II knock-out and/or knock down in the cells for use in “allogeneic” cell therapies will allow the cell product candidates to escape recognition and destruction by the host immune system. The reduction in allogeneic reactivity enabled by use of this technology will allow repeat dosing of the CAR-modified cell therapies to improve their therapeutic potential. In combination with the extended killing capability of optimized immune cells derived from single genetically engineered cell cloning, the cells will have the capacity for repeat dosing to maximize durability of response and efficacy. Additionally, this technology may permit dosing in patients with limited or no immune preconditioning regimens. Accordingly, in certain embodiments, an iPSC or derivative cell thereof of the application can be further modified by introducing a third exogenous polynucleotide sf-6638784 83 32429-20001.40 encoding one or more proteins related to immune evasion, such as non-classical HLA class I proteins (e.g., HLA-E and HLA-G). In certain embodiments, the iPSC or derivative cell thereof comprises a third exogenous polypeptide encoding at least one of a human leukocyte antigen E (HLA- E) and human leukocyte antigen G (HLA-G). In a particular embodiment, the HLA-E comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 65, preferably the amino acid sequence of SEQ ID NO: 65. In a particular embodiment, the HLA-G comprises an amino acid sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 68, preferably SEQ ID NO: 68. In certain embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-E via a linker. In a particular embodiment, the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 66. In other embodiments, the third exogenous polynucleotide encodes a polypeptide comprising a signal peptide operably linked to a mature B2M protein that is fused to an HLA-G via a linker. In a particular embodiment, the third exogenous polypeptide comprises an amino acid sequence at least sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 69. V. Other Optional Genome Edits In one embodiment of the above described cell, the genomic editing at one or more selected sites may comprise insertions of one or more exogenous polynucleotides encoding other additional artificial cell death polypeptides, targeting modalities, receptors, signaling molecules, transcription factors, pharmaceutically active proteins and peptides, drug target candidates, or proteins promoting engraftment, trafficking, homing, viability, self-renewal, persistence, and/or survival of the genome-engineered iPSCs or derivative cells thereof. In some embodiments, the exogenous polynucleotides for insertion are operatively linked to (1) one or more exogenous promoters comprising CMV, Efla, sf-6638784 84 32429-20001.40 PGK, CAG, UBC, or other constitutive, inducible, temporal-, tissue-, or cell type- specific promoters; or (2) one or more endogenous promoters comprised in the selected sites comprising AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1, or other locus meeting the criteria of a genome safe harbor. In some embodiments, the genome-engineered iPSCs generated using the above method comprise one or more different exogenous polynucleotides encoding proteins comprising caspase, thymidine kinase, cytosine deaminase, B-cell CD20, ErbB2 or CD79b wherein when the genome-engineered iPSCs comprise two or more suicide genes, the suicide genes are integrated in different safe harbor locus comprising AAVSl, CCR5, ROSA26, collagen, HTRP, Hll, Hll, beta-2 microglobulin, GAPDH, TCR or RUNX1. Other exogenous polynucleotides encoding proteins may include those encoding PET reporters, homeostatic cytokines, and inhibitory checkpoint inhibitory proteins such as PD1, PD-L1, and CTLA4 as well as proteins that target the CD47/signal regulatory protein alpha (SIRPα) axis. In some other embodiments, the genome-engineered iPSCs generated using the method provided herein comprise in/del at one or more endogenous genes associated with targeting modality, receptors, signaling molecules, transcription factors, drug target candidates, immune response regulation and modulation, or proteins suppressing engraftment, trafficking, homing, viability, self-renewal, persistence, and/or survival of the iPSCs or derivative cells thereof. In some aspects, the present disclosure describes genetically engineered iPSCs and cells derived therefrom that exogenously express recombinant CD16 and recombinant NKG2D. In some aspects, such cells also express one or more CARs (e.g., a CAR comprising a CD123 antigen-binding domain. For example, in some embodiments, such cells may express a single CAR comprising one or more antigen- binding domains that bind CD123 and another antigen. In another example, such cells may express two or more CARs, wherein each CAR comprises an antigen- binding domain that binds a tumor antigen, wherein one of the tumor antigens is CD123. Described herein is a method for exogenously expressing or overexpressing CD16 and NKG2D proteins and transgenes in cells, as well as such cells and therapeutic uses thereof. The surface receptor CD16 (FcγRIIIA) affects human NK cells during maturation. NK cells bind the Fc portion of IgG via CD16, and execute sf-6638784 85 32429-20001.40 ADCC, which is critical for the effectiveness of several anti-tumor monoclonal antibody therapies. NKG2D is a stimulatory/activating receptor that is mostly expressed on cells of the cytotoxic arm of the immune system including NK cells and subsets of T cells. NKG2D is crucial in diverse aspects of innate and adaptive immune functions. In some embodiments, CD16 and NKG2D are expressed from in a single polynucleotide construct as it is advantageous to reduce the number of gene edits of a cell. In certain aspects, provided is an iPSC or derivative cell thereof containing an exogenous or isolated polynucleotide construct encoding a CD16 protein and an NKG2D protein. In some embodiments, described herein is an iPSC or derivative cell thereof expressing recombinant CD16 proteins and recombinant NKG2D proteins. In some embodiments, the recombinant proteins are encoded by an exogenous or isolated polynucleotide construct. In some embodiments, the polynucleotide construct encoding the CD16 protein and the NKG2D protein also includes a polynucleotide sequence encoding an autoprotease peptide or self-cleaving peptide. In some embodiments, an exogenous polynucleotide construct encoding the CD16 protein, the NKG2D protein and the self-cleaving peptide is introduced into the iPSC or derivative cell thereof. The exogenous or isolated polynucleotide construct can be introduced into a gene locus of the iPSC or derivative cell thereof. In some embodiments, the iPSC or derivative cell thereof expressing recombinant CD16 proteins and recombinant NKG2D proteins also expresses chimeric antigen receptors (CARs). In some embodiments, the cell expressing recombinant CD16 proteins and recombinant NKG2D proteins also expresses either recombinant HLA-E, HLA-G, or both. In several embodiments, the iPSC or derivative cell thereof expressing recombinant CD16 proteins and recombinant NKG2D proteins also expresses CARs and either recombinant HLA-E, HLA-G, or both. In many embodiments, the cell expressing recombinant CD16 proteins, recombinant NKG2D proteins and CARs also expresses recombinant IL-15 proteins. In many embodiments, the cell expresses recombinant CD16 proteins, recombinant NKG2D proteins, CARs, recombinant IL-15 proteins, and either recombinant HLA-E, HLA-G, or both. In many embodiments, the cell expressing recombinant CD16 proteins, recombinant NKG2D proteins and CARs also expresses recombinant fusion proteins containing IL-15 and IL-15Ra. In many embodiments, the cell expresses recombinant sf-6638784 86 32429-20001.40 CD16 proteins, recombinant NKG2D proteins, CARs, recombinant fusion proteins containing IL-15 and IL-15Ra, and either recombinant HLA-E, HLA-G, or both. In some embodiments, the cell expressing recombinant CD16 proteins and recombinant NKG2D proteins also expresses recombinant IL-15 proteins. In some embodiments, the cell expressing recombinant CD16 proteins and recombinant NKG2D proteins also expresses recombinant fusion proteins containing IL-15 and IL-15Ra. In some embodiments, the cell expressing recombinant CD16 proteins, recombinant NKG2D proteins, and recombinant IL-15 proteins also expresses CARs. In some embodiments, the cell expressing recombinant CD16 proteins, recombinant NKG2D proteins, and recombinant fusion proteins containing IL-15 and IL-15Ra also expresses CARs. In one aspect, provided is an exogenous or isolated polynucleotide construct encoding a CD16 protein and an NKG2D protein. In some embodiments of the exogenous polynucleotide construct, the polynucleotide sequence encoding a CD16 protein and the polynucleotide sequence encoding an NKG2D protein are operably linked by a polynucleotide sequence encoding an autoprotease peptide or self- cleaving peptide. In some embodiments, the polynucleotide construct includes from 5’ to 3’ end: a polynucleotide sequence encoding a CD16 protein, a polynucleotide sequence encoding an autoprotease peptide or self-cleaving peptide and a polynucleotide sequence encoding a NKG2D protein. In some embodiments, the polynucleotide construct includes from 5’ to 3’ end: a polynucleotide sequence encoding an NKG2D protein, a polynucleotide sequence encoding an autoprotease peptide or self-cleaving peptide and a polynucleotide sequence encoding a CD16 protein. In some embodiments, the exogenous polynucleotide construct comprises the nucleic acid sequence of SEQ ID NO: 184. In some embodiments, the exogenous polynucleotide construct encodes for the amino acid sequence of SEQ ID NO: 185. In some embodiments, the CD16 protein (which is also referred to as “low affinity immunoglobulin gamma Fc region receptor III-A” or “Fc gamma receptor IIIa”) is a wildtype CD16 protein. In some embodiments, the human wildtype CD16 protein has the amino acid sequence set forth in NCBI Ref. Seq. No. NP_000560.7 or UniProt No. P08637. In some instance, the coding sequence of human wildtype CD16 is set forth in NCBI Ref. No. NM_000569.8. In some embodiments, the CD16 protein is a CD16 variant protein. In some instances, the CD16 variant protein comprises amino acids having at least 90%, e.g., sf-6638784 87 32429-20001.40 at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to wildtype CD16 such as that of SEQ ID NO: 186. In some instances, the CD16 variant is a high affinity CD16 variant. In other instances, the CD16 variant is a non-cleavable CD16 variant. In some instances, the CD16 variant is a high affinity and non-cleavable CD16 variant. In some embodiments, the CD16 variant comprises one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has an F158V substitution and one or more substitutions selected from F176V, S197P, D205A, S219A, T220A, and any combination thereof. In one embodiment, the CD16 variant has an F176V substitution and one or more substitutions selected from F158V, S197P, D205A, S219A, T220A, and any combination thereof. In many embodiments, the CD16 variant has an S197P, substitution and one or more substitutions selected from F158V, F176V, D205A, S219A, T220A, and any combination thereof. In various embodiments, the CD16 variant has a D205A substitution and one or more substitutions selected from F158V, F176V, S197P, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has a substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the CD16 variant has an S219A substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, T220A, and any combination thereof. In some embodiments, the CD16 variant has a T220A substitution and one or more substitutions selected from F158V, F176V, S197P, D205A, S219A, T220A, and any combination thereof. In some embodiments, the variant CD16 protein has the sequence of SEQ ID NO: 187. In some embodiments, the nucleic acid sequence encoding the variant CD16 protein has the sequence of SEQ ID NO: 188. In some embodiments, the wildtype CD16 protein has the sequence of SEQ ID NO: 186. In some embodiments, the NKG2D protein (which is also referred to as NKG2-D type II integral membrane protein, CD314, killer cell lectin-like receptor subfamily K1 member 1 or KLRK1) is a wildtype NKG2D protein. In some embodiments, the human wildtype NKG2D protein has the amino acid sequence set forth in NCBI Ref. Seq. Nos. NP_001186734.1 or NP_031386.2 or UniProt No. P26718. In some instance, the coding sequence of human wildtype NKG2D is set sf-6638784 88 32429-20001.40 forth in NCBI Ref. Nos. NM_001199805.1 or NM_007360.3. In some embodiments, the NKG2D protein is a NKG2D variant protein. In some instances, the NKG2D variant protein comprises amino acids having at least 90%, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to wildtype NKG2D such as that of SEQ ID NO: 189. In some embodiments, the NKG2D protein has the amino acid sequence of SEQ ID NO: 189. In some embodiments, the nucleic acid sequence encoding the NKG2D protein has sequence of SEQ ID NO: 190. As discussed above, provided herein are constructs containing autoprotease peptide sequences including 2A peptides that can induce ribosomal skipping during translation of a polypeptide. 2A peptides function to “cleave” an mRNA transcript by making the ribosome skip the synthesis of a peptide bond at the C-terminus, between the glycine (G) and proline (P) residues, thereby leading to separation between the end of the 2A sequence and the next peptide downstream. 2A peptides include, but are not limited to, a porcine tesehovirus-12A (P2A) peptide, a foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide. An exemplary P2A peptide can comprise amino acids having a sequence that has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 73 or 191. In some embodiment, the P2A peptide has the amino acid sequence of SEQ ID NO: 73 or 191. VI. CXCR4 Expression The terms “C-X-C CHEMOKINE RECEPTOR TYPE 4” and “CXCR4” refer to a G protein-coupled receptor that binds the chemokine CXCL12. It plays important roles in embryogenesis, hematopoiesis, organogenesis, vascularization, and tumorigenesis. The wild type CXCR4 receptor contains 352 amino acids and has seven transmembrane domains, with an extracellular N-terminus and intracellular C- terminus. Upon binding CXCL12, CXCR4 activates downstream signaling pathways such as Ras/MAPK and PI3K/Akt that regulate cell migration, proliferation, and survival. There are several known naturally occurring mutations in the CXCR4 gene that result in altered receptor function. For example, the WHIM syndrome mutation CXCR4-E343K leads to impaired receptor downregulation and enhanced cellular sf-6638784 89 32429-20001.40 responses to CXCL12. Patients with WHIM syndrome exhibit warts, hypogammaglobulinemia, recurrent bacterial infections, and myelokathexis (retention of mature neutrophils in bone marrow). The enhanced CXCL12/CXCR4 signaling causes abnormal neutrophil retention and B cell lymphopenia. Another noteworthy CXCR4 mutation is CXCR4-R334X, which causes truncation of the C-terminal tail of the receptor. This mutation impairs CXCL12-mediated chemotaxis and calcium flux, highlighting the importance of the C-tail in G protein coupling and downstream signaling. Truncation of the CXCR4 C-tail (CXCR4-T328) can reduce CXCL12 binding affinity but maintains signaling capacity. Mutating two extracellular loop glutamic acid residues (CXCR4-E179A/E262A) impairs CXCL12 binding and signaling. Substitution of the conserved DRY motif in the second intracellular loop (CXCR4-R144A) also abrogates CXCL12-induced signaling. In addition to natural and engineered mutations, CXCR4 signaling capacity and expression levels are affected in various disease states. For example, in warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, mutations lead to increased surface expression and impaired downregulation of CXCR4. In cancer, CXCR4 is upregulated in at least 23 different types of malignancies. The increased CXCR4 expression in cancer cells promotes metastasis and angiogenesis. Thus, CXCR4 is an important therapeutic target in both WHIM syndrome and various cancers. CXCR4 is also known to be specific to the chemokine stromal-derived-factor- 1 (SDF-1). Interaction of CXCR4 with SDF-1 can be involved in chemotaxis of lymphocytes specifically important for hematopoietic stem cell homing to bone marrow. Enforced human CXCR4 expression in primary derived NK or T cells has been shown to improve homing of the cells to the bone marrow. The present disclosure provides embodiments in which wild type human CXCR4 has been introduced into iPSC-derived cells expressing a CAR, demonstrating that the expression of CXCR4 in differentiated iPSC-derived effector cells (i) improves both homing of the effector cells to the bone marrow (e.g., in mice), and (ii) significantly improves tumor killing (e.g., in a Human leukemia xenograft mouse model). In certain embodiments, a primary cell, an iPSC or a derivative cell thereof according to the present disclosure can comprise one or more exogenous polynucleotides encoding CXCR4, and the CXCR4 or a fragment or variant thereof is selected from the group consisting of wild-type CXCR4 and a mutated variant of sf-6638784 90 32429-20001.40 CXCR4. In certain embodiments, the CXCR4 is wild-type CXCR4. In certain embodiments, the CXCR4 is wild-type CXCR4 and comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 192. In certain embodiments, the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 200. In other embodiments, the CXCR4 is a mutated variant of CXCR4. In certain embodiments, the CXCR4 is a mutated variant of CXCR4 and the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with a deletion of the C- terminal domain between 10 and 20 amino acid residues. In a particular embodiment, a CXCR4 with the deletion in the C-terminal domain corresponds to the deletion of the 10, 11, 12, 13, 14, 15, 16, 17, 18, 16, 17, 18, 19, or 20 amino acid residues in the C-terminal domain of the CXCR4 polypeptide. In certain embodiments, the CXCR4 is a mutated variant of CXCR4 and the mutated variant of CXCR4 comprises wild- type CXCR4 according to SEQ ID NO: 192 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A. In certain embodiments, the CXCR4 is a mutated variant of CXCR4 and the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 192-199. In some embodiments, the CXCR4 mutation is a CXCR4whim mutation. The term "CXCR4whim mutation" refers to the autosomal dominant mutation associated with the rare combined primary immunodeficiency Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) Syndrome (WS) which have been linked to inherited autosomal dominant gain-of-function mutations in CXCR4 (see, e.g., Kawai, T. & Malech, H. L. WHIM syndrome: congenital immune deficiency disease. Current Opinion in Hematology 16, 20-26 (2009); and Dotta, L., Tassone, L. & Badolato, R. Clinical and genetic features of Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) syndrome. Curr. Mol. Med. 11, 317-325 (2011), each of which is hereby incorporated by reference herein in its entirety). These mutations result in the distal truncation of the C-term of CXCR4 and a desensitization- and internalization-resistant receptor in response to CXCL12 (see, e.g., Hernandez, P. A. et al. Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease. Nat Genet 34, 70-74 (2003); and sf-6638784 91 32429-20001.40 Balabanian, K. et al. Proper desensitization of CXCR4 is required for lymphocyte development and peripheral compartmentalization in mice. Blood 119, 5722-5730 (2012), each of which is hereby incorporated by reference herein in its entirety). Patients also exhibit a severe, chronic pan-leukopenia with neutrophils, naive T cells and mature recirculating B cells being most affected (see, e.g., Gulino, A. V. Altered leukocyte response to CXCL12 in patients with warts hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome. Blood 104, 444-452 (2004), which is hereby incorporated by reference herein in its entirety). The different autosomal dominant gain-of-function mutations in CXCR4 associated with Whim syndrome include: (i) R334X (gene mutation 1000C>T) the most frequent mutation resulting in carboxy- terminal truncation of 19 residues (Hernandez, et al.); (ii) G336X, (gene mutation 1000C>T) resulting in carboxy-terminus truncation of 17 residues (Gulino et al., 2004); (iii) E343X (gene mutation 1027G>T) resulting in carboxy-terminus truncation of the receptor of 10 residues (Hernandez, et al.); (iv) S339fs342X, (gene mutation 1016-1017delCT) resulting in carboxy-terminus truncation of the receptor of 13 residues and addition of 3 additional amino acid residues (Hernandez, et al.; and Liu, Q. et al. WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4. Blood 120, 181-189 (2012), which is hereby incorporated by reference herein in its entirety); (v) S338X (gene mutation 1013OG), resulting in carboxy-terminus truncation of the receptor of 12 residues (Balabanian, K. et al. WHIM syndromes with different genetic anomalies are accounted for by impaired CXCR4 desensitization to CXCL12. Blood 105, 2449- 2457 (2005), which is hereby incorporated by reference herein in its entirety); and (vi) E343K (nucleic acid mutation 1027G>A) (Liu, et al.). The different autosomal dominant gain-of-function mutations in CXCR4 associated with Whim syndrome are located in the C-terminal domain of the CXCR4 receptor (see Table 3), a domain responsible for regulation of the receptor (internalization / inactivation). Accordingly, the C-terminal domain of the CXCR4 receptor, which is deleted in several Whim mutation could also be directly deleted to obtain the same biological effect (gain-of- function of CXCR4 associated with the long term CD8 memory response). Biological activity of CD8 cells according to the invention (gain-of-function of CXCR4) can be measured for example with chemokine receptor internalization assay or cell migration after adding a CXCR4 agonist. sf-6638784 92 32429-20001.40 Table 3. 93 sf-6638784 32429-20001.40 sf-6638784 94 32429-20001.40 sf-6638784 95 32429-20001.40 VII. Targeted Genome Editing at Selected Locus in iPSCs According to embodiments of the application, one or more of the exogenous polynucleotides are integrated at one or more loci on the chromosome of an iPSC. Genome editing, or genomic editing, or genetic editing, as used interchangeably herein, is a type of genetic engineering in which DNA is inserted, deleted, and/or replaced in the genome of a targeted cell. Targeted genome editing (interchangeable with “targeted genomic editing” or “targeted genetic editing”) enables insertion, deletion, and/or substitution at pre-selected sites in the genome. When an endogenous sequence is deleted or disrupted at the insertion site during targeted editing, an endogenous gene comprising the affected sequence can be knocked-out or knocked-down due to the sequence deletion or disruption. Therefore, targeted editing can also be used to disrupt endogenous gene expression with precision. Similarly used herein is the term “targeted integration,” referring to a process involving insertion of one or more exogenous sequences at pre-selected sites in the genome, with or without deletion of an endogenous sequence at the insertion site. Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach. In the nuclease-independent targeted editing approach, homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be inserted, through the enzymatic machinery of the host cell. Alternatively, targeted editing could be achieved with higher frequency through specific introduction of double strand breaks (DSBs) by specific rare-cutting endonucleases. Such nuclease-dependent targeted editing utilizes DNA repair mechanisms including non-homologous end joining (NHEJ), which occurs in response to DSBs. Without a donor vector containing exogenous genetic material, the NHEJ often leads to random insertions or deletions (in/dels) of a small number of endogenous nucleotides. In comparison, when a donor vector containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome during homology directed repair (HDR) by homologous recombination, resulting in a “targeted integration.” Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), RNA-guided CRISPR (Clustered Regular Interspaced 96 sf-6638784 32429-20001.40 Short Palindromic Repeats) systems. Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases is also a promising tool for targeted integration. ZFNs are targeted nucleases comprising a nuclease fused to a zinc finger DNA binding domain. By a “zinc finger DNA binding domain” or “ZFBD” it is meant a polypeptide domain that binds DNA in a sequence-specific manner through one or more zinc fingers. A zinc finger is a domain of about 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include, but not limited to, C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A “designed” zinc finger domain is a domain not occurring in nature whose design/composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496. A “selected” zinc finger domain is a domain not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. ZFNs are described in greater detail in U.S. Pat. No. 7,888,121 and U.S. Pat. No. 7,972,854, the complete disclosures of which are incorporated herein by reference. The most recognized example of a ZFN in the art is a fusion of the Fokl nuclease with a zinc finger DNA binding domain. A TALEN is a targeted nuclease comprising a nuclease fused to a TAL effector DNA binding domain. By “transcription activator-like effector DNA binding domain”, “TAL effector DNA binding domain”, or “TALE DNA binding domain” it is meant the polypeptide domain of TAL effector proteins that is responsible for binding of the TAL effector protein to DNA. TAL effector proteins are secreted by plant pathogens of the genus Xanthomonas during infection. These proteins enter the nucleus of the plant cell, bind effector-specific DNA sequences via their DNA binding domain, and activate gene transcription at these sequences via their transactivation domains. TAL effector DNA binding domain specificity depends on an effector-variable number of imperfect 34 amino acid repeats, which comprise polymorphisms at select repeat positions called repeat variable-diresidues (RVD). TALENs are described in greater detail in U.S. Patent Application No. 2011/0145940, which is herein incorporated by reference. The most recognized example of a TALEN sf-6638784 97 32429-20001.40 in the art is a fusion polypeptide of the Fokl nuclease to a TAL effector DNA binding domain. Another example of a targeted nuclease that finds use in the subject methods is a targeted Spoll nuclease, a polypeptide comprising a Spol l polypeptide having nuclease activity fused to a DNA binding domain, e.g., a zinc finger DNA binding domain, a TAL effector DNA binding domain, etc. that has specificity for a DNA sequence of interest. See, for example, U.S. Application No. 61/555,857, the disclosure of which is incorporated herein by reference. Additional examples of targeted nucleases suitable for the present application include, but not limited to Bxbl, phiC3 l, R4, PhiBTl, and Wp/SPBc/TP90l-l, whether used individually or in combination. Other non-limiting examples of targeted nucleases include naturally occurring and recombinant nucleases; CRISPR related nucleases from families including cas, cpf, cse, csy, csn, csd, cst, csh, csa, csm, and cmr; restriction endonucleases; meganucleases; homing endonucleases, and the like. As an example, CRISPR/Cas9 requires two major components: (1) a Cas9 endonuclease and (2) the crRNA- tracrRNA complex. When co-expressed, the two components form a complex that is recruited to a target DNA sequence comprising PAM and a seeding region near PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cas9 to target selected sequences. These two components can then be delivered to mammalian cells via transfection or transduction. As another example, CRISPR/Cpf1 comprises two major components: (1) a CPf1 endonuclease and (2) a crRNA. When co-expressed, the two components form a ribonucleoprotein (RNP) complex that is recruited to a target DNA sequence comprising PAM and a seeding region near PAM. The crRNA can be combined to form a chimeric guide RNA (gRNA) to guide Cpf1 to target selected sequences. These two components can then be delivered to mammalian cells via transfection or transduction. MAD7 is an engineered Cas12a variant originating from the bacterium Eubacterium rectale that has a preference for 5′-TTTN-3′ and 5′-CTTN-3′ PAM sites and does not require a tracrRNA. See, for example, PCT Publication No. 2018/236548, the disclosure of which is incorporated herein by reference. DICE mediated insertion uses a pair of recombinases, for example, phiC31 and Bxbl, to provide unidirectional integration of an exogenous DNA that is tightly restricted to each enzyme’s own small attB and attP recognition sites. Because these sf-6638784 98 32429-20001.40 target att sites are not naturally present in mammalian genomes, they must be first introduced into the genome, at the desired integration site. See, for example, U.S. Application Publication No. 2015/0140665, the disclosure of which is incorporated herein by reference. One aspect of the present application provides a construct comprising one or more exogenous polynucleotides for targeted genome integration. In one embodiment, the construct further comprises a pair of homologous arms specific to a desired integration site, and the method of targeted integration comprises introducing the construct to cells to enable site specific homologous recombination by the cell host enzymatic machinery. In some embodiments, the pair of homologous arms (e.g., left homology arm and right homology arm) are any of the left homology arms and right homology arms provided in Section VII.C or Table 4. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a ZFN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a ZFN-mediated insertion. In yet another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing a TALEN expression cassette comprising a DNA-binding domain specific to a desired integration site to the cell to enable a TALEN-mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cpf1 expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cpf1-mediated insertion. In another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more exogenous polynucleotides to the cell, introducing a Cas9 expression cassette, and a gRNA comprising a guide sequence specific to a desired integration site to the cell to enable a Cas9-mediated insertion. In still another embodiment, the method of targeted integration in a cell comprises introducing a construct comprising one or more att sites of a pair of DICE recombinases to a desired integration site in the cell, introducing a construct comprising one or more exogenous polynucleotides to the cell, and introducing an expression cassette for DICE recombinases, to enable DICE- mediated targeted integration. sf-6638784 99 32429-20001.40 In some embodiments, the guide sequence specific to a desired integration site is provided in Table 4. In one embodiment, the guide sequence is specific to an AAVS1 locus and set forth in SEQ ID NO: 225. In one embodiment, the guide sequence is specific to an AAVS1 locus and set forth in SEQ ID NO: 94. In one embodiment, the guide sequence is specific to a B2M locus and set forth in SEQ ID NO: 226 or SEQ ID NO: 227. In one embodiment, the guide sequence is specific to a B2M locus and set forth in SEQ ID NO: 238. In one embodiment, the guide sequence is specific to a B2M locus and set forth in SEQ ID NO: 93. In one embodiment, the guide sequence is specific to a CIITA locus and set forth in SEQ ID NO: 228. In one embodiment, the guide sequence is specific to a CIITA locus and set forth in SEQ ID NO: 229. In one embodiment, the guide sequence is specific to a CIITA locus and set forth in SEQ ID NO: 95. In one embodiment, the guide sequence is specific to a CD70 locus and set forth in SEQ ID NO: 230. In one embodiment, the guide sequence is specific to a CLYBL locus and set forth in SEQ ID NO: 231. In one embodiment, the guide sequence is specific to a NKG2A locus and set forth in SEQ ID NO: 232. In one embodiment, the guide sequence is specific to a TRAC locus and set forth in SEQ ID NO: 233. In one embodiment, the guide sequence is specific to a CD38 locus and set forth in SEQ ID NO: 234. In one embodiment, the guide sequence is specific to a CD33 locus and set forth in SEQ ID NO: 235 or SEQ ID NO: 236. In one embodiment, the guide sequence is specific to an IL2 locus and set forth in SEQ ID NO: 237. Sites for targeted integration include, but are not limited to, genomic safe harbors, which are intragenic or extragenic regions of the human genome that, theoretically, are able to accommodate predictable expression of newly integrated DNA without adverse effects on the host cell or organism. In certain embodiments, the genome safe harbor for the targeted integration is one or more loci of genes selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes. In other embodiments, the site for targeted integration is selected for deletion or reduced expression of an endogenous gene at the insertion site. As used herein, the term “deletion” with respect to expression of a gene refers to any genetic modification that abolishes the expression of the gene. Examples of “deletion” of expression of a gene include, e.g., a removal or deletion of a DNA sequence of the gene, an insertion sf-6638784 100 32429-20001.40 of an exogenous polynucleotide sequence at a locus of the gene, and one or more substitutions within the gene, which abolishes the expression of the gene. Genes for target deletion include, but are not limited to, genes of major histocompatibility complex (MHC) class I and MHC class II proteins. Multiple MHC class I and class II proteins must be matched for histocompatibility in allogeneic recipients to avoid allogeneic rejection problems. “MHC deficient”, including MHC- class I deficient, or MHC-class II deficient, or both, refers to cells that either lack, or no longer maintain, or have reduced level of surface expression of a complete MHC complex comprising a MHC class I protein heterodimer and/or a MHC class II heterodimer, such that the diminished or reduced level is less than the level naturally detectable by other cells or by synthetic methods. MHC class I deficiency can be achieved by functional deletion of any region of the MHC class I locus (chromosome 6p2l), or deletion or reducing the expression level of one or more MHC class-I associated genes including, not being limited to, beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene and Tapasin genes. For example, the B2M gene encodes a common subunit essential for cell surface expression of all MHC class I heterodimers. B2M null cells are MHC-I deficient. MHC class II deficiency can be achieved by functional deletion or reduction of MHC-II associated genes including, not being limited to, RFXANK, CIITA, RFX5 and RFXAP. CIITA is a transcriptional coactivator, functioning through activation of the transcription factor RFX5 required for class II protein expression. CIITA null cells are MHC-II deficient. In certain embodiments, one or more of the exogenous polynucleotides are integrated at one or more loci of genes selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby delete or reduce the expression of the gene(s) with the integration. In certain embodiments, the exogenous polynucleotides are integrated at one or more loci on the chromosome of the cell, preferably the one or more loci are of genes selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hl l, GAPDH, RUNX1, B2M, TAPI, 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, or TIGIT genes, provided at least one of the one or more loci is of a MHC gene, such as a gene selected from the group consisting of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. Preferably, the one or more exogenous polynucleotides are integrated sf-6638784 101 32429-20001.40 at a locus of an MHC class-I associated gene, such as a beta-2 microglobulin (B2M) gene, TAP 1 gene, TAP 2 gene or Tapasin gene; and at a locus of an MHC-II associated gene, such as a RFXANK, CIITA, RFX5, RFXAP, or CIITA gene; and optionally further at a locus of a safe harbor gene selected from the group consisting of AAVS1, CCR5, ROSA26, collagen, HTRP, Hll, GAPDH, TCR and RUNX1 genes. More preferably, the one or more of the exogenous polynucleotides are integrated at the loci of CIITA, AAVS1 and B2M genes. In certain embodiments, (i) the first exogenous polynucleotide is integrated at a locus of AAVS1 gene; (ii) the second exogenous polypeptide is integrated at a locus of CIITA gene; and (iii) the third exogenous polypeptide is integrated at a locus of B2M gene; wherein integrations of the exogenous polynucleotides delete or reduce expression of CIITA and B2M genes. In certain embodiments, (i) the first exogenous polynucleotide comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or both of SEQ ID NOs: 174 and 175; (ii) the second exogenous polynucleotide comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 75; and (iii) the third exogenous polynucleotide comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 67. In certain embodiments, (i) the first exogenous polynucleotide comprises polynucleotides having the sequence of one or both of SEQ ID NOs: 174 and 175; (ii) the second exogenous polynucleotide comprises polynucleotides having the sequence of SEQ ID NO: 75; and (iii) the third exogenous polynucleotide comprises polynucleotides having the sequence of SEQ ID NO: 67. B. Derivative Cells In another aspect, the invention relates to a cell derived from differentiation of an iPSC, a derivative cell. As described above, the genomic edits introduced into the iPSC cell are retained in the derivative cell. In certain embodiments of the derivative cell obtained from iPSC differentiation, the derivative cell is a hematopoietic cell, including, but not limited to, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, B cells, antigen presenting cells (APC), monocytes and sf-6638784 102 32429-20001.40 macrophages. In certain embodiments, the derivative cell is an immune effector cell, such as a NK cell or a T cell. In certain embodiments, the application provides a NK cell or a T cell comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) a deletion or reduced expression of an MHC class I associated gene and an MHC class II associated gene, such as an MHC class-I associated gene selected from the group consisting of a B2M gene, TAP 1 gene, TAP 2 gene and Tapasin gene, and an MHC-II associated gene selected from the group consisting of a RFXANK gene, CIITA gene, RFX5 gene, RFXAP gene, and CIITA gene, preferably the B2M gene and CIITA gene; and optionally (iii) an exogenous polynucleotide encoding a chimeric IL-15RA and an IL-15, wherein the IL-15RA and IL-15 are operably linked. In certain embodiments, the NK cell or T cell further comprises an exogenous polynucleotide encoding at least one of a human leukocyte antigen E (HLA-E) and a human leukocyte antigen G (HLA-G). Also provided is a NK cell or a T cell comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) having the amino acid sequence of one or both of SEQ ID NOs: 172 and 173; (ii) an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) having the amino acid sequence of SEQ ID NO: 66; and optionally (iii) an exogenous polynucleotide encoding a chimeric IL-15RA and an interleukin-15 (IL-15), wherein the IL-15RA and IL-15 are operably linked, having the amino acid sequence of SEQ ID NO:247, and wherein the exogenous polynucleotides are integrated at loci of AAVS1, B2M, and CIITA genes, respectively, to thereby delete or reduce expression of CIITA and B2M. In certain embodiments, the first exogenous polynucleotide comprises polynucleotides having the sequence of one or both of SEQ ID NOs: 174 and 175; the second exogenous polynucleotide comprises polynucleotides having the sequence of SEQ ID NO: 75; and the third exogenous polynucleotide comprises polynucleotides having the sequence of SEQ ID NO: 67. Also provided is a CD34+ hematopoietic progenitor cell (HPC) derived from an induced pluripotent stem cell (iPSC) comprising: (i) an exogenous polynucleotide encoding a chimeric antigen receptor (CAR); (ii) an exogenous polynucleotide encoding an inactivated cell surface receptor that comprises a monoclonal antibody- specific epitope and an IL-15, wherein the inactivated cell surface receptor and IL-15 sf-6638784 103 32429-20001.40 are operably linked by an autoprotease peptide sequence; and (iii) a deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. In certain embodiments, the CD34+ HPC further comprises an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and/or human leukocyte antigen G (HLA-G). In certain embodiments, the CAR comprises (i) optionally, a signal peptide; (ii) an extracellular domain comprising a binding domain that specifically binds to CD123; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and (vi) a co-stimulatory domain, such as a co-stimulatory domain comprising a CD28 signaling domain. Also provided is a method of manufacturing the derivative cell. The method comprises differentiating the iPSC under conditions for cell differentiation to thereby obtain the derivative cell. An iPSC of the application can be differentiated by any method known in the art. Exemplary methods are described in US8846395, US8945922, US8318491, WO2010/099539, WO2012/109208, WO2017/070333, WO2017/179720, WO2016/010148, WO2018/048828 and WO2019/157597, each of which are herein incorporated by reference in its entirety. The differentiation protocol may use feeder cells or may be feeder-free. As used herein, “feeder cells” or “feeders” are terms describing cells of one type that are co-cultured with cells of a second type to provide an environment in which the cells of the second type can grow, expand, or differentiate, as the feeder cells provide stimulation, growth factors and nutrients for the support of the second cell type. In another embodiment of the invention, the iPSC derivative cells of the invention are NK cells which are prepared by a method of differentiating an iPSC cell into an NK cell by subjecting the cells to a differentiation protocol including the addition of recombinant human IL-12p70 for the final 24 hours of culture. By including the IL-12 in the differentiation protocol, cells that are primed with IL-12 demonstrate more rapid cell killing compared to those that are differentiated in the absence of IL-12. In addition, the cells differentiated using the IL-12 conditions demonstrate improved cancer cell growth inhibition. C. Polynucleotides, vectors, and host cells sf-6638784 104 32429-20001.40 Table 4 provides an exemplary list of homology arm sequences (left homology arm (LHA) and right homology arm (RHA)) and corresponding guide sequences for facilitating integration of an exogenous polynucleotide at various loci. sf-6638784 105 32429-20001.40 Table 4. 106 sf-6638784 32429-20001.40 sf-6638784 107 32429-20001.40 sf-6638784 108 32429-20001.40 sf-6638784 109 32429-20001.40 sf-6638784 110 32429-20001.40 sf-6638784 111 32429-20001.40 sf-6638784 112 32429-20001.40 sf-6638784 113 32429-20001.40 sf-6638784 114 32429-20001.40 sf-6638784 115 32429-20001.40 sf-6638784 116 32429-20001.40 sf-6638784 117 32429-20001.40 sf-6638784 118 32429-20001.40 sf-6638784 119 32429-20001.40 B sf-6638784 120 32429-20001.40 (1) Nucleic acids encoding a CAR In another general aspect, the invention relates to an isolated nucleic acid encoding a chimeric antigen receptor (CAR) useful for an invention according to embodiments of the application. It will be appreciated by those skilled in the art that the coding sequence of a CAR can be changed (e.g., having nucleotides substituted) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding CARs of the application can be altered without changing the amino acid sequences of the proteins. In certain embodiments, the isolated nucleic acid encodes a CAR targeting CD123. In a particular embodiment, the isolated nucleic acid encoding the CAR comprises a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to one of SEQ ID NOs: 174 and 175. In another general aspect, the application provides a vector comprising a polynucleotide encoding a CAR useful for an invention according to embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a CAR in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application. In a particular aspect, the application provides vectors for targeted integration of a CAR useful for an invention according to embodiments of the application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding a CAR according to an embodiment of the application; and (c) a terminator/polyadenylation signal. 121 sf-6638784 32429-20001.40 In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 63. Other promoters can also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta actin. In certain embodiments, the terminator/ polyadenylation signal is a SV40 signal. In certain embodiments, the SV40 signal is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 64. Other terminator sequences can also be used, examples of which include, but are not limited to, BGH, hGH, and PGK. In certain embodiments, the polynucleotide sequence encoding a CAR comprises polynucleotides having a sequence that is at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to one or more of SEQ ID NOs: 149-171, 174, and 175. In some embodiment, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide. As used herein, “left homology arm” and “right homology arm” refers to a pair of nucleic acid sequences that flank an exogenous polynucleotide and facilitate the integration of the exogenous polynucleotide into a specified chromosomal locus. Sequences of the left and right arm homology arms can be designed based on the integration site of interest. In some embodiment, the left or right arm homology arm is homologous to the left or right side sequence of the integration site. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 90. In certain embodiments, the left homology arm is a polynucleotide set forth in SEQ ID NO: 90. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 91. In certain embodiments, the right homology arm is a polynucleotide set forth in SEQ ID NO: 91. In certain embodiments, the left homology arm and right homology arms comprise any of the nucleotide sequences provided in Table 4. sf-6638784 122 32429-20001.40 In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 201. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 201. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 213. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 213. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 202. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 202. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 214. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 214. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 203. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 203. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 215. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 215. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 204. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 204. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 216. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 216. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, sf-6638784 123 32429-20001.40 98%, 99% or 100%, identical to SEQ ID NO: 205. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 205. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 217. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 217. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 206. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 206. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 218. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 218. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 207. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 207. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 219. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 219. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 208. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 208. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 220. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 220. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 209. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 209. In sf-6638784 124 32429-20001.40 certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 221. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 221. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 210. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 210. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 222. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 222. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 211. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 211. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 223. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 223. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 212. In certain embodiments, the left homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 212. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 224. In certain embodiments, the right homology arm is a polynucleotide having a sequence set forth in SEQ ID NO: 224. In a particular embodiment, the vector comprises polynucleotides having a sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 92, preferably the polynucleotide sequence of SEQ ID NO: 92. (2) Nucleic acids encoding an inactivated cell surface receptor sf-6638784 125 32429-20001.40 In another general aspect, the invention relates to an isolated nucleic acid encoding an inactivated cell surface receptor useful for an invention according to embodiments of the application. It will be appreciated by those skilled in the art that the coding sequence of an inactivated cell surface receptor can be changed (e.g., having nucleotides substituted) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding an inactivated cell surface receptor of the application can be altered without changing the amino acid sequences of the protein. In certain embodiments, an isolated nucleic acid encodes any inactivated cell surface receptor described herein, such as that comprises a monoclonal antibody- specific epitope, and a cytokine, such as an IL-15 or IL-2, wherein the monoclonal antibody-specific epitope and the cytokine are operably linked by an autoprotease peptide sequence. In some embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor comprising an epitope specifically recognized by an antibody, such as abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab, infliximab, ipilimumab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, polatuzumab vedotin, ranibizumab, rituximab, tiuxetan, tocilizumab, tositumomab, trastuzumab, tremelimumab, ustekinumab, or vedolizumab. In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having a truncated epithelial growth factor (tEGFR) variant. Preferably, the inactivated cell surface receptor comprises an epitope specifically recognized by cetuximab, matuzumab, necitumumab or panitumumab, preferably cetuximab. In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD79b, such as an epitope specifically recognized by polatuzumab vedotin. In certain embodiments, the isolated nucleic acid encodes an inactivated cell surface receptor having one or more epitopes of CD20, such as an epitope specifically recognized by rituximab. In certain embodiments, the isolated nucleic acid encodes an sf-6638784 126 32429-20001.40 inactivated cell surface receptor having one or more epitopes of Her 2 receptor, such as an epitope specifically recognized by trastuzumab. In certain embodiments, the autoprotease peptide sequence is porcine tesehovirus-12A (P2A). In certain embodiments, the truncated epithelial growth factor (tEGFR) variant consists of amino acids having a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71. In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by polatuzumab vedotin consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 78. In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by rituximab consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 80. In certain embodiments, the monoclonal antibody-specific epitope specifically recognized by trastuzumab consists of amino acids having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 82. In certain embodiments, the IL-15 comprises amino acids having a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 72. In certain embodiments, the autoprotease peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 73. In certain embodiments, the inactivated cell surface receptor comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74. In a particular embodiment, the isolated nucleic acid encoding the inactivated cell surface receptor is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 75, preferably the polynucleotide sequence of SEQ ID NO: 75. sf-6638784 127 32429-20001.40 In another general aspect, the application provides a vector comprising a polynucleotide sequence encoding an inactivated cell surface receptor useful for an invention according to embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of an inactivated cell surface receptor in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application. In a particular aspect, the application provides a vector for targeted integration of an inactivated cell surface receptor useful for an invention according to embodiments of the application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an inactivated cell surface receptor, such as an inactivated cell surface receptor comprising a truncated epithelial growth factor (tEGFR) variant and an (IL-15, wherein the tEGFR variant and IL-15 are operably linked by an autoprotease peptide sequence, such as porcine tesehovirus-12A (P2A), and (c) a terminator/polyadenylation signal. In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 63. Other promoters can also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta actin. In certain embodiments, the terminator/polyadenylation signal is a SV40 signal. In certain embodiments, the SV40 signal is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 64. Other terminator sf-6638784 128 32429-20001.40 sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK. In certain embodiments, the polynucleotide sequence encoding an inactivated cell surface receptor is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 75. In some embodiments, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide. In some embodiments, the left homology arm and the light homology arm comprise the sequence set forth in Table 4 or sequences set forth in Section VII.C(1) above. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 84. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 85. In a particular embodiment, the vector is a polynucleotide having a sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 86, preferably the polynucleotide sequence of SEQ ID NO: 86. (3) Nucleic acids encoding an HLA construct In another general aspect, the invention relates to an isolated nucleic acid encoding an HLA construct useful for an invention according to embodiments of the application. It will be appreciated by those skilled in the art that the coding sequence of an HLA construct can be changed (e.g., having nucleotides substituted) without changing the amino acid sequence of the protein. Accordingly, it will be understood by those skilled in the art that nucleic acid sequences encoding an HLA construct of the application can be altered without changing the amino acid sequences of the proteins. In certain embodiments, the isolated nucleic acid encodes an HLA construct comprising a signal peptide, such as an HLA-G signal peptide, operably linked to an HLA coding sequence, such as a coding sequence of a mature B2M, and/or a mature HLA-E. In some embodiments, the HLA coding sequence encodes the HLA-G and sf-6638784 129 32429-20001.40 B2M, which are operably linked by a 4X GGGGS linker, and/or the B2M and HLA- E, which are operably linked by a 3X GGGGS linker. In a particular embodiment, the isolated nucleic acid encoding the HLA construct is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the isolated nucleic acid encoding the HLA construct is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70. In another general aspect, the application provides a vector comprising a polynucleotide sequence encoding a HLA construct useful for an invention according to embodiments of the application. Any vector known to those skilled in the art in view of the present disclosure can be used, such as a plasmid, a cosmid, a phage vector or a viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any element to establish a conventional function of an expression vector, for example, a promoter, ribosome binding element, terminator, enhancer, selection marker, and origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. A number of expression vectors capable of delivering nucleic acids to a cell are known in the art and can be used herein for production of a HLA construct in the cell. Conventional cloning techniques or artificial gene synthesis can be used to generate a recombinant expression vector according to embodiments of the application. In a particular aspect, the application provides vectors for targeted integration of a HLA construct useful for an invention according to embodiments of the application. In certain embodiments, the vector comprises an exogenous polynucleotide having, in the 5’ to 3’ order, (a) a promoter; (b) a polynucleotide sequence encoding an HLA construct; and (c) a terminator/polyadenylation signal. In certain embodiments, the promoter is a CAG promoter. In certain embodiments, the CAG promoter is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 63. Other promoters can also be used, examples of which include, but are not limited to, EF1a, UBC, CMV, SV40, PGK1, and human beta actin. sf-6638784 130 32429-20001.40 In certain embodiments, the terminator/ polyadenylation signal is a SV40 signal. In certain embodiments, the SV40 signal is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 64. Other terminator sequences can also be used, examples of which include, but are not limited to BGH, hGH, and PGK. In certain embodiments, a polynucleotide sequence encoding a HLA construct comprises a signal peptide, such as a HLA-G signal peptide, a mature B2M, and a mature HLA-E, wherein the HLA-G and B2M are operably linked by a 4X GGGGS linker (SEQ ID NO: 31) and the B2M transgene and HLA-E are operably linked by a 3X GGGGS linker (SEQ ID NO: 25). In particular embodiments, the HLA construct is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 67, preferably the polynucleotide sequence of SEQ ID NO: 67. In another embodiment, the HLA construct is encoded by a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 70, preferably the polynucleotide sequence of SEQ ID NO: 70. In some embodiment, the vector further comprises a left homology arm and a right homology arm flanking the exogenous polynucleotide. In certain embodiments, the left homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 87. In certain embodiments, the right homology arm is a polynucleotide having a sequence at least 90%, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 88. In a particular embodiment, the vector comprises polynucleotides having a sequence at least 85%, such as at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, identical to SEQ ID NO: 89, preferably the polynucleotide sequence of SEQ ID NO: 89. (4) Host cells In another general aspect, the application provides a host cell comprising a vector of the application and/or an isolated nucleic acid encoding a construct of the sf-6638784 131 32429-20001.40 application. Any host cell known to those skilled in the art in view of the present disclosure can be used for recombinant expression of exogenous polynucleotides of the application. According to particular embodiments, the recombinant expression vector is transformed into host cells by conventional methods such as chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed. Examples of host cells include, for example, recombinant cells containing a vector or isolated nucleic acid of the application useful for the production of a vector or construct of interest; or an engineered iPSC or derivative cell thereof containing one or more isolated nucleic acids of the application, preferably integrated at one or more chromosomal loci. A host cell of an isolated nucleic acid of the application can also be an immune effector cell, such as a T cell or NK cell, comprising the one or more isolated nucleic acids of the application. The immune effector cell can be obtained by differentiation of an engineered iPSC of the application. Any suitable method in the art can be used for the differentiation in view of the present disclosure. The immune effector cell can also be obtained transfecting an immune effector cell with one or more isolated nucleic acids of the application. D. Compositions In another general aspect, the application provides a composition comprising an isolated polynucleotide of the application, a host cell and/or an iPSC or derivative cell thereof of the application. In certain embodiments, the composition further comprises one or more therapeutic agents 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), siRNA, oligonucleotide, mononuclear blood cells, a vector comprising one or more polynucleic acids of interest, an antibody, a chemotherapeutic agent, a radioactive moiety, or an immunomodulatory drug (iMiD). In certain embodiments, the composition is a pharmaceutical composition comprising an isolated polynucleotide of the application, a host cell and/or an iPSC or derivative cell thereof of the application and a pharmaceutically acceptable carrier. The term “pharmaceutical composition” as used herein means a product comprising an isolated polynucleotide of the application, an isolated polypeptide of the application, a host cell of the application, and/or an iPSC or derivative cell thereof of sf-6638784 132 32429-20001.40 the application together with a pharmaceutically acceptable carrier. Polynucleotides, polypeptides, host cells, and/or iPSCs or derivative cells thereof of the application and compositions comprising them are also useful in the manufacture of a medicament for therapeutic applications mentioned herein. As used herein, the term “carrier” refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microsphere, liposomal encapsulation, or other material well known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term “pharmaceutically acceptable carrier” refers to a non-toxic material that does not interfere with the effectiveness of a composition described herein or the biological activity of a composition described herein. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in a polynucleotide, polypeptide, host cell, and/or iPSC or derivative cell thereof can be used. The formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005), and any later editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity regulating agents, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carrier may be used in formulating the pharmaceutical compositions of the application. E. Methods of use Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a “clinically detectable” tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation on physical examination, and/or which is detectable because of the sf-6638784 133 32429-20001.40 expression of one or more cancer-specific antigens in a sample obtainable from a patient. Cancer conditions may be characterized by the abnormal proliferation of malignant cancer cells and may include leukemias, such as AML, CML, ALL and CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP). Cancer cells within an individual may be immunologically distinct from normal somatic cells in the individual. For example, the cancerous tumor may be immunogenic, i.e., the cancer cells may be capable of eliciting a systemic immune response in the individual against one or more antigens expressed by the cancer cells. The tumor antigens that elicit the immune response may be specific to cancer cells or may be shared by one or more normal cells in the individual. The cancer cells of an individual suitable for treatment as described herein may express the antigen and/or may be of correct HLA type to bind the antigen receptor expressed by the Τ cells. An individual suitable for treatment as described above may be a mammal. In preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, primate, porcine, canine, or rabbit animals) may be employed. In some embodiments, the individual may have minimal residual disease (MRD) after an initial cancer treatment. In some embodiments, the individual may have no minimal residual disease after one or more cancer treatments or repeated dosing. An individual with cancer may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of cancer in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine such as Harrison’s Principles of Internal Medicine, 15th Ed., Fauci AS et al., eds., McGraw-Hill, New York, 2001. In some instances, a sf-6638784 134 32429-20001.40 diagnosis of a cancer in an individual may include identification of a particular cell type (e.g., a cancer cell) in a sample of a body fluid or tissue obtained from the individual. An anti-tumor effect is a biological effect which can be manifested by a reduction in the rate of tumor growth, decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies, also Τ cells which may be obtained according to the methods of the present invention, as described herein in prevention of the occurrence of tumors in the first place. Treatment may be any treatment and/or therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of a subject or patient beyond that expected in the absence of treatment. Treatment may also be prophylactic (e.g., prophylaxis). For example, an individual susceptible to or at risk of the occurrence or re-occurrence of cancer may be treated as described herein. Such treatment may prevent or delay the occurrence or re-occurrence of cancer in the individual. In particular, treatment may include inhibiting cancer growth, including complete cancer remission, and/or inhibiting cancer metastasis. Cancer growth generally refers to any one of a number of indices that indicate change within the cancer to a more developed form. Thus, indices for measuring an inhibition of cancer growth include a decrease in cancer cell survival, a decrease in tumor volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumor growth, a destruction of tumor vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of Τ cells, and a decrease in levels of tumor-specific antigens. Administration of Τ cells modified as described herein may improve the capacity of sf-6638784 135 32429-20001.40 the individual to resist cancer growth, in particular growth of a cancer already present the subject and/or decrease the propensity for cancer growth in the individual. This application provides a method of treating a disease or a condition in a subject in need thereof. The methods comprise administering to the subject in need thereof a therapeutically effective amount of cells of the application and/or a composition of the application. In certain embodiments, the disease or condition is cancer. The cancer can, for example, be a solid or a liquid cancer. The cancer, can, for example, be selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a liver cancer, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, an endometrial cancer, a prostate cancer, a thyroid cancer, a glioma, a glioblastoma, and other solid tumors, and a non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma/disease (HD), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors. In a preferred embodiment, the cancer is a non-Hodgkin’s lymphoma (NHL). According to embodiments of the application, the composition comprises a therapeutically effective amount of an isolated polynucleotide, an isolated polypeptide, a host cell, and/or an iPSC or derivative cell thereof. As used herein, the term “therapeutically effective amount” refers to an amount of an active ingredient or component that elicits the desired biological or medicinal response in a subject. A therapeutically effective amount can be determined empirically and in a routine manner, in relation to the stated purpose. As used herein with reference to a cell of the application and/or a pharmaceutical composition of the application a therapeutically effective amount means an amount of the cells and/or the pharmaceutical composition that modulates an immune response in a subject in need thereof. According to particular embodiments, a therapeutically effective amount refers to the amount of therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder or condition to be treated or a symptom associated therewith; (ii) reduce the duration of the disease, disorder or condition to be treated, or a symptom associated therewith; (iii) prevent the progression of the disease, disorder or condition to be sf-6638784 136 32429-20001.40 treated, or a symptom associated therewith; (iv) cause regression of the disease, disorder or condition to be treated, or a symptom associated therewith; (v) prevent the development or onset of the disease, disorder or condition to be treated, or a symptom associated therewith; (vi) prevent the recurrence of the disease, disorder or condition to be treated, or a symptom associated therewith; (vii) reduce hospitalization of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (viii) reduce hospitalization length of a subject having the disease, disorder or condition to be treated, or a symptom associated therewith; (ix) increase the survival of a subject with the disease, disorder or condition to be treated, or a symptom associated therewith; (xi) inhibit or reduce the disease, disorder or condition to be treated, or a symptom associated therewith in a subject; and/or (xii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy. The therapeutically effective amount or dosage can vary according to various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, e.g., age, body weight, health), whether the subject is a human or an animal, other medications administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy. According to particular embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration. The cells of the application and/or the pharmaceutical compositions of the application can be administered in any convenient manner known to those skilled in the art. For example, the cells of the application can be administered to the subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and/or transplantation. The compositions comprising the cells of the application can be administered transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrapleurally, by intravenous (i.v.) injection, or intraperitoneally. In certain embodiments, the cells of the application can be administered with or without lymphodepletion of the subject. The pharmaceutical compositions comprising cells of the application can be provided in sterile liquid preparations, typically isotonic aqueous solutions with cell suspensions, or optionally as emulsions, dispersions, or the like, which are typically sf-6638784 137 32429-20001.40 buffered to a selected pH. The compositions can comprise carriers, for example, water, saline, phosphate buffered saline, and the like, suitable for the integrity and viability of the cells, and for administration of a cell composition. Sterile injectable solutions can be prepared by incorporating cells of the application in a suitable amount of the appropriate solvent with various other ingredients, as desired. Such compositions can include a pharmaceutically acceptable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like, that are suitable for use with a cell composition and for administration to a subject, such as a human. Suitable buffers for providing a cell composition are well known in the art. Any vehicle, diluent, or additive used is compatible with preserving the integrity and viability of the cells of the application. The cells of the application and/or the pharmaceutical compositions of the application can be administered in any physiologically acceptable vehicle. A cell population comprising cells of the application can comprise a purified population of cells. Those skilled in the art can readily determine the cells in a cell population using various well known methods. The ranges in purity in cell populations comprising genetically modified cells of the application can be from about 50% to about 55%, from about 55% to about 60%, from about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%, from about 85% to about 90%, from about 90% to about 95%, or from about 95% to about 100%. Dosages can be readily adjusted by those skilled in the art, for example, a decrease in purity could require an increase in dosage. The cells of the application are generally administered as a dose based on the number cells per kilogram (cells/kg) of body weight of the subject to which the cells and/or pharmaceutical compositions comprising the cells are administered. Generally, the cell doses are in the range of about 104 to about 1010 cells/kg of body weight, for example, about 105 to about 109, about 105 to about 108, about 105 to about 107, or about 105 to about 106, depending on the mode and location of administration. In general, in the case of systemic administration, a higher dose is used than in regional administration, where the immune cells of the application are administered in the region of a tumor and/or cancer. Exemplary dose ranges include, but are not limited to, 1 x 104 to 1 x 108, 2 x 104 to 1 x 108, 3 x 104 to 1 x 108, 4 x 104 to 1 x 108, 5 x 104 to 6 x 108, 7 x 104 to 1 x 108, 8 x 104 to 1 x 108, 9 x 104 to 1 x 108, 1 x 105 to 1 x 108, 1 x 105 to 9 x 107, 1 x 105 to 8 x 107, 1 x 105 to 7 x 107, 1 x 105 to 6 x 107, 1 x sf-6638784 138 32429-20001.40 105 to 5 x 107, 1 x 105 to 4 x 107, 1 x 105 to 4 x 107, 1 x 105 to 3 x 107, 1 x 105 to 2 x 107, 1 x 105 to 1 x 107, 1 x 105 to 9 x 106, 1 x 105 to 8 x 106, 1 x 105 to 7 x 106, 1 x 105 to 6 x 106, 1 x 105 to 5 x 106, 1 x 105 to 4 x 106, 1 x 105 to 4 x 106, 1 x 105 to 3 x 106, 1 x 105 to 2 x 106, 1 x 105 to 1 x 106, 2 x 105 to 9 x 107, 2 x 105 to 8 x 107, 2 x 105 to 7 x 107, 2 x 105 to 6 x 107, 2 x 105 to 5 x 107, 2 x 105 to 4 x 107, 2 x 105 to 4 x 107, 2 x 105 to 3 x 107, 2 x 105 to 2 x 107, 2 x 105 to 1 x 107, 2 x 105 to 9 x 106, 2 x 105 to 8 x 106, 2 x 105 to 7 x 106, 2 x 105 to 6 x 106, 2 x 105 to 5 x 106, 2 x 105 to 4 x 106, 2 x 105 to 4 x 106, 2 x 105 to 3 x 106, 2 x 105 to 2 x 106, 2 x 105 to 1 x 106, 3 x 105 to 3 x 106 cells/kg, and the like. In certain embodiments, a fixed dosage of cells can be administered to a patient. Exemplary fixed dosages include, but are not limited to about 105, about 106, about 107, or about 108 cells. Additionally, the fixed or weigh- based dose can be adjusted to account for whether a single dose is being administered or whether multiple doses are being administered. The precise determination of what would be considered an effective dose can be based on factors individual to each subject. As used herein, the terms “treat,” “treating,” and “treatment” are all intended to refer to an amelioration or reversal of at least one measurable physical parameter related to a cancer, which is not necessarily discernible in the subject, but can be discernible in the subject. The terms “treat,” “treating,” and “treatment,” can also refer to causing regression, preventing the progression, or at least slowing down the progression of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an alleviation, prevention of the development or onset, or reduction in the duration of one or more symptoms associated with the disease, disorder, or condition, such as a tumor or more preferably a cancer. In a particular embodiment, “treat,” “treating,” and “treatment” refer to prevention of the recurrence of the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to an increase in the survival of a subject having the disease, disorder, or condition. In a particular embodiment, “treat,” “treating,” and “treatment” refer to elimination of the disease, disorder, or condition in the subject. The cells of the application and/or the pharmaceutical compositions of the application can be administered in combination with one or more additional therapeutic agents. In certain embodiments 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), siRNA, sf-6638784 139 32429-20001.40 oligonucleotide, mononuclear blood cells, 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 Abbreviations BLI Bioluminescent imaging BSA Bovine serum albumin C Celsius CAR Chimeric antigen receptor CD Cluster of differentiation CXCL12 C-X-C motif chemokine 12 E:T Effector to target ratio FACS Fluorescence activated cell sorting HDR Homology-directed-repair HPC Hematopoietic stem cell Ig Immunoglobulin IHC Immunohistochemistry iNK iPSC-derived NK cell iPSC Induced pluripotent stem cell iT iPSC-derived T cell M Molar mAb Monoclonal antibody mM Micromolar NBF Neutral buffered formalin NLR NucLight Red nM Nanomolar NSG NOD scid gamma RBC Red blood cell RPMI Roswell Park Memorial Institute medium scFv Single-chain variable fragment SDF-1 Stromal cell-derived factor 1 TGI Tumor growth inhibition UTD untransduced VHH Single variable domain on a heavy chain WT Wild-type sf-6638784 140 32429-20001.40 Example 1. Engineering Cells Expressing CD33-specific CARs CARs were constructed with binding domains on the extracellular side of the protein construct, singly or in multiples. scFv proteins were tested as scFv-IgG1 Fc fusions for protein affinity and specific binding to CD33+ cells. Epitope mapping was performed by binding competition and using CD33 protein variants with selected domains deleted from the full-length protein. VH and VL fragments were cloned into lentiviral vectors in frame with a hinge domain (IgG4 Fc or CD8 hinge), a transmembrane domain (CD28tm), a co- stimulatory domain (41BB, CD28, or DAP10) and a primary signaling domain from CD3z. Lentivirus was produced for the various constructs and transduced into primary T-cells to examine antigen-dependent activation of this T-cell line. scFv-CAR transduced cells were cultured for two weeks, then injected into tumor-bearing female NSG mice (FIG. 1). Clones with tumor growth inhibition (TGI) of 90% and higher were selected for further functional assessment. Example 2. Development of cells expressing CD33-specific VHH-CARs CARs were constructed with VHH binding domains on the extracellular side of the protein construct, singly or in multiples. The VHH antibody binding fragments were isolated through phage display of VHH libraries and selection on the soluble CD33 protein. VHH proteins were tested as VHH-IgG1 Fc fusions for protein affinity and specific binding to CD33+ cells. VHH sequences were fused to the human IgG1 Fc domain in a mammalian expression construct and expressed as VHH-Fc fusion proteins in Expi293 cells and purified with Protein A (MabSelect SuRe) resin. VHH- Fc proteins were eluted with 0.1M sodium acetate (pH=3.5) and neutralized with 2.5M sodium acetate (pH=7.5). VHH-Fc fusion proteins were assessed for binding to CD33 expressed on the cell surface. VHH-Fc proteins were serially diluted and added to CHO-CD33 and CHO-K1 cells and incubated at 4C for 30 minutes. Cells were washed once in BD staining buffer, BSA (BD Pharmingen), then PE-labeled anti- human IgG Fc (Jackson Immunoresearch) was added to detect VHH-Fc cell binding and incubated at 4C for 30 minutes. Cells were washed once, then resuspended in staining buffer with 0.1% pluronic acid and run through a flow cytometer to detect the fluorescent labeling of the VHH-Fc cells. Binding data was displayed as a ratio of specific to background binding (FIG. 2). 141 sf-6638784 32429-20001.40 Example 3. Development of cells expressing CD123-specific VHH-CARs CARs were constructed with VHH binding domains on the extracellular side of the protein construct, singly or in multiples. The VHH antibody binding fragments were isolated through phage display of VHH libraries and selection on the soluble CD123 protein. VHH proteins were tested as VHH-IgG1 Fc fusions for protein affinity and specific binding to CD33+ cells. VHH sequences were fused to the human IgG1 Fc domain in a mammalian expression construct and expressed as VHH- Fc fusion proteins in Expi293 cells and purified with Protein A (MabSelect SuRe) resin. VHH-Fc proteins were eluted with 0.1M sodium acetate (pH=3.5) and neutralized with 2.5M sodium acetate (pH=7.5). VHH-Fc fusion proteins were assessed for binding to CD123 expressed on the cell surface. VHH-Fc proteins were serially diluted and added to: 1) HL-60, KASUMI-3, MOLM-13 and Jurkat cells (FIGs. 3A-3B and FIGs. 5A-5B); or 2) CHO-CD123 and CHO-K1 cells (FIG.4) and incubated at 4C for 30 minutes. Cells were washed once in BD staining buffer, BSA (BD Pharmingen), then PE-labeled anti-human IgG Fc (Jackson Immunoresearch) was added to detect VHH- Fc cell binding and incubated at 4C for 30 minutes. Cells were washed once, then resuspended in staining buffer with 0.1% pluronic acid and run through a flow cytometer to detect the fluorescent labeling of the VHH-Fc cells. Binding data is displayed as either individual cell line binding or a ratio of specific to background binding. CD123 antigen expression on target cell lines was assessed using FACS (FIGs. 6A-6F). VHH fragments were cloned into lentiviral vectors in frame with a hinge domain (IgG4 Fc or CD8 hinge), a transmembrane domain (CD28tm), a co- stimulatory domain (41BB, CD28, DAP10) and a primary signaling domain from CD3z. Lentivirus was produced for the various constructs and transduced into primary T-cells to examine antigen-dependent activation of this T-cell line. VHH-CARs were tested in primary T-cells for killing CD123+ tumor target cells. Lentivirus for each of the CARs described was transduced into primary donor T-cells and grown for 14 days. Expression of the CARs was assessed by flow cytometry. CAR expression confirmed for all constructs, some ranging from 70-80% and others around 30-50%. sf-6638784 142 32429-20001.40 CD123+ cells (FIGs. 6A-6F) were labeled with CellTrace Violet (ThermoFisher) and co-cultured with the CAR-T cells at various E:T ratios for 48 hours. Target cell viability was then assessed by flow cytometry (FIGs. 7A-7F). Anti-CD123 VHH binders demonstrated CAR cytotoxic activity against CD123+ tumor cell lines. VHH-CAR transduced cells were cultured for two weeks, then injected into tumor-bearing female NSG mice (FIG. 8). Clones with TGI of 90% and higher were selected for further functional assessment. Example 4. Cytotoxicity Assays Using Therapeutic Cells Expressing Mono- and Dual-targeting CARs Against CD33 and/or CD123 Target Cells Dual-targeting CAR ectodomains were engineered with select CD33 binders in combination with selected CD123 binders and with various combinations of hinge, transmembrane and co-stimulatory endodomains (FIGs. 9A-9D, FIGs. 10A-10E, and FIG. 11). Selected CARs were expressed and tested for cytotoxicity activity in iNK cells. CAR expression was confirmed using a FACS assay (FIGs. 12A-12D), and CAR-iNK cells were subject to in vivo efficacy study. CAR-iNK cells (IPSC3081, IPSC3082, IPSC3083 derived from the iPSC1631 parental line and IPSC3084, IPSC3085, and IPSC3086 derived from the parental iPSC1650 parental line) were injected to tumor bearing female NSG mice, and the resulting cytotoxic effect was assessed by chemiluminescence readout at days 6 and 8 (FIG. 13; Table 5). Table 5. sf-6638784 143 32429-20001.40 Example 5. Development of cells expressing CXCR4 An iPSC cell line engineered to express a CAR against a tumor cell antigen was further engineered, through homology-directed-repair (HDR) into the CLYBL safe harbor site, to express the human wild type CXCR4 protein. The iPSC cell lines were differentiated to iNK cells, and evaluated for CXCR4 expression through the process. At iPSC stage (following engineering), HPC stage, Day 14 (D14) of iNK differentiation, and Day 21 (D21) of iNK differentiation, cells were analyzed for CXCR4 levels by flow cytometry (FIG. 14). Briefly, cells were collected, centrifuged, and stained with antibody detecting human CXCR4 (PE mouse anti- human CD184 [CXCR4] mAb). FACS was conducted on the BD Symphony flow cytometer, and results depicted as % CXCR4 positive cells of live cells. CXCR4 was found to be expressed in 42.4% of the engineered Day 21 iNK cells (IPSC1613) versus 2.4% of the iNK cells not engineered to express CXCR4 (iPSC611). The IPSC lines were differentiated at larger scale for use in functional assays, and CXCR4 expression was evaluated as previously indicated. At scale, the CXCR4-engineered iNK cells maintained 47.7% expression of CXCR4 (FIG. 15). iNK cells differentiated from iPSC611 (CAR+/CXCR4-), iPSC1613 (CAR+/CXCR4+), and a CAR-CXCR4- iT cell line (iTCAR-CXCR4-) were tested for activity in a cytotoxicity assay against NALM-6 CD19+ tumor cell line as shown in FIG. 16. 2x105 effector cells were co-cultured with 2x105 NucLight Red (NLR) labeled NALM-6 tumor cells in flatbottom 96-well tissue culture treated plates and placed in an Incucyte live cell imager. Whole well images were taken every 3 hours for 72 hours total collecting phase contrast and NLR images. Raw data was normalized to NLR count of tumor cell alone condition. To generate CXCR4-expressing iNK lines for evaluation in a second tumor model, IPSC cells expressing a dual-targeting CAR to two tumor targets (IPSC2984) was transduced with a lentivirus containing sequence encoding for WT CXCR4 upon integration into the cell line genome. Hematopoietic progenitor cells (HPCs) derived from IPSC2984 were transduced with the WT CXCR4 lentivirus and then differentiated 14 days to iNK cells. CXCR4 expression was evaluated as previously described, and the lentivirus- transduced cells determined to be 54.5% CXCR4 positive (versus 3.29% for non-transduced cells) (FIG. 17). To assess the functionality of the CXCR4 transgene in iPSC2984, CXCR4 positive cells (CAR-iNKCXCR4+) and CXCR4 negative cells (CAR-iNKCXCR4-) were sf-6638784 144 32429-20001.40 assessed in a transwell migration assay and compared to Jurkat (CXCR4+) and MOLM13 (CXCR4-) tumor cells as positive and negative controls as shown in FIGs. 18A-18B. Transwell plates were coated with rat tail collagen at a concentration of 50 µg/mL for 2 hours. After coating, rat tail collagen solution was removed, and plates were allowed to dry for 30 minutes. In the lower chamber of the assay plate, 1 mL of assay media with or without 400 ng/mL C-X-C motif chemokine 12 (CXCL12) was added. The insert of the assay plate was loaded with 200 µL of cells at a concentration of 1x106 cells/mL in assay media. Assay plates were transferred to a tissue culture incubator at 37°C and incubated for 4 hours. After 4 hours cells were removed from the lower chamber of the transwell plate and stained for live / dead (near-IR), CD56 (BV786), and CXCR4 (BV421). Samples were collected on a BD Symphony A3 cytometer using absolute flow count beads to quantify the number of total cells in the well. Raw cell counts are displayed in FIG. 18A. Chemotactic index was calculated as the number of cells in experimental wells (400 ng CXCL12) divided by the average of cells in the baseline wells (0 ng CXCL12) and is shown in FIG. 18B. CAR-iNKCXCR4+ and CAR-iNKCXCR4- were tested for functionality in a cytotoxicity assay against NucLight Red-labeled antigen-positive and antigen-knock- out tumor cell lines as shown in FIGS. 19A-19B. 2x105 target cells were co-cultured with either 2x105 or 4x105 effector cells for effector to target ratios (E:T) of 1:1 (FIG. 19A) and 2:1 (FIG. 19B), respectively. Target cell growth was measured by Total Integrated Intensity readings from Incucyte Live-Cell Imaging system every 3 hours for 72 hours total. Figure curves represent target cell growth curves when co-cultured with effector cells, normalized to target cell growth curves when target cells are cultured alone. CAR-iNK cells with (CAR-iNKCXCR4+) and without (CAR-iNKCXCR4-) CXCR4 engineering were subject to in vivo functional studies. As shown in FIG. 20, CAR-iNK cells were injected to tumor bearing female NSG mice, and the resulting cytotoxic effect was assessed over 9 days. MOLM13-Luc cells were implanted intravenously (IV) at 1x105 cells/mouse 4 days prior to intravenous injection of CAR- iNK at 1x107 cells/mouse. Day 9 blood and tissue sampling were conducted to evaluate CAR-iNK presence by FACS and histology. FIG. 21 shows the efficacy results of in vivo efficacy screening of CAR-iNK cells with (CAR-iNKCXCR4+) and without (CAR-iNKCXCR4-) CXCR4 engineering using the disseminated MOLM13-Luc xenograft model in NSG mice. MOLM13-Luc cells were implanted intravenously sf-6638784 145 32429-20001.40 (IV) at 1x105 cells/mouse 4 days prior to intravenous injection of CAR-iNK at 1x107 cells/mouse. Tumor burden was measured using bioluminescent imaging (BLI). Tumor growth inhibition (TGI) was calculated on Day 8, the final day of measurements for the tumor control group. Significant TGI compared to tumor control was observed only for CAR-iNKCXCR4+ (82% TGI, p=0.0061). On Day 9, mice were humanely euthanized and sampled for blood, bone marrow, and tissue analysis by FACS and histology. Blood was collected into lithium heparin coated tubes, and bone marrow aspirate was collected and kept in serum-free RPMI until FACS analysis. Briefly, samples were centrifuged to pellet cells and medium discarded. Samples underwent RBC lysis and were resuspended for FACS antibody staining. Samples were stained with Near-IR Live/Dead stain (Thermo cat# L34976A) and FC block (Innovex# NB309) added to samples. Samples were then stained with the following antibodies: FITC (CD33), BV421 (CD45), BV786 (CD56), APC (CD184/CXCR4), incubated, and FACS conducted on the BD Symphony A3 cytometer. FIG. 22 shows bone marrow presence of CAR-iNK in the presence and absence of tumor, assessed by FACS. Both tumor-bearing and non-tumor-bearing mice that received CAR-iNKCXCR4- did not have iNK present in the bone marrow. Significant numbers of iNK were present in the bone marrow of both tumor-bearing (p<0.0001) and non-tumor-bearing mice (p=0.0018) that received CAR-iNKCXCR4+ compared with tumor-bearing and non-tumor-bearing mice (respectively) that received CAR-iNKCXCR4-. Femur, spleen, liver, and lung were collected from mice, and fixed in 10% NBF, then transferred to 70% ethanol. Tissues were then processed, paraffin-embedded, and cut for immunohistochemical (IHC) staining. Immunohistochemistry of normal NSG mouse bone marrow stained using CXCL12 antibody (SDF1/CXCL12 [D8G6H] Rabbit mAb, 1:100 dilution) is shown in FIG. 23. Femur, spleen, liver, and lung were stained with CD3 antibody (NCL-L-CD3-565 [LN10] Mouse mAb) to detect iNK. CAR-iNKCXCR4- and CAR-iNKCXCR4+ tissue infiltration of femur, spleen, liver, and lung is shown in FIG. 24. It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description. sf-6638784 146

Claims

32429-20001.40 CLAIMS It is claimed: 1. An induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising: at least one exogenous polynucleotide encoding one or more chimeric antigen receptors (CARs), said one or more CARs comprising an antigen- binding domain targeting a Cluster of Differentiation 123 (CD123) antigen; and at least one of: (i) a deletion or reduced expression of one or more of Beta 2 Microglobulin (B2M), Transporter Associated with Antigen Processing (TAP) 1, TAP 2, Tapasin, Regulatory Factor X Associated Ankyrin Containing Protein (RFXANK), Class II Major Histocompatibility Complex Transactivator (CIITA), Regulatory Factor X5 (RFX5), and Regulatory Factor X Associated Protein (RFXAP) genes; (ii) an exogenous polynucleotide encoding a human leukocyte antigen E (HLA-E) and/or a human leukocyte antigen G (HLA- G); (iii) an exogenous polynucleotide encoding a cluster of differentiation 16 (CD16; natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII)) protein and/or a Natural Killer Group 2 Member D (NKG2D) protein; (iv) a deletion or reduced expression of one or both of Natural Killer Group 2 Member A (NKG2A) and Cluster of Differentiation 70 (CD70) genes; (v) an exogeneous polynucleotide encoding a cytokine; (vi) an exogenous polynucleotide encoding a safety switch; (vii) an exogenous polynucleotide encoding C-X-C chemokine receptor type 4 (CXCR4) or a fragment or variant thereof; and (viii) an exogeneous polynucleotide encoding a Prostate-Specific Membrane Antigen (PSMA) cell tracer. 2. The iPSC or the derivative cell thereof according to claim 1, wherein, sf-6638784 147 32429-20001.40 the antigen-binding domain targeting the CD123 antigen is a first antigen- binding domain, and wherein: (i) the one or more CARs consists of a dual-targeting CAR comprising the first antigen-binding domain and a second antigen-binding domain; or (ii) the one or more CARs comprises a plurality of CARs including a first CAR and a second CAR, wherein the first CAR comprises the first antigen-binding domain, and wherein the second CAR comprises a second antigen-binding domain. 3. The iPSC or the derivative cell thereof according to claim 2, wherein the first antigen-binding domain comprises an anti-CD123 VHH single domain antibody. 4. The iPSC or the derivative cell thereof according to claim 2 or 3 comprising the dual-targeting CAR, wherein the dual-targeting CAR comprises: (i) optionally, a signal peptide; (ii) an extracellular domain comprising the first antigen-binding domain and the second antigen-binding domain; (vi) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and/or (vi) a co-stimulatory domain. 5. The iPSC or the derivative cell thereof according to claim 2 or 3 comprising the plurality of CARs, wherein each of the first CAR and the second CAR comprises: (i) optionally, a signal peptide; (ii) an extracellular domain comprising the first antigen-binding domain or the second antigen-binding domain; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and/or (vi) a co-stimulatory domain. sf-6638784 148 32429-20001.40 6. The iPSC or the derivative cell thereof according to claim 4 or 5, wherein the first antigen-binding domain comprises an anti-CD123 VHH single domain antibody. 7. The iPSC or the derivative cell thereof according to any one of claims 1-6, wherein the antigen-binding domain targeting the CD123 antigen comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 126-148. 8. The iPSC or the derivative cell thereof according to any one of claims 1-7, wherein the antigen-binding domain targeting the CD123 antigen is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 149-171. 9. The iPSC or the derivative cell thereof according to claim 6, wherein in the first CAR in the plurality of CARs: a. the signal peptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1 or 103; b. the extracellular domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 126-148; c. the hinge region comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21, 22, 61, 99, 100, or 102; d. the transmembrane domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 23 or 24; and e. the intracellular signaling or co-stimulatory domains comprise amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 6, 8-20, 243, and 244. sf-6638784 149 32429-20001.40 10. The iPSC or the derivative cell thereof according to claim 6 or 9, wherein in the first CAR in the plurality of CARs: a. the signal peptide comprises amino acids having the sequence of SEQ ID NO: 1 or 103; b. the extracellular domain comprises amino acid having the sequence of one of SEQ ID NOs: 126-148; c. the hinge region comprises amino acids having the sequence of SEQ ID NO: 21, 22, 61, 99, 100, or 102; d. the transmembrane domain comprises amino acids having the sequence of SEQ ID NO: 23 or 24; and e. the intracellular signaling or co-stimulatory domains comprise amino acids having the sequence of one or more of SEQ ID NOs: 6, 8-20, 243, and 244. 11. The iPSC or the derivative cell thereof according to any one of claims 1-10, wherein the cytokine comprises Interleukin-15 (IL-15). 12. The iPSC or the derivative cell thereof according to claim 11, further comprising an inactivated cell surface receptor that comprises a monoclonal antibody- specific epitope, wherein the inactivated cell surface receptor and the IL-15 are operably linked by an autoprotease peptide. 13. The iPSC or the derivative cell thereof according to claim 11, wherein the IL- 15 comprises (i) an IL-15 and an IL-15 receptor alpha (IL-15Rα) fusion polypeptide, or (ii) a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 247. 14. The iPSC or the derivative cell thereof according to any one of claims 11-13, wherein the IL-15 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 72. sf-6638784 150 32429-20001.40 15. The iPSC or the derivative cell thereof according to any one of claims 1-14, comprising the deletion or reduced expression of one or more of B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes. 16. The iPSC or the derivative cell thereof according to any one of claims 1-15, comprising the exogenous polynucleotide encoding the HLA-E and/or the HLA-G. 17. The iPSC or the derivative cell thereof according to any one of claims 1-16, wherein the CD16 protein is a CD16 variant protein. 18. The iPSC or the derivative cell thereof according to claim 17, wherein the CD16 variant protein is a high affinity CD16 variant. 19. The iPSC or the derivative cell thereof according to claim 17 or 18, wherein the CD16 variant protein is a non-cleavable CD16 variant. 20. The iPSC or the derivative cell thereof according to any one of claims 17-19, wherein the CD16 variant protein comprises wild-type CD16 comprising amino acids having the sequence of SEQ ID NO: 186 and one or more amino acid substitutions selected from the group consisting of F158V, F176V, S197P, D205A, S219A, and T220A. 21. The iPSC or the derivative cell thereof according to any one of claims 17-20, wherein the CD16 variant protein comprises amino acids having at least 90% sequence identity to SEQ ID NO: 187. 22. The iPSC or the derivative cell thereof according to any one of claims 1-21 comprising the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein, wherein the CD16 protein and the NKG2D protein are operably linked by an autoprotease peptide. 23. The iPSC or the derivative cell thereof according to claim 22, wherein the NKG2D protein comprises amino acids having at least 90%, 91%, 92%, 93%, sf-6638784 151 32429-20001.40 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a wildtype NKG2D protein. 24. The iPSC or the derivative cell thereof according to claim 22, wherein the NKG2D protein comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 189. 25. The iPSC or the derivative cell thereof according to any one of claims 22-24, wherein the autoprotease peptide is selected from the group consisting of a porcine tesehovirus-12A (P2A) peptide, a foot-and-mouth disease virus 2A (F2A) peptide, an Equine Rhinitis A Virus (ERAV) 2A (E2A) peptide, a Thosea asigna virus 2A (T2A) peptide, a cytoplasmic polyhedrosis virus 2A (BmCPV2A) peptide, and a Flacherie Virus 2A (BmIFV2A) peptide. 26. The iPSC or the derivative cell thereof according to claim 25, wherein the autoprotease peptide is a P2A peptide comprising amino acids having at least 90% sequence identity to SEQ ID NO: 73 or 191. 27. The iPSC or the derivative cell thereof according to any one of claims 22-26, wherein the exogenous polynucleotide encoding the CD16 protein and the NKG2D protein comprises a nucleic acid having at least 90% sequence identity to SEQ ID NO: 184. 28. The iPSC or the derivative cell thereof according to any one of claims 1-27, wherein the exogenous polynucleotide(s) are integrated into the chromosome of the cell at one or more loci selected from the group consisting of Adeno- associated Virus Integration Site 1 (AAVS1), B2M, Cbl Proto-oncogene B (CBL-B), C-C Motif Chemokine Receptor 5 (CCR5), CD33, Cluster of Differentiation 38 (CD38), CD70, CIITA, CIS, Cytokine Inducible SH2 Containing Protein (CISH), Citramalyl-CoA Lyase (CLYBL), Collagen, Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4), Glyceraldehyde-3- Phosphate Dehydrogenase (GAPDH), HTRP, Lymphocyte Activating 3 (LAG3), NKG2A, NKG2D, NLR Family CARD Domain Containing 5 (NLRC5), Programmed Cell Death 1 (PD1), RFX5, RFXANK, RFXAP, sf-6638784 152 32429-20001.40 Reverse Orientation Splice Acceptor 26 (ROSA26), RUNX Family Transcription Factor 1 (RUNX1), Suppressor Of Cytokine Signaling 2 (SOCS2), TAP 1, TAP 2, Tapasin, TAP Binding Protein (TAPBP), TCR a or b constant region, T Cell Immunoreceptor With Ig And ITIM Domains (TIGIT), T-cell immunoglobulin and mucin-domain containing-3 (TIM3), and T Cell Receptor Alpha Constant (TRAC) genes, provided at least one of the exogenous polynucleotides is integrated at a locus of a gene selected from the group consisting of AAVS1, B2M, TAP 1, TAP 2, Tapasin, RFXANK, CIITA, RFX5 and RFXAP genes to thereby result in a deletion or reduced expression of the gene. 29. The iPSC or the derivative cell thereof according to claim 28, wherein the exogenous polynucleotide(s) are integrated into the chromosome of the cell at one or more loci selected from the group consisting of AAVS1, B2M, CIITA, CD33, and CLYBL. 30. The iPSC or the derivative cell according to any one of claims 1-29 having a deletion or reduced expression of one or both of the B2M and the CIITA genes. 31. The iPSC or the derivative cell thereof according to claim 30, having a deletion or reduced expression of both of the B2M and the CIITA genes. 32. The iPSC according to any one of claims 1-31, wherein the iPSC is reprogrammed from whole peripheral blood mononuclear cells (PBMCs). 33. The iPSC according to any one of claims 1-31, wherein the iPSC is derived from a re-programmed T-cell. 34. The iPSC according to any one of claims 1-33, or the derivative cell thereof according to any one of claims 1-31, further comprising an exogenous polynucleotide encoding a safety switch. sf-6638784 153 32429-20001.40 35. The iPSC or the derivative cell thereof of claim 34, wherein the safety switch comprises an inactivated cell surface receptor that comprises a monoclonal antibody-specific epitope. 36. The iPSC or the derivative cell thereof according to claim 35, wherein the monoclonal antibody specific epitope is selected from the group of epitopes specifically recognized by abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, brentuximab vedotin, canakinumab, certolizumab pegol, cetuximab, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab, infliximab, ipilimumab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, polatuzumab vedotin, ranibizumab, rituximab, tiuxetan, tocilizumab, tositumomab, trastuzumab, tremelimumab, ustekinumab, and vedolizumab. 37. The iPSC or the derivative cell thereof according to claim 35, wherein the inactivated cell surface receptor is a truncated epithelial growth factor (tEGFR) variant. 38. The iPSC or the derivative cell thereof according to claim 37, wherein the tEGFR variant consists of amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71. 39. The iPSC or the derivative cell thereof according to any one of claims 1-38, wherein the safety switch comprises (i) an intracellular domain comprising a herpes simplex virus thymidine kinase (HSV-TK) or (ii) an inducible Caspase 9 (iCasp9). 40. The iPSC or the derivative cell thereof according to any one of claims 1-39 comprising the exogeneous polynucleotide encoding the PSMA cell tracer, wherein the safety switch further comprises an extracellular domain comprising a PSMA cell tracer or fragment thereof. sf-6638784 154 32429-20001.40 41. The iPSC or the derivative cell thereof according to claim 40 comprising the HSV-TK domain, wherein the HSV-TK is operatively linked to the extracellular domain comprising the PSMA cell tracer or fragment thereof by a linker to form a combined artificial cell death/reporter system polypeptide. 42. The iPSC or the derivative cell thereof according to any one of claims 39-41, wherein (i) the HSV-TK comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 248 or 249, or (ii) the iCasp9 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 245 or 246. 43. The iPSC or the derivative cell thereof according to claim 41, wherein the combined artificial cell death/reporter system polypeptide comprises the HSV- TK fused to a truncated variant PSMA polypeptide via the linker. 44. The iPSC or the derivative cell thereof according to claim 43, wherein the truncated variant PSMA polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 250. 45. The iPSC or the derivative cell thereof according to any one of claims 41-44, wherein the linker comprises an autoprotease peptide selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and a F2A peptide. 46. The iPSC or the derivative cell thereof according to any one of claims 41-45, wherein the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 251. 47. The iPSC or the derivative cell thereof according to claim 46, wherein the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 252-254. sf-6638784 155 32429-20001.40 48. The iPSC or the derivative cell thereof according to any one of claims 41-47, wherein the artificial cell death/reporter system polypeptide comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 255-257. 49. The iPSC or the derivative cell thereof according to any one of claims 1-48, wherein the HLA-E comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 66, and/or the HLA-G comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 69. 50. The iPSC or the derivative cell thereof according to any one of claims 1-49, wherein: (i) the at least one exogenous polynucleotide encoding one or more CARs comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to at least one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 174 and 175; (ii) the exogenous polynucleotide encoding the HLA-E and/or the HLA-G comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 67 and 70; (iii) the exogenous polynucleotide encoding the CD16 protein and/or the NKG2D protein comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 184, 188, and 190; (iv) the exogeneous polynucleotide encoding the cytokine comprises polynucleotides having at least 90%, 91%, 92%, sf-6638784 156 32429-20001.40 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 258; (v) the exogenous polynucleotide encoding the safety switch comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more of SEQ ID NOs: 255-257; and/or (vi) the exogeneous polynucleotide encoding the PSMA cell tracer comprises polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 250. 51. The iPSC or the derivative cell thereof according to any one of claims 1-50, wherein: (i) the at least one exogenous polynucleotide encoding one or more CARs comprises polynucleotides having at least one sequence selected from the group consisting of SEQ ID NOs: 174 and 175; (ii) the exogenous polynucleotide encoding the HLA-E and/or the HLA-G comprises polynucleotides having the sequence of one or more of SEQ ID NOs: 67 and 70; (iii) the exogenous polynucleotide encoding the CD16 protein and/or the NKG2D protein comprises polynucleotides having the sequence of one or more of SEQ ID NOs: 184, 188, and 190; (iv) the exogeneous polynucleotide encoding the cytokine comprises polynucleotides having the sequence of SEQ ID NO: 258; and/or (v) the exogenous polynucleotide encoding the safety switch comprises polynucleotides having the sequence of one of SEQ ID NOs: 255-257. 52. The iPSC or the derivative cell thereof according to claim 50 or 51, wherein each of the exogenous polynucleotides are independently integrated into a gene locus selected from the group consisting of AAVS1, B2M, CBL-B, sf-6638784 157 32429-20001.40 CCR5, CD33, CD38, CD70, CIITA, CIS, CISH, CLYBL, Collagen, CTLA4 , GAPDH, HTRP, LAG3, NKG2A, NKG2D, NLRC5, PD1, RFX5, RFXANK, RFXAP, ROSA26, RUNX1, SOCS2, TAP 1, TAP 2, Tapasin, TAPBP, TCR a or b constant region, TIGIT, TIM3, and TRAC. 53. The iPSC or the derivative cell thereof according to claim 52, wherein: (i) the at least one exogenous polynucleotide encoding the one or more CARs is integrated at a locus of the AAVS1 gene, the CD33 gene, or the CLYBL gene; (ii) the exogenous polynucleotide encoding the HLA-E and/or the HLA-G is integrated at a locus of the B2M gene; (iii) the exogenous polynucleotide encoding the CD16 protein and/or the NKG2D protein is integrated at a locus of the CD70 gene; (iv) the exogeneous polynucleotide encoding the cytokine is integrated at the locus of the NKG2A gene; (v) the iPSC or the derivative cell thereof comprises the deletion or reduced expression of the CIITA gene; and, optionally (vi) the exogenous polynucleotide encoding the safety switch or the PSMA cell tracer is integrated at the locus of the CIITA gene. 54. The iPSC or the derivative cell thereof according to any one of claims 2-4, 6-8, and 11-53 comprising the dual-targeting CAR, wherein the dual-targeting CAR comprises a CD123 loop. 55. The iPSC or the derivative cell thereof according to any one of claims 2-4, 6-8, and 11-54 comprising the dual-targeting CAR, wherein the dual-targeting CAR comprises one or more polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 126-148. 56. The iPSC or the derivative cell thereof according to any one of claims 2-4, 6-8, and 11-55 comprising the dual-targeting CAR, wherein the dual-targeting CAR comprises one or more polynucleotides having at least 90%, 91%, 92%, 93%, sf-6638784 158 32429-20001.40 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 149-171. 57. The iPSC or the derivative cell thereof according to any one of claims 1-56, comprising the exogenous polynucleotide encoding CXCR4 or the fragment or variant thereof. 58. The iPSC or the derivative cell thereof according to claim 57, wherein the CXCR4 or the fragment or variant thereof is selected from the group consisting of a wild-type CXCR4 and a mutated variant of CXCR4. 59. The iPSC or the derivative cell thereof according to claim 57 comprising the wild-type CXCR4, wherein the wild-type CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:192. 60. The iPSC or the derivative cell thereof according to claim 57 comprising the wild-type CXCR4, wherein the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:200. 61. The iPSC or the derivative cell thereof according to claim 57 comprising the mutated variant of CXCR4, wherein the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with a deletion of the C- terminal domain between 10 and 20 amino acid residues. 62. The iPSC or the derivative cell thereof according to claim 57 comprising the mutated variant of CXCR4, wherein the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A. 63. The iPSC or the derivative cell thereof according to claim 57 comprising the mutated variant of CXCR4, wherein the mutated variant of CXCR4 comprises sf-6638784 159 32429-20001.40 amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 193-199. 64. The derivative cell thereof according to any one of claims 1-31, and 34-63, wherein the derivative cell is a NK cell or a T cell. 65. The derivative cell thereof according to claim 64, wherein the derivative cell is a NK cell. 66. The derivative cell thereof according to claim 64, wherein the derivative cell is a T cell. 67. The derivative cell thereof according to claim 66, wherein the T cell is a gamma delta T cell. 68. The derivative cell thereof according to claim 66 or 67, wherein the T cell is a gamma delta Vγ9/Vδ1 T cell. 69. A composition comprising the iPSC according to any one of the claims 1-63, or the derivative cell thereof of any one of claims 1-31 and 34-68. 70. The composition according to claim 69, further comprising or being used in combination with, one or more therapeutic agents selected from the group consisting of a small-molecule therapeutic agent, a biologic, a peptide, a cytokine, a checkpoint inhibitor, a mitogen, a growth factor, a small RNA, a dsRNA (double stranded RNA), a siRNA, an oligonucleotide, mononuclear blood cells, an antibody, a chemotherapeutic agent, a radioactive moiety, and an immunomodulatory drug (IMiD). 71. An induced pluripotent stem cell (iPSC) or a derivative cell thereof comprising: one or more exogenous polynucleotides encoding: (i) a dual-targeting chimeric antigen receptor (CAR) targeting a CD123 antigen integrated into a CD33 gene locus; (ii) a human leukocyte antigen E (HLA-E) and a human leukocyte antigen G (HLA-G) integrated into a B2M gene locus; sf-6638784 160 32429-20001.40 (iii) a cluster of differentiation 16 (CD16; natural killer (NK) cell receptor immunoglobulin gamma Fc region receptor III (FcγRIII)) protein and/or an NKG2D protein integrated into a CD70 gene locus; (iv) an IL-15 and an IL-15 receptor alpha (IL-15Rα) fusion polypeptide integrated into a NKG2A gene locus; and (v) a C-X-C chemokine receptor type 4 (CXCR4) or a fragment or variant thereof integrated into a CLYBL gene locus; and a deletion or reduced expression of CD33, B2M, CD70, NKG2A, CLYBL, and CIITA genes. 72. The iPSC or the derivative cell thereof according to claim 71, wherein the dual-targeting CAR comprises a CD123 loop. 73. The iPSC or the derivative cell thereof according to claim 71 or 72, wherein the at least one exogenous polynucleotide encoding the dual-targeting CAR comprises (i) nucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 149-171; or (ii) nucleotides encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one or more sequences selected from the group consisting of SEQ ID NOs: 126-148. 74. The iPSC or the derivative cell thereof according to any one of claims 71-73, wherein the exogenous polynucleotide encoding the HLA-E and the HLA-G is integrated into exon 2 of the B2M gene locus. 75. The iPSC or the derivative cell thereof according to any one of claims 71-74, wherein the exogeneous polynucleotide encoding the IL-15 and the IL-15 receptor alpha (IL-15Rα) fusion polypeptide is integrated into exon 3 of the NKG2A gene locus. 76. The iPSC or the derivative cell thereof according to any one of claims 71-75, wherein the CXCR4 or the fragment or variant thereof is selected from the group consisting of a wild-type CXCR4 and a mutated variant of CXCR4. sf-6638784 161 32429-20001.40 77. The iPSC or the derivative cell thereof according to claim 76 comprising the wild-type CXCR4, wherein the wild-type CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 192. 78. The iPSC or the derivative cell thereof according to claim 76 comprising the wild-type CXCR4, wherein the wild-type CXCR4 is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 200. 79. The iPSC or the derivative cell thereof according to claim 76 comprising the mutated variant of CXCR4, wherein the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with a deletion of the C- terminal domain between 10 and 20 amino acid residues. 80. The iPSC or the derivative cell thereof according to claim 76 comprising the mutated variant of CXCR4, wherein the mutated variant of CXCR4 comprises wild-type CXCR4 according to SEQ ID NO: 192 with one or more mutations selected from the group consisting of R334X, G336X, E343X, S341fs, S339fs342X, S338X, E343K, T328X, R144A, E179A, and E262A. 81. The iPSC or the derivative cell thereof according to claim 76 comprising the mutated variant of CXCR4, wherein the mutated variant of CXCR4 comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 193-199. 82. A dual-targeting chimeric antigen receptor (CAR) polypeptide comprising an antigen-binding domain targeting a CD123 antigen. 83. The dual-targeting CAR according to claim 82, wherein the antigen-binding domain targeting the CD123 antigen is a first antigen-binding domain and wherein the first antigen-binding domain comprises an anti-CD123 VHH single domain antibody. sf-6638784 162 32429-20001.40 84. The dual-targeting CAR according to claim 82 or 83, wherein the dual- targeting CAR comprises: (i) optionally, a signal peptide; (ii) an extracellular domain comprising the first antigen-binding domain and the second antigen-binding domain; (iii) a hinge region; (iv) a transmembrane domain; (v) an intracellular signaling domain; and/or (vi) a co-stimulatory domain. 85. The dual-targeting CAR according to any one of claims 82-84, wherein the first antigen-binding domain comprises amino acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 126-148. 86. The dual-targeting CAR according to any one of claims 82-85, wherein the second antigen-binding domain is encoded by a polynucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 149-171. 87. A method of treating cancer in a subject in need thereof, comprising administering the derivative cell thereof according to any one of claims 1-31, 34-68, and 71-81, or the composition according to claim 69 or 70 to a subject in need thereof. 88. The method of treatment according to claim 87, wherein the cancer is selected from the group consisting of leukemias, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), and chronic lymphocytic leukemia (CLL), lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, and follicular lymphoma. sf-6638784 163 32429-20001.40 89. The method of treatment according to claim 87 or 88, wherein the cancer is a B-cell malignancy, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), non- Hodgkin lymphoma, or follicular lymphoma. 90. The method of treatment according to claim 87, wherein the cancer is a myeloid cell malignancy. 91. The method of treatment according to claim 90, wherein the myeloid cell malignancy is acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). 92. A method of manufacturing a derivative cell comprising differentiating the iPSC according to any one of claims 1-56, and 71-81 under conditions for cell differentiation to thereby obtain the derivative cell. 93. The method according to claim 92, wherein the iPSC is obtained by genomic engineering an unmodified iPSC, wherein the genomic engineering comprises targeted editing. 94. The method according to claim 93, wherein the targeted editing comprises deletion, insertion, or in/del carried out by CRISPR, ZFN, TALEN, homing nuclease, or homology recombination. sf-6638784 164
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