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WO2025076196A2 - Chimeric antigen receptors comprising toll-like receptor 2 (tlr2) and cd3 zeta signaling domains and uses thereof - Google Patents

Chimeric antigen receptors comprising toll-like receptor 2 (tlr2) and cd3 zeta signaling domains and uses thereof Download PDF

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Publication number
WO2025076196A2
WO2025076196A2 PCT/US2024/049757 US2024049757W WO2025076196A2 WO 2025076196 A2 WO2025076196 A2 WO 2025076196A2 US 2024049757 W US2024049757 W US 2024049757W WO 2025076196 A2 WO2025076196 A2 WO 2025076196A2
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seq
cell
car
amino acid
acid sequence
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WO2025076196A3 (en
Inventor
Lee Andrew Boland SWANSON
Spencer Matthew GOODMAN
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Shoreline Biosciences Inc
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Shoreline Biosciences Inc
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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/21—Transmembrane domain
    • 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
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • 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
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma

Definitions

  • the ligand-binding domain comprises an antibody-like domain. In some embodiments, the ligand-binding domain comprises an antibody domain. In some embodiments, the ligand-binding domain comprises a variable heavy (VH) domain and/or a variable light (VL) domain. In some embodiments, the ligand-binding domain comprises a single- chain variable fragment (scFv). [0020] In some embodiments, the ligand-binding domain specifically binds a CD19 antigen.
  • the ligand-binding domain comprises the a CDR-H1 according to SEQ ID NO:86; a CDR-H2 according to SEQ ID NO:87; a CDR-H3 according to SEQ ID NO:88; a CDR-L1 according to SEQ ID NO:89; a CDR-L2 according to SEQ ID NO:32; and/or a CDR-L3 according to SEQ ID NO:33.
  • a CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 16.
  • a CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 18.
  • a CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:19.
  • provided herein is a method of treating cancer in a subject in need thereof, comprising administering the pharmaceutical compositions of the present disclosure to the subject.
  • provided herein are one or more polynucleotides encoding the CAR of the present disclosure.
  • one or more vectors comprising the one or more polynucleotides of the present disclosure.
  • FIG. 1D illustrate exemplary CARs with generic ligand-binding and transmembrane domains, and specified hinge (CD8 ⁇ ) and signaling domains (TLR2 and CD3 ⁇ ) (FIG. 1C), and an embodiment of a CAR in the membrane of a cell with a generic ligand-binding and transmembrane domains, and specified hinge (CD8 ⁇ ) and signaling domains (TLR2 and CD3 ⁇ ) (FIG.1D).
  • FIG. 2A – FIG. 2L illustrate expression of CARs in iNK cells on day 17 post- transduction following FACS sorting.
  • the CARs have a cytoplasmic portion that includes a TLR2 signaling domain, and a CD3 ⁇ signaling domain, and either an NKG2D TM (FIG.2B), a CD3 TM (FIG.2C), a CD64 TM (FIG.2D), a CD32a TM (FIG.2E), a CD32c TM (FIG.2F), a CD4 TM (FIG.2G), a CD28 TM (FIG.2H), a TLR2 TM (FIG.2I), a 2B4 TM (FIG.2J), a NTBA TM (FIG. 2K), or a DNAM1 TM (FIG.2L).
  • FIG.4 illustrates results of luciferase release assay for various CD19 CARs expressing the indicated transmembrane domain, and a cytoplasmic portion that includes a TLR2 signaling domain, and a CD3 ⁇ signaling domain at a 0.5:1 effector NK cells to target cell ratio (SupB15, left column; Raji, right column).
  • FIG.5A and FIG.5B illustrate results of luciferase release assays after four hours of incubation for various CD19 CARs expressing the indicated transmembrane domain, and a cytoplasmic portion that includes a TLR2 signaling domain, and a CD3 ⁇ signaling domain at effector NK cell to target cell ratio ranging from 0.25:1 to 15:1 against SupB15 target cells (FIG. 5A), and Raji cells (FIG.5B).
  • FIG.6A – FIG.6J illustrate results of luciferase release assay for untransduced iNK cells (FIG.
  • the term “functional variant” refers to a homology (by sequence or structure) of a domain that retains sufficient signaling activity to activate an NK cell.
  • the terms “percent (%) identity” and “percent (%) identical” as used herein has the same meaning as commonly understood to one of ordinary skill in the art. A representative way to determine percent identity is by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100.
  • the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 27 or a functional variant thereof. In some embodiments, the TLR2 signaling domain consists of an amino acid sequence set forth in SEQ ID NO:26 or SEQ ID NO:27 or a functional variant thereof. In some embodiments, the TLR2 signaling domain consists of the amino acid sequence set forth in SEQ ID NO:26 or a functional variant thereof. In some embodiments, the TLR2 signaling domain consists of the amino acid sequence set forth in SEQ ID NO:27 or a functional variant thereof.
  • the TLR2 signaling domain comprises an amino acid sequence at least 90%, identical to SEQ ID NO: 27. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 95%, identical to SEQ ID NO: 27.
  • Functional variants and/or some or all of the conserved residues of the TLR2 signaling domain may be retained. Functional variants may include N to C terminal truncations, insertions, and/or mutations. Functional variants may comprise a polypeptide sequence sufficient to promote signal propagation using the TLR2 signaling pathway.
  • conserveed residues of the TLR2 signaling domain may comprise residues 698-707 of human TLR2 (NP_001305716.1). (See, e.g., Toshchakov, V.
  • the CD3 ⁇ signaling domain comprises the amino acid sequence SEQ ID NO:30 or a functional variant thereof. In some embodiments, the CD3 ⁇ signaling domain consists of amino acid sequence set forth in SEQ ID NO:30 or a functional variant thereof. In some embodiments, the CD3 ⁇ signaling domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:30. In some embodiments, the CD3 ⁇ signaling domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:30. In some embodiments, the CD3 ⁇ signaling domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:30.
  • the cytoplasmic portion consists of an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the cytoplasmic portion consists of an amino acid sequence at least 99% identical to SEQ ID NO:1. In some embodiments, the cytoplasmic portion consists of the amino acid sequence as set forth in SEQ ID NO: 1.
  • the CD3 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:76.
  • the CD4 transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO: 77. In some embodiments, the CD4 transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO:77.
  • a CD28 transmembrane domain is encoded by the CD28 nucleotide sequence set forth in Table 2. In some embodiments, a CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD28 nucleotide sequence set forth in Table 2. In some embodiments, a CD28 transmembrane domain is encoded by the CD28 nucleotide sequence set forth Table 2.
  • the CD28 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:78.
  • a TLR2 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, a TLR2 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:5.
  • a TLR2 transmembrane domain is encoded by the TLR2 nucleotide sequence set forth in Table 2.
  • a TLR2 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the TLR2 nucleotide sequence set forth in Table 2.
  • a TLR2 transmembrane domain is encoded by the TLR2 nucleotide sequence set forth Table 2.
  • a CD32a transmembrane domain is encoded by the CD32a nucleotide sequence set forth in Table 2.
  • a CD32a transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD32a nucleotide sequence set forth in Table 2.
  • a CD32a transmembrane domain is encoded by the CD32a nucleotide sequence set forth Table 2.
  • the CD32a transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:82.
  • the 2B4 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 10.
  • a 2B4 transmembrane domain is encoded by the 2B4 nucleotide sequence set forth in Table 2.
  • a 2B4 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the 2B4 nucleotide sequence set forth in Table 2.
  • a 2B4 transmembrane domain is encoded by the 2B4 nucleotide sequence set forth Table 2.
  • a 2B4 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the 2B4 nucleotide sequence set forth Table 2.
  • the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 84.
  • the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:84.
  • the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO: 84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:84.
  • the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:81.
  • the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO:81.
  • the NTBA transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:11. [0081] In some embodiments, a NTBA transmembrane domain is encoded by the NTBA nucleotide sequence set forth in Table 2.
  • the ligand- binding domain of the instant disclosure may comprise an antibody-based binding agent that specifically binds an antigen of interest via an antigen or antigenic fragment thereof.
  • the term “antigen” refers to a peptide, polypeptide, glycoprotein, glycan, lipid, glycolipid, or other biomolecule that identifies target cells for treatment by immunotherapy with NK cells.
  • the ligand-binding domain comprises a variable heavy (VH) domain and/or a variable light (VL) domain.
  • the ligand-binding domain comprises a single-chain variable fragment (scFv).
  • the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 90% identity to SEQ ID NO:47. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 95% identity to SEQ ID NO:47.
  • the ligand binding domain comprises an scFv having a polypeptide sequence of SEQ ID NO:41.
  • the extracellular domain of a chimeric receptor comprises an amino acid sequence set forth in SEQ ID NO:51.
  • the extracellular domain of a chimeric receptor consists of an amino acid sequence set forth in SEQ ID NO:51.
  • the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:51.
  • the ligand binding domain comprises an scFv having a polypeptide sequence at least 55% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 70% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 75% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 80% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 90% identical to SEQ ID NO:90.
  • the ligand binding domain comprises an scFv having a polypeptide sequence at least 95% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence of SEQ ID NO:90.
  • the ligand-binding domain comprises an scFv according to SEQ ID NO:43 or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto.
  • the ligand binding domain comprises an scFv having a polypeptide sequence at least 55% identical to SEQ ID NO:43.
  • the ligand binding domain comprises an scFv having a polypeptide sequence at least 70% identical to SEQ ID NO:43.
  • the ligand binding domain comprises an scFv having a polypeptide sequence at least 75% identical to SEQ ID NO:43.
  • the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs:53-55. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:53.
  • the hinge domain is isolated or derived from IgD. [0099] In some embodiments, the hinge domain is isolated or derived from the human CD8 ⁇ molecule.
  • the hinge domain may comprise the polypeptide sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:12) or a variant with 1, 2, 3, 4, 5 or more substitutions.
  • the hinge domain is isolated or derived from the human CD8 molecule.
  • the hinge domain may comprise the polypeptide sequence: TTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:105) or a variant with 1, 2, 3, 4, 5 or more substitutions.
  • the hinge domain is isolated or derived from the human CD28 molecule.
  • the hinge domain may comprise the polypeptide sequence: CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:13) or a variant with 1, 2, 3, 4, 5 or more substitutions.
  • the hinge domain may comprise the polypeptide sequence: LDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:14) or a variant with 1, 2, 3, 4, 5 or more substitutions.
  • the hinge domain is isolated or derived from the human IgG4 molecule.
  • the hinge domain may comprise the polypeptide sequence: ESKYGPPCPPCP (SEQ ID NO:15) or a variant with 1, 2, 3, 4, 5 or more substitutions. 7.1.5.
  • Linkers [0102]
  • a chimeric receptor described herein comprises one or more linkers between the components of the receptors.
  • a chimeric receptor described herein comprises a linker between the extracellular domain and the transmembrane domain.
  • a chimeric receptor described herein comprises a linker between the transmembrane domain and the cytoplasmic domain.
  • a chimeric receptor described herein comprises a linker between two or more intracellular signaling domains of the cytoplasmic domain.
  • the IL-18 polypeptide sequence is at least 80% identical to SEQ ID NO:66. In some embodiments, the IL-18 polypeptide sequence is at least 90% identical to SEQ ID NO:66. In some embodiments, the IL-18 polypeptide sequence is at least 95% identical to SEQ ID NO:66.
  • the co-expressed proteins of the NK cells of the instant disclosure may be fusion proteins.
  • a “fusion protein,” as used herein, refers to an expression product resulting from the fusion of at least two genes.
  • the fusion protein may be a chimeric polypeptide comprising two or more unrelated protein, or protein fragments, conjugated to one another.
  • the IL15/IL15R fusion protein may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:69. In some embodiments, the IL15/IL15R fusion protein may have a polypeptide sequence at least 55% identical to SEQ ID NO:69. In some embodiments, the IL15/IL15R fusion protein may have a polypeptide sequence at least at least 70% identical to SEQ ID NO:69. In some embodiments, the IL15/IL15R fusion protein may have a polypeptide sequence at least 75% identical to SEQ ID NO:69.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno- associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Introduction of a polynucleotide encoding a CAR may be achieved by transduction of either the NK cell or an iPSC or ESC precursor with a viral vector, such as a lentiviral vector or a retroviral vector.
  • a viral vector such as a lentiviral vector or a retroviral vector.
  • Suitable lentiviral vectors are known in the art and are described in, for example, Zufferey et al., (1997); Dull et al., (1998), US6013516A, and US5994136A.
  • Suitable retroviral vectors are also known in the art.
  • the ESC is differentiated into an NK cell (i.e., an ESC-derived NK cell).
  • the NK cell or pluripotent cell for differentiation into an NK cell e.g., an iPSC-derived NK cell, ESC-derived NK cell
  • the cell comprises homozygous inactivating mutations of the cytokine-inducible SH2-containing protein (CISH) genes (e.g., CISH -/- ).
  • CISH cytokine-inducible SH2-containing protein
  • the cell comprises inactivating mutations of the CISH and TGFBR2 genes (e.g., CISH -/- ; TGFBR2 -/- ), and overexpression of a gene encoding CD16.
  • the cell comprises expression and/or overexpression of a gene encoding CD16.
  • the cell comprises inactivating mutations of the CISH and TGFBR2 genes (e.g., CISH -/- ; TGFBR2 -/- ), overexpression of a gene encoding a soluble IL-15 or membrane-bound IL-15, and overexpression of a gene encoding CD16.
  • the disclosure provides a method of killing a target cell comprising contacting a population of target cells with a population of natural killer cells of the disclosure, wherein the chimeric antigen receptors of the natural killer cell comprise a ligand binding domain that specifically binds an antigen on the target cell, and wherein the natural killer cells induce specific killing of the target cells.
  • the disclosure provides method of killing a target cell comprising contacting a population of target cells with a population of NK cells.
  • Cell killing may be assessed by assays known in the art, including but not limited to those described in the examples.
  • the disclosure provides methods of treating cancer in a subject in need thereof, comprising administering a population of NK cells, or pharmaceutical compositions comprising a population of NK cells.
  • subject refers to a vertebrate, such as a mammal, e.g., a human.
  • treatment embraces at least an amelioration of the symptoms associated with a disease or condition in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom associated with the condition being treated.
  • amelioration also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from happening) or stopped (e.g., terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.
  • the CAR NK cells and pharmaceutical compositions comprising the same described herein are used as part of a combination therapy.
  • the CAR NK cells or pharmaceutical compositions of the instant disclosure are co-administered to the subject with tumor targeting antibodies.
  • the disclosure provides a method of treating cancer with the chimeric receptor expressing cells provided herein or pharmaceutical compositions or formulations thereof.
  • the method comprises administering the chimeric receptor expressing cells provided herein, pharmaceutical compositions or formulations described herein to a subject in need thereof.
  • administration and “administering,” as used herein, refer to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and intramuscular, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.
  • a cell expressing a chimeric receptor described herein exhibits enhanced anti-tumor efficacy relative to a cell lacking the chimeric receptor.
  • Methods for measuring anti-tumor efficacy in vitro and in vivo are known to those of skill in the art and include, for example, in vitro killing assays and implanting tumors into mouse models.
  • a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 90% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 100% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 1000% compared to a cell that that lacks the chimeric receptor.
  • a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 40% to 50% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 50% to 60% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 60% to 70% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 70% to 80% compared to a cell that that lacks the chimeric receptor.
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and/or non-human mammals.
  • the pluripotent cell comprises homozygous inactivating mutations of the CISH, TGFBR2, and CIITA genes (e.g., CISH -/- ; TGFBR2 -/- ; CIITA -/- ). In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the CISH, TGFBR2, B2M,, and CIITA genes (e.g., CISH -/- ; TGFBR2 -/- ; B2M -/- ; CIITA -/- ).
  • the pluripotent cell comprises expression and/or overexpression of a gene encoding a soluble IL-15 or membrane-bound IL-15. In some embodiments, the pluripotent cell comprises expression and/or overexpression of a gene encoding CD16. In some embodiments, the pluripotent cell comprises expression and/or overexpression of a gene encoding HLA-E. In some embodiments, the pluripotent cell comprises expression and/or overexpression of a gene encoding a chemokine receptor (e.g., CXCR2).
  • a chemokine receptor e.g., CXCR2
  • a chimeric antigen receptor comprising a ligand-binding domain, a transmembrane domain, and a cytoplasmic portion, wherein the cytoplasmic portion comprises an amino acid sequence at least 90% identical to SEQ ID NO:1 and wherein the transmembrane domain is selected from the group consisting of a CD3, a CD4, a CD28, a TLR2, a CD64, a CD32a, a CD32c, a 2B4, a DNAM1, and a NTBA transmembrane domain, or functional variant thereof.
  • the CAR of embodiment A1, wherein the cytoplasmic portion comprises an amino acid sequence of SEQ ID NO: 1.
  • A20 The CAR of any one of embodiments A16 to A19, wherein the ligand-binding domain specifically binds a CD 19 antigen.
  • the CAR of embodiment A20, wherein the ligand-binding domain comprises the a CDR- H1 according to SEQ ID NO:86; a CDR-H2 according to SEQ ID NO:87; a CDR-H3 according to SEQ ID NO:88; a CDR-L1 according to SEQ ID NO:89; a CDR-L2 according to SEQ ID NO:32; and/or a CDR-L3 according to SEQ ID NO:33.
  • the ligand-binding domain comprises the a CDR- H1 according to SEQ ID NO:86; a CDR-H2 according to SEQ ID NO:87; a CDR-H3 according to SEQ ID NO:88; a CDR-L1 according to SEQ ID NO:89; a CDR-L2 according to SEQ ID NO:32
  • the CAR of embodiment A29, wherein the CD28 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 13 or SEQ ID NO: 14.
  • a CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 16.
  • a CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 19.
  • a cell comprising the CAR of any one of embodiments A1 to A36, wherein the cell is a pluripotent cell.
  • a pharmaceutical composition comprising the cell of any one of embodiments A39 to A41, and a pharmaceutically acceptable solution.
  • a method of making a natural killer cell comprising providing a pluripotent cell with the one or more polynucleotides of embodiment A46 and differentiating the pluripotent cell into a natural killer cell.
  • Example 1 Screening of Chimeric Antigen Receptors Designs
  • TLR2 toll-like receptor 2
  • CD3 ⁇ CD3 zeta
  • iPSC-NK cells are then stimulated with 2:1 aAPCs (irradiated at 10,000 Gy) to NK cells at 350,000 NK cells/mL of media containing CTS NK Xpander media with supplement and 10% human AB serum), supplemented with 50-100 U/mL IL-2.
  • aAPCs irradiated at 10,000 Gy
  • NK cells 350,000 NK cells/mL of media containing CTS NK Xpander media with supplement and 10% human AB serum
  • 50-100 U/mL IL-2 50-100 U/mL IL-2.
  • Transduction of iPSC-NK cells Non-treated 24-well plates were coated with Retronectin (Takara). Equal amount of concentrated retrovirus was added to plates and centrifuged at 2000xg at 32°C for 2 hrs.
  • iPSC-derived NK cells (3e5 cells) were added to each well in media containing IL-2 (50 U/ml) and centrifuged at 800xg at 32°
  • CAR expression was checked by flow cytometry using the CD19 CAR detection reagent. Following CAR expression check, cells were stimulated with 2:1 aAPCs (irradiated at 10,000 Gy) to NK cells at 350,000 NK cells/mL of media containing CTS NK Xpander media with supplement and 10% human AB serum), supplemented with 50-100 U/mL IL-2. On day 9 cells were FACS sorted using CD19 CAR detection reagents. Sorted CAR+ were then stimulated again 2:1 with sAPC.
  • iPSC derived NK cells expressing functional CAR constructs are tested for in vivo efficacy using a CD19 expressing model of B cell malignancies.
  • 10-12 week-old NOD-SCID-gamma -/- (NSG) mice (Jackson Laboratory) are injected intravenously with luciferase expressing Raji cells (CD19 positive).
  • mice After 1 day, the mice are administered iPSC-derived NK cells engineered to express (a) no CAR (negative control); (b) a 2 nd generation T cell CAR (benchmark control); or (c) an NK CAR (test group). Subject mice are administered IL-2 every other day for the remainder of the experiment. Disease progression is monitored by survival, weight loss, and bioluminescent imaging of tumor burden (measured weekly). To assess the number of infused NK cells present during the course of the experiment, CD45 + CD56 + CD3- cells from peripheral blood are quantified weekly.

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Abstract

Provided is a chimeric antigen receptor (CAR) having a ligand-binding domain, a transmembrane domain, and a cytoplasmic portion (e.g., a cytoplasmic portion that includes a toll-like receptor 2 (TLR2) signaling domain, and a CD3 zeta (CD3ζ) signaling domain). Further provided is a cell (e.g., a pluripotent cell, or a NK cell derived from a pluripotent cell) comprising the CAR, and methods for making and using such cells in immunotherapy for cancer. The CAR-NK cells may be CISH-/- iPSC-NK cells.

Description

CHIMERIC ANTIGEN RECEPTORS COMPRISING TOLL-LIKE RECEPTOR 2 (TLR2) AND CD3 ΖETA SIGNALING DOMAINS AND USES THEREOF 1. CROSS REFERENCE [0001] This application claims the benefit of US Provisional Application No.63/587,904 filed October 4, 2023, the entirety of which is hereby incorporated by reference herein in its entirety. 2. SEQUENCE LISTING [0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14735-041-228_SEQLISTING.xml”, was created on September 30, 2024, and is 142,801 bytes in size. 3. FIELD [0003] The present disclosure relates, in part, to chimeric antigen receptors (CARs) comprising a toll-like receptor 2 (TLR2) signaling domain, and a CD3 zeta (CD3ζ) signaling domain, and uses thereof. In some embodiments, the CAR is expressed on a natural killer (NK) cell. 4. BACKGROUND [0004] NK cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. The innate targeting capacity of NK cells can be redirected to a selected cell population by engineering the NK cells to express a chimeric antigen receptor (CAR). Chimeric antigen receptors have a ligand-binding domain designed to specifically bind a target antigen; a transmembrane domain; and a cytoplasmic portion that includes one or more signaling domains. Provided herein are NK cells expressing a CAR with a cytoplasmic portion that includes a TLR2 signaling domain, and a CD3ζ signaling domain, with improved function relative to NK cells that do not express the CAR. 5. SUMMARY [0005] Provided are chimeric antigen receptors for use in engineered natural killer (NK) cells and uses thereof. In one aspect, the disclosure provides chimeric antigen receptors (CARs) having a cytoplasmic portion that includes a toll-like receptor 2 (TLR2) signaling domain, and a CD3 zeta (CD3ζ) signaling domain. [0006] In one aspect, provided herein is a chimeric antigen receptor (CAR) comprising a ligand-binding domain, a transmembrane domain, and a cytoplasmic portion, wherein the cytoplasmic portion comprises an amino acid sequence at least 90% identical to SEQ ID NO:1, and wherein the transmembrane domain is selected from the group consisting of a CD3, a CD4, a CD28, a TLR2, a CD64, a CD32a, a CD32c, a 2B4, a DNAM1, and a NTBA transmembrane domain, or functional variant thereof. [0007] In some embodiments, the cytoplasmic portion comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the cytoplasmic portion consists of an amino acid sequence at least 90% identical to SEQ ID NO:1. In some embodiments, the cytoplasmic portion consists of the amino acid sequence of SEQ ID NO:1. [0008] In some embodiments, the transmembrane domain is selected from the group consisting of the CD32a, the CD32c, the 2B4, and the NTBA transmembrane domain. [0009] In some embodiments, the CD3 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:2. [0010] In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:3. [0011] In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4. [0012] In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:5. [0013] In some embodiments, the CD64 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:6. [0014] In some embodiments, the DNAM1 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:7. [0015] In some embodiments, the CD32a transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:8. [0016] In some embodiments, the CD32c transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:9. [0017] In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:10. [0018] In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:11. [0019] In some embodiments, the ligand-binding domain comprises an antibody-like domain. In some embodiments, the ligand-binding domain comprises an antibody domain. In some embodiments, the ligand-binding domain comprises a variable heavy (VH) domain and/or a variable light (VL) domain. In some embodiments, the ligand-binding domain comprises a single- chain variable fragment (scFv). [0020] In some embodiments, the ligand-binding domain specifically binds a CD19 antigen. [0021] In some embodiments, the ligand-binding domain comprises the a CDR-H1 according to SEQ ID NO:86; a CDR-H2 according to SEQ ID NO:87; a CDR-H3 according to SEQ ID NO:88; a CDR-L1 according to SEQ ID NO:89; a CDR-L2 according to SEQ ID NO:32; and/or a CDR-L3 according to SEQ ID NO:33. In some embodiments, the ligand-binding domain comprises a VH according to SEQ ID NO:38 and/or a VL according to SEQ ID NO:39, or functional variants having polypeptide sequences at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. In some embodiments, the ligand-binding domain comprises an scFv according to SEQ ID NO:40, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto.
[0022] In some embodiments, the ligand-binding domain specifically binds a CD20 antigen. In some embodiments, the ligand-binding domain comprises an scFv according to SEQ ID NO:41, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto.
[0023] In some embodiments, the ligand-binding domain specifically binds a HER2 antigen. In some embodiments, the ligand-binding domain comprises an scFv according to SEQ ID NOs:42, 43, or 90, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto.
[0024] In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the hinge domain selected from the group consisting of a CD8, a CD28, or an IgG4 hinge domain. In some embodiments, the CD8 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 12. In some embodiments, the CD28 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 13 or SEQ ID NO: 14. In some embodiments, the IgG4 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 15.
[0025] In one aspect, provided herein is a CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 16.
[0026] In another aspect, provided herein is a CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID N0:17.
[0027] In a further aspect, provided herein is a CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 18. [0028] In yet another aspect, provided herein is a CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:19.
[0029] In some embodiments, provided herein is a cell comprising the CAR disclosed herein, wherein the cell is a pluripotent cell. In some embodiments, the pluripotent cell is an induced pluripotent cell. In some embodiments, provided herein is a cell comprising the CAR disclosed herein, wherein the cell is a natural killer cell. In some embodiments, the cell is an induced pluripotent stem cell-derived natural killer (iPSC-NK) cell. In some embodiments, the cell comprises homozygous inactivating mutations in the cytokine-inducible SH2-containing protein (CISH) genes of the cell.
[0030] In one aspect, provided herein is a pharmaceutical composition comprising the natural killer cell (e.g., an iPSC-NK cell) disclosed herein, and a pharmaceutically acceptable solution.
[0031] In another aspect, provided herein is a method of killing a target cell comprising contacting a population of target cells with a population of natural killer cells according to the present disclosure, wherein the chimeric antigen receptors of the natural killer cell comprise a ligand-binding domain that specifically binds on antigen on the target cell, and wherein the natural killer cells induce specific killing of the target cells.
[0032] In one aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering the pharmaceutical compositions of the present disclosure to the subject.
[0033] In some aspect, provided herein is use of the natural killer cell of the present disclosure, or the pharmaceutical composition of the present disclosure for treatment of cancer.
[0034] In one aspect, provided herein are one or more polynucleotides encoding the CAR of the present disclosure. In another aspect, provided herein are one or more vectors comprising the one or more polynucleotides of the present disclosure.
[0035] In yet another aspect, provided herein is a cell comprising the one or more polynucleotides of the present disclosure, and the cell is a pluripotent cell or a natural killer cell. In some embodiments, the pluripotent cell is an induced pluripotent cell. In some embodiments, the cell is an induced pluripotent stem cell-derived natural killer (iPSC-NK) cell. [0036] In a further aspect, provided herein is a method of making a natural killer cell, comprising providing a pluripotent cell with the one or more polynucleotides of the present disclosure and differentiating the pluripotent cell into a natural killer cell. [0037] Other aspects and advantages of the invention, will be apparent from the following description and the accompanying drawings. 6. BRIEF DESCRIPTION OF THE DRAWINGS [0038] FIG.1A – FIG.1D illustrate exemplary CARs. FIG.1A illustrates an exemplary CAR with generic ligand-binding, transmembrane, hinge, and signaling domains (FIG. 1A), and an embodiment of a CAR in the membrane of a cell with a generic ligand-binding, transmembrane, hinge, and signaling domains (FIG.1B). FIG. 1C and FIG. 1D illustrate exemplary CARs with generic ligand-binding and transmembrane domains, and specified hinge (CD8α) and signaling domains (TLR2 and CD3ζ) (FIG. 1C), and an embodiment of a CAR in the membrane of a cell with a generic ligand-binding and transmembrane domains, and specified hinge (CD8α) and signaling domains (TLR2 and CD3ζ) (FIG.1D). [0039] FIG. 2A – FIG. 2L illustrate expression of CARs in iNK cells on day 17 post- transduction following FACS sorting. The CARs have a cytoplasmic portion that includes a TLR2 signaling domain, and a CD3ζ signaling domain, and either an NKG2D TM (FIG.2B), a CD3 TM (FIG.2C), a CD64 TM (FIG.2D), a CD32a TM (FIG.2E), a CD32c TM (FIG.2F), a CD4 TM (FIG.2G), a CD28 TM (FIG.2H), a TLR2 TM (FIG.2I), a 2B4 TM (FIG.2J), a NTBA TM (FIG. 2K), or a DNAM1 TM (FIG.2L). [0040] FIG.3A-FIG.3C illustrate results of an NK cell impedance assay for various CD19 CARs expressing the indicated transmembrane domain, and a cytoplasmic portion that includes a TLR2 signaling domain, and a CD3ζ signaling domain at a 5:1 ratio (FIG.3A), a 2.5:1 ratio (FIG. 3B), and a 1.25:1 ratio (FIG.3C) of effector NK cells to target Raji 2.0 cells. [0041] FIG.4 illustrates results of luciferase release assay for various CD19 CARs expressing the indicated transmembrane domain, and a cytoplasmic portion that includes a TLR2 signaling domain, and a CD3ζ signaling domain at a 0.5:1 effector NK cells to target cell ratio (SupB15, left column; Raji, right column). [0042] FIG.5A and FIG.5B illustrate results of luciferase release assays after four hours of incubation for various CD19 CARs expressing the indicated transmembrane domain, and a cytoplasmic portion that includes a TLR2 signaling domain, and a CD3ζ signaling domain at effector NK cell to target cell ratio ranging from 0.25:1 to 15:1 against SupB15 target cells (FIG. 5A), and Raji cells (FIG.5B). [0043] FIG.6A – FIG.6J illustrate results of luciferase release assay for untransduced iNK cells (FIG. 6A), or iNK cells transduced with various CD19 CARs expressing a cytoplasmic portion that includes a TLR2 signaling domain, and a CD3ζ signaling domain and either a NKG2D TM (FIG.6B), a CD4 TM (FIG.6C), a CD32a TM (FIG.6D), a CD32c TM (FIG.6E), a 2B4 TM (FIG.6F), a TLR2 TM (FIG.6G), a CD64 TM (FIG.6H), a NTBA TM (FIG.6I), or a DNAM1 TM (FIG.6J), at effector NK cell to target cell ratios ranging from 0.25:1 to 15:1 against SupB15 target cells (black columns), and Raji target cells (grey columns, where indicated). 7. DETAILED DESCRIPTION [0044] The present disclosure relates, in part, to chimeric antigen receptors (CARs) comprising a TLR2 signaling domain (such as a TLR2 signaling domain described in Section 7.1.1) and a CD3ζ signaling domain (such as a CD3ζ signaling domain described in Section 7.1.1). In some embodiments, the CAR is expressed on a natural killer (NK) cell or a pluripotent cell that is differentiated into an NK cell (such an NK cell described in Section 7.4). Also provided herein, are methods of treating cancer comprising administering the NK cells of the present disclosure, and methods of making the CAR expressing NK cells. [0045] 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 the invention pertains. Otherwise, certain terms used herein have the meanings as set forth in the specification. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control. [0046] As used herein, the abbreviations for the genetically encoded amino acids are conventional and are as follows:
Figure imgf000009_0001
[0047] It should 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.
[0048] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise,” “comprising,” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to.”
[0049] The term “functional variant” refers to a homology (by sequence or structure) of a domain that retains sufficient signaling activity to activate an NK cell. [0050] The terms “percent (%) identity” and “percent (%) identical” as used herein has the same meaning as commonly understood to one of ordinary skill in the art. A representative way to determine percent identity is by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. [0051] As used herein, the terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range. [0052] As used herein, the term “inactivating mutation” refers to a mutation (e.g., deletion, insertion, substitution) in a genomic sequence that disrupts a function of a gene. The inactivating mutation can be in any sequence region (e.g., coding or non-coding) that contributes to gene expression. Examples include, but are not limited to, cis-acting elements (enhancers) or sequences that are subject to transcription (e.g., mRNA transcript sequences). An inactivating mutation includes mutations that render a gene or its encoded protein non-functional or that reduce the function of the gene or its encoded protein. [0053] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney. ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle. J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and CC. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C A. Janeway and P. Travers.1997); Antibodies (P. Finch. 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988- 1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press. 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al., eds., J. B. Lippincott Company, 1993). [0054] In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections, or is directed to various embodiments of the application. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples. The application contemplates use of any of the applicable components in any combination, whether or not a particular combination is expressly described. 7.1. Chimeric Antigen Receptors (CARs) [0055] Provided herein are CARs for use in natural killer (NK) cells, such as an NK cell described in Section 7.4, and uses thereof. In some embodiments, the CARs of the present disclosure comprise a ligand binding domain (such as an ligand-binding domain described in Section Error! Reference source not found.), a transmembrane domain (such as a transmembrane domain described in Section 7.1.2), and a cytoplasmic portion (such as a cytoplasmic portion described in Section 7.1.1). In certain embodiments, the cytoplasmic portion includes a TLR2 signaling domain, such as a TLR2 signaling domain described in Section 7.1.1, and a CD3ζ signaling domain, such as a CD3ζ signaling domain described in Section 7.1.1. In some embodiments, the CAR also comprises a hinge domain (such as a hinge domain described in Section 7.1.4) (FIG.1A and FIG.1B). 7.1.1. Cytoplasmic Portion [0056] In certain aspects, a chimeric receptor described herein comprises a cytoplasmic portion. In some embodiments, the cytoplasmic portion includes a TLR2 signaling domain. In some embodiments, the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:26 or SEQ ID NO:27 or a functional variant thereof. In some embodiments, the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO:26 or a functional variant thereof. In some embodiments, the TLR2 signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 27 or a functional variant thereof. In some embodiments, the TLR2 signaling domain consists of an amino acid sequence set forth in SEQ ID NO:26 or SEQ ID NO:27 or a functional variant thereof. In some embodiments, the TLR2 signaling domain consists of the amino acid sequence set forth in SEQ ID NO:26 or a functional variant thereof. In some embodiments, the TLR2 signaling domain consists of the amino acid sequence set forth in SEQ ID NO:27 or a functional variant thereof. The TLR2 signaling domain may comprise an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:26 or SEQ ID NO:27 or a functional variant thereof. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 55%, identical to SEQ ID NO:26. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 70%, identical to SEQ ID NO:26. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 75%, identical to SEQ ID NO:26. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 80%, identical to SEQ ID NO:26. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 90%, identical to SEQ ID NO:26. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 95%, identical to SEQ ID NO:26. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 55%, identical to SEQ ID NO:27. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 70%, identical to SEQ ID NO: 27. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 75%, identical to SEQ ID NO: 27. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 80%, identical to SEQ ID NO: 27. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 90%, identical to SEQ ID NO: 27. In some embodiments, the TLR2 signaling domain comprises an amino acid sequence at least 95%, identical to SEQ ID NO: 27. Functional variants and/or some or all of the conserved residues of the TLR2 signaling domain may be retained. Functional variants may include N to C terminal truncations, insertions, and/or mutations. Functional variants may comprise a polypeptide sequence sufficient to promote signal propagation using the TLR2 signaling pathway. Conserved residues of the TLR2 signaling domain may comprise residues 698-707 of human TLR2 (NP_001305716.1). (See, e.g., Toshchakov, V. and Neuwald, A., 2020). Further conserved residues of TLR2 may comprise P681, K709, K714, KK742-743, K751, K754, S636, R677, Y715, and/or R753 (Xu, et al., 2000; Mckelvey, et al., 2016; Ben-Ali, et al., 2011; Kang, et al., 2001; Bochud, et al., 2003; Georgel, et al.2009). In some embodiments, the TLR2 signaling domain is encoded by a TLR2 nucleotide sequence set forth in Table 1. In some embodiments, the TLR2 signaling domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a TLR2 nucleotide sequence set forth in Table 1. [0057] In some embodiments, the cytoplasmic portion includes a CD3ζ signaling domain. In some embodiments, the CD3ζ signaling domain comprises the amino acid sequence SEQ ID NO:28 or a functional variant thereof. In some embodiments, the CD3ζ signaling domain consists of amino acid sequence set forth in SEQ ID NO:28 or a functional variant thereof. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:28. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:28. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:28. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:28. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:28. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:28. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:28. In some embodiments, the CD3ζ signaling domain comprises the amino acid sequence SEQ ID NO:29 or a functional variant thereof. In some embodiments, the CD3ζ signaling domain consists of amino acid sequence set forth in SEQ ID NO:29 or a functional variant thereof. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:29. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:29. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:29. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:29. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:29. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:29. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:29. In some embodiments, the CD3ζ signaling domain comprises the amino acid sequence SEQ ID NO:30 or a functional variant thereof. In some embodiments, the CD3ζ signaling domain consists of amino acid sequence set forth in SEQ ID NO:30 or a functional variant thereof. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:30. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:30. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:30. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:30. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:30. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:30. In some embodiments, the CD3ζ signaling domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:30. In some embodiments, the CD3ζ signaling domain is encoded by a nucleotide sequence set forth in Table 1. In some embodiments, the CD3ζ signaling domain is encoded by a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a CD3ζ nucleotide sequence set forth in Table 1. [0058] In some embodiments, the cytoplasmic portion includes a TLR2 signaling domain, and a CD3ζ signaling domain. In specific embodiments, the cytoplasmic portion comprises, in N- to C-terminal order, a TLR2 signaling domain and a CD3ζ signaling domain. In specific embodiments, the cytoplasmic portion consists essentially of, in N- to C-terminal order, a TLR2 signaling domain and a CD3ζ signaling domain. In certain embodiments, the cytoplasmic portion comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1. In certain embodiments, the cytoplasmic portion comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1. In certain embodiments, the cytoplasmic portion comprises an amino acid sequence at least 99% identical to SEQ ID NO:1. In some embodiments, the cytoplasmic portion comprises the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the cytoplasmic portion consists of an amino acid sequence at least 90% identical to SEQ ID NO:1. In some embodiments, the cytoplasmic portion consists of an amino acid sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the cytoplasmic portion consists of an amino acid sequence at least 99% identical to SEQ ID NO:1. In some embodiments, the cytoplasmic portion consists of the amino acid sequence as set forth in SEQ ID NO: 1.
Table 1: Signaling domains.
Figure imgf000015_0001
Figure imgf000016_0001
7.1.2. Transmembrane Domain. [0059] In certain aspects, a chimeric receptor described herein comprises a transmembrane domain. In some embodiments, the transmembrane domain is operably linked to the extracellular domain with or without a linker. In some embodiments, the transmembrane domain is operably linked to the extracellular domain via a hinge domain (such as a hinge domain described in Section 7.1.4). In some embodiments, the transmembrane domain is a naturally occurring transmembrane domain derived from a protein. In some embodiments, the transmembrane domain is a synthetic transmembrane domain. In some embodiments, the transmembrane domain comprises a full transmembrane domain of a polypeptide. In some embodiments, the transmembrane domain comprises a fragment of a transmembrane domain of a polypeptide. [0060] The transmembrane domain may be derived either from a natural or from a synthetic source. In some embodiments, the transmembrane domain is derived from a human source. In some embodiments, the transmembrane domain is derived from a non-human source. Transmembrane regions that may be used include the transmembrane domains of CD3, CD4, CD28, TLR2, CD64, CD32a, CD32c, 2B4, DNAM1, and NTBA, or a functional variant thereof. In certain embodiments, the transmembrane domain is selected from the group consisting of the CD32a, the CD32c, the 2B4, and the NTBA transmembrane domain. In certain embodiments, the transmembrane domain is selected from the group consisting of the CD32a, the CD28, the 2B4, and the NTBA transmembrane domain. In specific embodiments, the transmembrane domain is not an NKG2D transmembrane domain. In some embodiments, the transmembrane domain is derived either from a human source. Illustrative transmembrane domains are provided in Table 2. [0061] Table 2: Transmembrane (TM) domains
Figure imgf000017_0001
Figure imgf000018_0001
[0062] In some embodiments, the transmembrane domain is a transmembrane domain of CD3. In some embodiments, the transmembrane domain is a fragment of a CD3 transmembrane domain. In some embodiments, a CD3 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, a CD3 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, a CD3 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2. In some embodiments, the CD3 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:2. In some embodiments, the CD3 transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:2. In some embodiments, the CD3 transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:2. In some embodiments, the CD3 transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:2. In some embodiments, the CD3 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:2. In some embodiments, the CD3 transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:2. In some embodiments, the CD3 transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:2. In some embodiments, the CD3 transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:2. [0063] In some embodiments, a CD3 transmembrane domain is encoded by the CD3 nucleotide sequence set forth in Table 2. In some embodiments, a CD3 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD3 nucleotide sequence set forth in Table 2. In some embodiments, a CD3 transmembrane domain is encoded by the CD3 nucleotide sequence set forth Table 2. In some embodiments, a CD3 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD3 nucleotide sequence set forth Table 2. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:76. In some embodiments, the CD3 transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO:76. [0064] In some embodiments, the transmembrane domain is a transmembrane domain of CD4. In some embodiments, the transmembrane domain is a fragment of a CD4 transmembrane domain. In some embodiments, a CD4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, a CD4 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, a CD4 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:3. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:3. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:3. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:3. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:3. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:3. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:3. In some embodiments, the CD4 transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:3.
[0065] In some embodiments, a CD4 transmembrane domain is encoded by the CD4 nucleotide sequence set forth in Table 2. In some embodiments, a CD4 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD4 nucleotide sequence set forth in Table 2. In some embodiments, a CD4 transmembrane domain is encoded by the CD4 nucleotide sequence set forth Table 2. In some embodiments, a CD4 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD4 nucleotide sequence set forth Table 2. In some embodiments, the CD4 transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO:77. In some embodiments, the CD4 transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:77. In some embodiments, the CD4 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:77. In some embodiments, the CD4 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:77. In some embodiments, the CD4 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:77. In some embodiments, the CD4 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:77. In some embodiments, the CD4 transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO: 77. In some embodiments, the CD4 transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO:77.
[0066] In some embodiments, the transmembrane domain is a transmembrane domain of CD28. In some embodiments, the transmembrane domain is a fragment of a CD28 transmembrane domain. In some embodiments, a CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, a CD28 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, a CD28 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:4. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:4. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:4. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:4. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:4. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:4. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:4.
[0067] In some embodiments, a CD28 transmembrane domain is encoded by the CD28 nucleotide sequence set forth in Table 2. In some embodiments, a CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD28 nucleotide sequence set forth in Table 2. In some embodiments, a CD28 transmembrane domain is encoded by the CD28 nucleotide sequence set forth Table 2. In some embodiments, a CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD28 nucleotide sequence set forth Table 2. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:78. In some embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO:78. [0068] In some embodiments, the transmembrane domain is a transmembrane domain of TLR2. In some embodiments, the transmembrane domain is a fragment of a TLR2 transmembrane domain. In some embodiments, a TLR2 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, a TLR2 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, a TLR2 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:5. In some embodiments, the TLR2 transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:5. [0069] In some embodiments, a TLR2 transmembrane domain is encoded by the TLR2 nucleotide sequence set forth in Table 2. In some embodiments, a TLR2 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the TLR2 nucleotide sequence set forth in Table 2. In some embodiments, a TLR2 transmembrane domain is encoded by the TLR2 nucleotide sequence set forth Table 2. In some embodiments, a TLR2 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the TLR2 nucleotide sequence set forth Table 2. In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO:79. In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:79. In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:79. In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:79. In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:79. In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:79. In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:79. In some embodiments, the TLR2 transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO:79. [0070] In some embodiments, the transmembrane domain is a transmembrane domain of CD64. In some embodiments, the transmembrane domain is a fragment of a CD64 transmembrane domain. In some embodiments, a CD64 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, a CD64 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, a CD64 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6. In some embodiments, the CD64 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:6. In some embodiments, the CD64 transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:6. In some embodiments, the CD64 transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:6. In some embodiments, the CD64 transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:6. In some embodiments, the CD64 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:6. In some embodiments, the CD64 transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:6. In some embodiments, the CD64 transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:6. In some embodiments, the CD64 transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:6. [0071] In some embodiments, a CD64 transmembrane domain is encoded by the CD64 nucleotide sequence set forth in Table 2. In some embodiments, a CD64 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD64 nucleotide sequence set forth in Table 2. In some embodiments, a CD64 transmembrane domain is encoded by the CD64 nucleotide sequence set forth Table 2. In some embodiments, a CD64 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD64 nucleotide sequence set forth Table 2. In some embodiments, the CD64 transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO:80. In some embodiments, the CD64 transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:80. In some embodiments, the CD64 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:80. In some embodiments, the CD64 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:80. In some embodiments, the CD64 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:80. In some embodiments, the CD64 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:80. In some embodiments, the CD64 transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:80. In some embodiments, the CD64 transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO:80. [0072] In some embodiments, the transmembrane domain is a transmembrane domain of CD32a. In some embodiments, the transmembrane domain is a fragment of a CD32a transmembrane domain. In some embodiments, a CD32a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, a CD32a transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, a CD32a transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:8. In some embodiments, the CD32a transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:8. In some embodiments, the CD32a transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:8. In some embodiments, the CD32a transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:8. In some embodiments, the CD32a transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:8. In some embodiments, the CD32a transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 8. In some embodiments, the CD32a transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 8. In some embodiments, the CD32a transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 8. In some embodiments, the CD32a transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:8.
[0073] In some embodiments, a CD32a transmembrane domain is encoded by the CD32a nucleotide sequence set forth in Table 2. In some embodiments, a CD32a transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD32a nucleotide sequence set forth in Table 2. In some embodiments, a CD32a transmembrane domain is encoded by the CD32a nucleotide sequence set forth Table 2. In some embodiments, a CD32a transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD32a nucleotide sequence set forth Table 2. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:82. In some embodiments, the CD32a transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO: 82.
[0074] In some embodiments, the transmembrane domain is a transmembrane domain of CD32c. In some embodiments, the transmembrane domain is a fragment of a CD32c transmembrane domain. In some embodiments, a CD32c transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, a CD32c transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, a CD32c transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:9. In some embodiments, the CD32c transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:9. In some embodiments, the CD32c transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:9. In some embodiments, the CD32c transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:9. In some embodiments, the CD32c transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:9. In some embodiments, the CD32c transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:9. In some embodiments, the CD32c transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:9. In some embodiments, the CD32c transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:9. In some embodiments, the CD32c transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:9.
[0075] In some embodiments, a CD32c transmembrane domain is encoded by the CD32c nucleotide sequence set forth in Table 2. In some embodiments, a CD32c transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD32c nucleotide sequence set forth in Table 2. In some embodiments, a CD32c transmembrane domain is encoded by the CD32c nucleotide sequence set forth Table 2. In some embodiments, a CD32c transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the CD32c nucleotide sequence set forth Table 2. In some embodiments, the CD32c transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO:83. In some embodiments, the CD32c transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:83. In some embodiments, the CD32c transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:83. In some embodiments, the CD32c transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:83. In some embodiments, the CD32c transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:83. In some embodiments, the CD32c transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:83. In some embodiments, the CD32c transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:83. In some embodiments, the CD32c transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO: 83.
[0076] In some embodiments, the transmembrane domain is a transmembrane domain of 2B4. In some embodiments, the transmembrane domain is a fragment of a 2B4 transmembrane domain. In some embodiments, a 2B4 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, a 2B4 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, a 2B4 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence identical to SEQ ID NO: 10.
[0077] In some embodiments, a 2B4 transmembrane domain is encoded by the 2B4 nucleotide sequence set forth in Table 2. In some embodiments, a 2B4 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the 2B4 nucleotide sequence set forth in Table 2. In some embodiments, a 2B4 transmembrane domain is encoded by the 2B4 nucleotide sequence set forth Table 2. In some embodiments, a 2B4 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the 2B4 nucleotide sequence set forth Table 2. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO: 84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO: 84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:84. In some embodiments, the 2B4 transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO: 84.
[0078] In some embodiments, the transmembrane domain is a transmembrane domain of DNAM1. In some embodiments, the transmembrane domain is a fragment of a DNAM1 transmembrane domain. In some embodiments, a DNAM1 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, a DNAM1 transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, a DNAM1 transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:7. In some embodiments, the DNAM1 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:7. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:7. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:7. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:7. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:7. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:7. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:7. In some embodiments, the 2B4 transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:7.
[0079] In some embodiments, a DNAM1 transmembrane domain is encoded by the DNAM1 nucleotide sequence set forth in Table 2. In some embodiments, aDNAMl transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the DNAM1 nucleotide sequence set forth in Table 2. In some embodiments, a DNAM1 transmembrane domain is encoded by the DNAM1 nucleotide sequence set forth Table 2. In some embodiments, a DNAM1 transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the DNAM1 nucleotide sequence set forth Table 2. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:81. In some embodiments, the DNAM1 transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO:81.
[0080] In some embodiments, the transmembrane domain is a transmembrane domain of NTBA. In some embodiments, the transmembrane domain is a fragment of a NTBA transmembrane domain. In some embodiments, a NTBA transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, a NTBA transmembrane domain consists of the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, a NTBA transmembrane domain comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence at least 55% identical to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence at least 70% identical to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence at least 75% identical to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence at least 80% identical to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence at least 90% identical to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:11. In some embodiments, the NTBA transmembrane domain comprises an amino acid sequence identical to SEQ ID NO:11. [0081] In some embodiments, a NTBA transmembrane domain is encoded by the NTBA nucleotide sequence set forth in Table 2. In some embodiments, a NTBA transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the NTBA nucleotide sequence set forth in Table 2. In some embodiments, a NTBA transmembrane domain is encoded by the NTBA nucleotide sequence set forth Table 2. In some embodiments, a NTBA transmembrane domain is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the NTBA nucleotide sequence set forth Table 2. In some embodiments, the NTBA transmembrane domain is encoded by a nucleotide sequence at least 80% identical to SEQ ID NO:85. In some embodiments, the NTBA transmembrane domain is encoded by a nucleotide sequence at least 85% identical to SEQ ID NO:85. In some embodiments, the NTBA transmembrane domain is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:85. In some embodiments, the NTBA transmembrane domain is encoded by a nucleotide sequence at least 95% identical to SEQ ID NO:85. In some embodiments, the NTBA transmembrane domain is encoded by a nucleotide sequence at least 97% identical to SEQ ID NO:85. In some embodiments, the NTBA transmembrane domain is encoded by a nucleotide sequence at least 98% identical to SEQ ID NO:85. In some embodiments, the NTBA transmembrane domain is encoded by a nucleotide sequence at least 99% identical to SEQ ID NO:85. In some embodiments, the NTBA transmembrane domain is encoded by a nucleotide sequence identical to SEQ ID NO:85. [0082] In some embodiments, the transmembrane is not an NKG2D transmembrane domain. 7.1.3. Ligand-Binding Domain [0083] In certain aspects, the disclosure provides CARs having a ligand-binding domain. In some embodiments, the ligand-binding domain comprises an antibody-like domain. The ligand- binding domain of the instant disclosure may comprise an antibody-based binding agent that specifically binds an antigen of interest via an antigen or antigenic fragment thereof. As used herein, the term “antigen” refers to a peptide, polypeptide, glycoprotein, glycan, lipid, glycolipid, or other biomolecule that identifies target cells for treatment by immunotherapy with NK cells. [0084] In some embodiments, the ligand-binding domain comprises a variable heavy (VH) domain and/or a variable light (VL) domain. In some embodiments, the ligand-binding domain comprises a single-chain variable fragment (scFv). An scFv protein is a fusion protein in which a light chain variable region (VL) of an antibody and a heavy chain variable region (VH) of an antibody expressed as fusion polypeptide, optionally with an intervening link, such as a Gly-Ser linker, in either VH-VL or VL-VH orientation. The scFv may be an dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. [0085] In certain embodiments, the ligand-binding domain specifically binds a CD19 antigen. In some embodiments, the ligand-binding domain comprises a CDR-H1 according to SEQ ID NO:86; a CDR-H2 according to SEQ ID NO:87; a CDR-H3 according to SEQ ID NO:88; a CDR- L1 according to SEQ ID NO:89; a CDR-L2 according to SEQ ID NO:32; and/or a CDR-L3 according to SEQ ID NO:33. In some embodiments, the ligand-binding domain comprises a VH according to SEQ ID NO:38 and/or a VL according to SEQ ID NO:39, or functional variants having polypeptide sequences at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. In some embodiments, the ligand-binding domain comprises an scFv according to SEQ ID NO:40, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. In some embodiments, the ligand-binding domain comprises an scFv having a polypeptide sequence at least 55% identity to SEQ ID NO:40. In some embodiments, the ligand-binding domain comprises an scFv having a polypeptide sequence at least 70% identity to SEQ ID NO:40. In some embodiments, the ligand-binding domain comprises an scFv having a polypeptide sequence at least 80% identity to SEQ ID NO:40. In some embodiments, the ligand-binding domain comprises an scFv having a polypeptide sequence at least 90% identity to SEQ ID NO:40. In some embodiments, the ligand-binding domain comprises an scFv having a polypeptide sequence at least 95% identity to SEQ ID NO:40. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence set forth in SEQ ID NO:47. In some embodiments, the extracellular domain of a chimeric receptor consists of an amino acid sequence set forth in SEQ ID NO:47. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:47. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80% identity to SEQ ID NO:47. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 85% identity to SEQ ID NO:47. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 90% identity to SEQ ID NO:47. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 95% identity to SEQ ID NO:47.
[0086] In some embodiments, the extracellular domain of a chimeric receptor is encoded by the nucleotide sequence set forth in SEQ ID NO:48. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:48. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 80% identity to SEQ ID NO:48. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 85% identity to SEQ ID NO:48. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 90% identity to SEQ ID NO:48. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 95% identity to SEQ ID NO:48.
[0087] In certain embodiments, the ligand-binding domain specifically binds a CD20 antigen. CD20 is a B-cell lineage-specific antigens expressed on the cell surface of most B-cell lymphomas, particularly in late stages of B-cell lymphogenesis. In some embodiments, the ligand-binding domain comprises an scFv according to SEQ ID NO:41, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 55% identical to SEQ ID NO:41. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 70% identical to SEQ ID NO:41. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 75% identical to SEQ ID NO:41. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 80% identical to SEQ ID NO:41. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 90% identical to SEQ ID NO:41. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 95% identical to SEQ ID NO:41. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence of SEQ ID NO:41. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence set forth in SEQ ID NO:51. In some embodiments, the extracellular domain of a chimeric receptor consists of an amino acid sequence set forth in SEQ ID NO:51. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:51. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80% identity to SEQ ID NO:51. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 85% identity to SEQ ID NO:51. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 90% identity to SEQ ID NO:51. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 95% identity to SEQ ID NO:51. The ligand binding domain may comprise a VH sequence according to SEQ ID NO:49 and/or a VL sequence according to SEQ ID NO:50, or a functional variants thereof having a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:49 and SEQ ID NO:50, respectively. In some embodiments, the ligand binding domain comprises a VH having a polypeptide sequence at least 55% identical to SEQ ID NO:49. In some embodiments, the ligand binding domain comprises a VH having a polypeptide sequence at least 55% identical to SEQ ID NO:49. In some embodiments, the ligand binding domain comprises a VH having a polypeptide sequence at least 70% identical to SEQ ID NO:49. In some embodiments, the ligand binding domain comprises a VH having a polypeptide sequence at least 75% identical to SEQ ID NO:49. In some embodiments, the ligand binding domain comprises a VH having a polypeptide sequence at least 80% identical to SEQ ID NO:49. In some embodiments, the ligand binding domain comprises a VH having a polypeptide sequence at least 90% identical to SEQ ID NO:49. In some embodiments, the ligand binding domain comprises a VH having a polypeptide sequence at least 95% identical to SEQ ID NO:49. In some embodiments, the ligand binding domain comprises a VH having a polypeptide sequence of SEQ ID NO:49. In some embodiments, the ligand binding domain comprises a VL having a polypeptide sequence at least 55% identical to SEQ ID NO:50. In some embodiments, the ligand binding domain comprises a VL having a polypeptide sequence atleast 70% identical to SEQ ID NO:50. In some embodiments, the ligand binding domain comprises a VL having a polypeptide sequence at least 75% identical to SEQ ID NO:50. In some embodiments, the ligand binding domain comprises a VL having a polypeptide sequence at least 80% identical to SEQ ID NO: 50. In some embodiments, the ligand binding domain comprises a VL having a polypeptide sequence at least 90% identical to SEQ ID NO:50. In some embodiments, the ligand binding domain comprises a VL having a polypeptide sequence at least 95% identical to SEQ ID NO: 50. In some embodiments, the ligand binding domain comprises a VL having a polypeptide sequence of SEQ ID NO:50.
[0088] In some embodiments, the extracellular domain of a chimeric receptor is encoded by the nucleotide sequence set forth in SEQ ID NO: 52. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:52. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 80% identity to SEQ ID NO:52. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 85% identity to SEQ ID NO:52. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 90% identity to SEQ ID NO: 52. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 95% identity to SEQ ID NO:52.
[0089] In certain embodiments, the ligand-binding domain may bind a cancer antigen. In specific embodiments, the ligand-binding domain may bind an antigen associated with a solid cancer, e.g., an antigen present on a tumor cell. For example, functional activation of human epidermal growth factor receptor 2 (HER2) has been shown to strongly promote carcinogenesis. In some embodiments, the ligand-binding domain specifically binds a HER2 antigen. In some embodiments, the ligand-binding domain comprises an scFv according to SEQ ID NO:90 or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 55% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 70% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 75% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 80% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 90% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 95% identical to SEQ ID NO:90. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence of SEQ ID NO:90.
[0090] In some embodiments, the ligand-binding domain comprises an scFv according to SEQ ID NO:42 or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 55% identical to SEQ ID NO:42. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 70% identical to SEQ ID NO:42. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 75% identical to SEQ ID NO:42. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 80% identical to SEQ ID NO:42. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 90% identical to SEQ ID NO:42. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 95% identical to SEQ ID NO:42. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence of SEQ ID NO:42. In some embodiments, the ligand-binding domain comprises an scFv according to SEQ ID NO:43 or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 55% identical to SEQ ID NO:43. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 70% identical to SEQ ID NO:43. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 75% identical to SEQ ID NO:43. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 80% identical to SEQ ID NO:43. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 90% identical to SEQ ID NO:43. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence at least 95% identical to SEQ ID NO:43. In some embodiments, the ligand binding domain comprises an scFv having a polypeptide sequence of SEQ ID NO:43.
[0091] In some embodiments, a chimeric receptor described herein comprises an extracellular domain comprising an ligand binding domain or binding fragment thereof of BCMA. BCMA is preferentially expressed in mature B lymphocytes and is important for B cell development.
[0092] In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence set forth in SEQ ID NOs:53-55. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence set forth in SEQ ID NO:53. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence set forth in SEQ ID NO:54. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence set forth in SEQ ID NO:55. In some embodiments, the extracellular domain of a chimeric receptor consists of an amino acid sequence set forth in SEQ ID NOs:53-55. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NOs:53-55. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:53. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:54. In some embodiments, the extracellular domain of a chimeric receptor comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 55.
[0093] In some embodiments, the extracellular domain of a chimeric receptor is encoded by the nucleotide sequence set forth in SEQ ID NOs:57-60. In some embodiments, the extracellular domain of a chimeric receptor is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID Nos:57-60.
[0094] Table 3: Exemplary Ligand Binding Domain Sequences
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
7.1.4. Hinge Domain [0095] In an aspect, the disclosure provides CARs having a hinge domain. The hinge domain of the CAR of the disclosure may comprise a linker or spacer sequence between the extracellular antigen binding domain and the transmembrane domain. One of ordinary skill in the art will appreciate that a hinge domain (e.g., sequence) is a polypeptide segment that, in at least some instances, facilitates flexibility (see, e.g., Woof et al., Nat. Rev. Immunol., 4(2): 89-99 (2004)). The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. [0096] The hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In certain embodiments, the spacer domain includes at least a portion of an IgG1, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains. In some embodiments, the spacer domain may also include at least a portion of a corresponding immunoglobulin hinge region. [0097] In some embodiments, a portion of the immunoglobulin constant region serves as a hinge region between the antigen binding domain, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. Exemplary hinges include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a hinge has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. In some embodiments, the hinge is at or about 12 amino acids in length. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number WO2014031687, U.S. Pat. Pub. No. US 2014/0271635. [0098] The CARs of the disclosure may comprise a hinge domain isolated or derived from CD4, CD8α, CD28, IgG1, IgG2, IgG4, or IgD. In some embodiments, the hinge domain is isolated or derived from CD4. In some embodiments, the hinge domain is isolated or derived from CD8α. In some embodiments, the hinge domain is isolated or derived from CD28. In some embodiments, the hinge domain is isolated or derived from IgG1. In some embodiments, the hinge domain is isolated or derived from IgG2. In some embodiments, the hinge domain is isolated or derived from IgG4. In some embodiments, the hinge domain is isolated or derived from IgD. [0099] In some embodiments, the hinge domain is isolated or derived from the human CD8α molecule. The hinge domain may comprise the polypeptide sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:12) or a variant with 1, 2, 3, 4, 5 or more substitutions. In some embodiments, the hinge domain is isolated or derived from the human CD8 molecule. The hinge domain may comprise the polypeptide sequence: TTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:105) or a variant with 1, 2, 3, 4, 5 or more substitutions. [0100] In some embodiments, the hinge domain is isolated or derived from the human CD28 molecule. The hinge domain may comprise the polypeptide sequence: CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:13) or a variant with 1, 2, 3, 4, 5 or more substitutions. The hinge domain may comprise the polypeptide sequence: LDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO:14) or a variant with 1, 2, 3, 4, 5 or more substitutions. [0101] In some embodiments, the hinge domain is isolated or derived from the human IgG4 molecule. The hinge domain may comprise the polypeptide sequence: ESKYGPPCPPCP (SEQ ID NO:15) or a variant with 1, 2, 3, 4, 5 or more substitutions. 7.1.5. Linkers [0102] In some embodiments, a chimeric receptor described herein comprises one or more linkers between the components of the receptors. In some embodiments, a chimeric receptor described herein comprises a linker between the extracellular domain and the transmembrane domain. In some embodiments, a chimeric receptor described herein comprises a linker between the transmembrane domain and the cytoplasmic domain. In some embodiments, a chimeric receptor described herein comprises a linker between two or more intracellular signaling domains of the cytoplasmic domain. Gly-Ser Linkers [0103] Linkers suitable for joining polypeptide sequences are known to those of skill in the art. Exemplary linkers include gly-ser polypeptide linkers, glycine -proline polypeptide linkers, and proline- alanine polypeptide linkers. In certain embodiments, the linker is a gly-ser polypeptide linker, i.e., a peptide that consists of glycine and serine residues. [0104] As used herein, the term “gly-ser linker” refers to a peptide that consists of glycine and serine residues. An exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n (SEQ ID NO: 106). In some embodiments, n=1. In certain embodiments, n=2. In certain embodiments, n=3, i.e., Ser(Gly4Ser)3 (SEQ ID NO: 107). In some embodiments, n=4, i.e., Ser(Gly4Ser)4 (SEQ ID NO: 108). In certain embodiments, n=5. In certain embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In certain embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence (Gly4Ser)n (SEQ ID NO: 109). In some embodiments, n=1. In certain embodiments, n=2. In certain embodiments, n=3. In some embodiments, n=4. In certain embodiments, n=5. In certain embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence (Gly3Ser)n (SEQ ID NO: 110). In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Exemplary gly-ser polypeptide linkers comprise the amino acid sequence Ser(Gly4Ser)n (SEQ ID NO: 106). In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly4Ser)3 (SEQ ID NO: 107). In some embodiments, n=4, i.e., Ser(Gly4Ser)4 (SEQ ID NO: 108). In some embodiments, n=5. In certain embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In certain embodiments, n=9. In some embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n (SEQ ID NO: 111). In certain embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In certain embodiments, n=4. In some embodiments, n=5. In certain embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly4Ser)n (SEQ ID NO: 109). In some embodiments, n=1. In some embodiments, n=2. In certain embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In certain embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly3Ser)n (SEQ ID NO: 110). In some embodiments, n=1. In some embodiments, n=2. In certain embodiments, n=3. In certain embodiments, n=4. In some embodiments, n=5. In some embodiments n=6. Additional Linkers [0105] Additional linkers suitable for joining polypeptide sequences to allow bi-cistronic expression are known to those of skill in the art. Exemplary linkers include Furin GSG-T2A. 7.1.6. Signal peptides [0106] The chimeric antigen receptor may be expressed from a polynucleotide sequence that encodes an N-terminal signal peptide. Signal peptides may direct their fusion partners to be integrated into the plasma membrane of the cell. For example, the signal peptide comprises the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO:63), or a functional variant with 1, 2, 3 or more amino acid substitutions. 7.1.7. Exemplary Chimeric Antigen Receptors [0107] In some embodiments, a chimeric antigen receptor described herein comprises a ligand binding domain, a transmembrane domain, and a cytoplasmic portion that comprises an amino acid sequence at least 90% identical to SEQ ID NO:1. In some embodiments, the transmembrane domain is selected from the group consisting of a CD3, a CD4, a CD28, a TLR2, a CD64, a CD32a, a CD32c, a 2B4, a DNAM1, and a NTBA transmembrane domain, or functional variant thereof. In some embodiments, the CAR is a CAR provided in Table 4.
[0108] Table 4: Exemplary CARs
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
7.2. Co-Expressed Partners [0109] In an aspect, the NK CAR cells of the disclosure are engineered to co-express additional proteins (either membrane bound or secreted). Exemplary compositions and methods are provided in US 2017/0073638 A1. Co-expressed partners [0110] Activity of NK cells may be increased by co-expressing various factors, including but not limited to those provided in Table 7. [0111] Table 5: Co-expression partners
Figure imgf000063_0001
Figure imgf000064_0001
[0112] In some variations, the NK cells of the disclosure are engineered to express Interleukin- 15 (IL-15) (either membrane bound or secreted). IL-15 is a cytokine that plays a role in regulating immune response. Without being bound by theory, expression of IL- 15 may improve NK cell function. The IL-15 may comprise the polypeptide sequence set forth in SEQ ID NO:65. The IL- 15 may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:65. In some embodiments, the IL-15 polypeptide sequence is at least 55% identical to SEQ ID NO:65. In some embodiments, the IL- 15 polypeptide sequence is at least 70% identical to SEQ ID NO:65. In some embodiments, the IL-15 polypeptide sequence is at least 75% identical to SEQ ID NO:65. In some embodiments, the IL-15 polypeptide sequence is at least 80% identical to SEQ ID NO:65. In some embodiments, the IL-15 polypeptide sequence is at least 90% identical to SEQ ID NO:65. In some embodiments, the IL-15 polypeptide sequence is at least 95% identical to SEQ ID NO:65. In some embodiments, the IL-15 is encoded by the nucleotide sequence set forth in SEQ ID NO:71. In some embodiments, the IL-15 is encoded by a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:71. In some embodiments, the IL-15 nucleotide sequence is at least 55% identical to SEQ ID NO:71. In some embodiments, the IL-15 nucleotide sequence is at least 70% identical to SEQ ID NO:71. In some embodiments, the IL-15 nucleotide sequence is at least 75% identical to SEQ ID NO:71. In some embodiments, the IL-15 nucleotide sequence is at least 80% identical to SEQ ID NO:71. In some embodiments, the IL-15 nucleotide sequence is at least 90% identical to SEQ ID NO:71. In some embodiments, the IL-15 nucleotide sequence is at least 95% identical to SEQ ID NO:71. atgagaatttcgaaaccacatttgagaagtatttccatccagtgctacttgtgtttacttctaaacagtcattttctaactgaagctggc attcatgtcttcattttgggctgtttcagtgcagggcttcctaaaacagaagccaactgggtgaatgtaataagtgatttgaaaaaaat tgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagt gctttctcttggagttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaac aacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttg cagagttttgtacatattgtccaaatgttcatcaacacttct (SEQ ID NO:71) [0113] Alternatively, the IL-15 may comprise the polypeptide sequence set forth in SEQ ID NO:64. The IL-15 may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:64. In some embodiments, the IL-15 polypeptide sequence is at least 55% identical to SEQ ID NO:64. In some embodiments, the IL-15 polypeptide sequence is at least 70% identical to SEQ ID NO:64. In some embodiments, the IL-15 polypeptide sequence is at least 75% identical to SEQ ID NO:64. In some embodiments, the IL-15 polypeptide sequence is at least 80% identical to SEQ ID NO:64. In some embodiments, the IL-15 polypeptide sequence is at least 90% identical to SEQ ID NO:64. In some embodiments, the IL-15 polypeptide sequence is at least 95% identical to SEQ ID NO:64. [0114] In some variations, the NK cells of the disclosure are engineered to express Interleukin- 18 (IL-18) (either membrane bound or secreted). IL-18 is a pro-inflammatory cytokine that plays a role in immune cell activation and response. Without being bound by theory, expression of IL- 18 may enhance NK cell proliferation, maturation, and cytotoxicity. The IL-18 may comprise the polypeptide sequence set forth in SEQ ID NO: 67. The IL-18 may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 67. In some embodiments, the IL-18 polypeptide sequence is at least 55% identical to SEQ ID NO:67. In some embodiments, the IL-18 polypeptide sequence is at least 70% identical to SEQ ID NO:67. In some embodiments, the IL-18 polypeptide sequence is at least 75% identical to SEQ ID NO:67. In some embodiments, the IL-18 polypeptide sequence is at least 80% identical to SEQ ID NO:67. In some embodiments, the IL-18 polypeptide sequence is at least 90% identical to SEQ ID NO:67. In some embodiments, the IL-18 polypeptide sequence is at least 95% identical to SEQ ID NO:67. Alternatively, the IL-18 may comprise the polypeptide sequence set forth in: SEQ ID NO: 66. The IL-18 may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:66. In some embodiments, the IL-18 polypeptide sequence is at least 55% identical to SEQ ID NO:66. In some embodiments, the IL-18 polypeptide sequence is at least 70% identical to SEQ ID NO:66. In some embodiments, the IL-18 polypeptide sequence is at least 75% identical to SEQ ID NO:66. In some embodiments, the IL-18 polypeptide sequence is at least 80% identical to SEQ ID NO:66. In some embodiments, the IL-18 polypeptide sequence is at least 90% identical to SEQ ID NO:66. In some embodiments, the IL-18 polypeptide sequence is at least 95% identical to SEQ ID NO:66. [0115] The co-expressed proteins of the NK cells of the instant disclosure may be fusion proteins. A “fusion protein,” as used herein, refers to an expression product resulting from the fusion of at least two genes. The fusion protein may be a chimeric polypeptide comprising two or more unrelated protein, or protein fragments, conjugated to one another. The co-expressed polypeptides may be linked to the polypeptide encoding the CAR of the disclosure via a self- cleaving peptide, such as a self-cleaving (2A) peptide, or functional variant thereof. [0116] In some variations, the NK cells of the disclosure are engineered to express Interleukin- 15/Interleukin-15R (IL15/IL15R) fusion protein. Interleukin- 15 Receptor (IL-15R) has been shown to play a role in stimulation of proliferation of immune cells. Without being bound by theory, expression of IL-15/IL15R may improve NK cell function. The IL15/IL15R fusion protein may comprise the polypeptide sequence set forth in SEQ ID NO:69. The IL15/IL15R fusion protein may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:69. In some embodiments, the IL15/IL15R fusion protein may have a polypeptide sequence at least 55% identical to SEQ ID NO:69. In some embodiments, the IL15/IL15R fusion protein may have a polypeptide sequence at least at least 70% identical to SEQ ID NO:69. In some embodiments, the IL15/IL15R fusion protein may have a polypeptide sequence at least 75% identical to SEQ ID NO:69. In some embodiments, the IL15/IL15R fusion protein may have a polypeptide sequence at least 80% identical to SEQ ID NO:69. In some embodiments, the IL15/IL15R fusion protein may have a polypeptide sequence at least 90% identical to SEQ ID NO:69. In some embodiments, the IL15/IL15R fusion protein may have a polypeptide sequence at least 95% identical to SEQ ID NO:69.
[0117] In some variations, the NK cells of the disclosure are engineered to express Interleukin- 18/Interleukin-18R (IL18/IL18R) fusion protein. Interleukin- 18 Receptor (IL-18R) has been shown to play a role in immune cell activation and promotion of cytotoxic activity. Without being bound by theory, expression of IL18/IL18R may enhance NK cell proliferation, maturation, and cytotoxicity. The IL18/IL18R fusion protein may comprise the polypeptide sequence set forth in: SEQ ID NO:70. The IL18/IL18R fusion protein may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:70. In some embodiments, the IL18/IL18R fusion protein may have a polypeptide sequence at least 55% identical to SEQ ID NO:70. In some embodiments, the IL18/IL18R fusion protein may have a polypeptide sequence at least at least 70% identical to SEQ ID NO:70. In some embodiments, the IL18/IL18R fusion protein may have a polypeptide sequence at least 75% identical to SEQ ID NO:70. In some embodiments, the IL18/IL18R fusion protein may have a polypeptide sequence at least 80% identical to SEQ ID NO:70. In some embodiments, the IL18/IL18R fusion protein may have a polypeptide sequence at least 90% identical to SEQ ID NO:70. In some embodiments, the IL18/IL18R fusion protein may have a polypeptide sequence at least 95% identical to SEQ ID NO:70. [0118] In some variations, the NK cells of the disclosure are engineered to express OX40. OX40 (CD134) is a member of the tumor necrosis factor receptor (TNFR) family that plays a role in signal transduction and priming of immune cells. Without being bound by theory, expression of OX40 may enhance activation of MAPK/ERK, NFkB, and AKT pathways; and/or induce proliferation and cytokine release. The OX40 may comprise the polypeptide sequence set forth in SEQ ID NO:68. The OX40 may have a polypeptide sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:68. In some embodiments, the OX40 polypeptide sequence is at least 55% identical to SEQ ID NO:68. In some embodiments, the OX40 polypeptide sequence is at least 70% identical to SEQ ID NO:68. In some embodiments, the OX40 polypeptide sequence is at least 75% identical to SEQ ID NO:68. In some embodiments, the OX40 polypeptide sequence is at least 80% identical to SEQ ID NO:68. In some embodiments, the OX40 polypeptide sequence is at least 90% identical to SEQ ID NO:68. In some embodiments, the OX40 polypeptide sequence is at least 95% identical to SEQ ID NO:68. [0119] Any combination of extracellular ligand-binding domain, hinge domain, transmembrane domain, intracellular/cytoplasmic signaling domain, and co-expressed proteins described herein are envisaged within the scope of the CARs of the instant disclosure. 7.3. Polynucleotides and Vectors [0120] Polynucleotides of the disclosure may be delivered to cells as an isolated nucleic acid or in a vector. In an aspect, the disclosure provides a vector comprising polynucleotides comprising the CARs of the disclosure. In some embodiments, the vector comprises a polynucleotide encoding the co-expressed partners of the disclosure. Additional elements present in the vectors of the disclosure may also comprise a selectable marker and other components, such as enhancers, promoters, and termination sequences, necessary for the expression of proteins. The polynucleotides of the disclosure may comprise flanking left and right arms for homology-directed repair. The polynucleotide may comprise a promoter operatively linked to the polynucleotide sequence encoding the CAR. [0121] Polynucleotide of the disclosure may also be used for gene-editing. For example, homology directed repair may be used to insert a polynucleotide into a desired genomic loci. The polynucleotide may include a promoter and other genetic elements, or the repair template may omit these and instead rely on the host genome sequences for transcription.
[0122] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well- known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
[0123] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno- associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
[0124] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g. , an artificial membrane vesicle).
[0125] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or other assays.
[0126] Polynucleotides comprising the CARs, or parts thereof, are provided. Polynucleotides encoding any combination of extracellular ligand-binding domain, hinge domain, transmembrane domain, intracellular/cytoplasmic signaling domain, and co-expressed proteins described herein are envisaged within the scope of the instant disclosure. 7.4. Natural Killer Cells [0127] In one aspect, the disclosure provides natural killer (NK) cells engineered to express the disclosed CARs (such as the CARs described in Section 7.1). The NK cells may be primary NK cells. Primary NK cell mays be isolated from umbilical cord blood or peripheral blood using well-known methods. A polynucleotide encoding the CAR may be introduced into the primary NK cell using a viral vector, a non-viral vector, or electroporation. The NK cells may alternatively be induced pluripotent stem cell (iPSC)-derived NK cells or human embryonic stem cell (hESC)- derived NK cells. Methods for making iPSC-NK cells may be obtained by differentiating iPSCs using known methods. In one variation, iPSC-NK cells are made using embryoid bodies. Exemplary methods are provided in US 2013/0287751 A1 and Zhu et al. Methods Mol. Biol. 2048:107-119 (2019). In another variation, hESC-NK cells are made using single cell differentiation, as described, for example, in Woll et al. Blood 113:6094-6101 (2009). In further variations, iPSC-NK cells are made by differentiating single cells. Exemplary methods are provided in US 2021/0024891 A1 and US 2016/0097035 A1. hESC-/iPSC-derived NK cells may be phenotyped by measuring surface marker expression such as CD16, NKG2D, NKp44, NKp46, TRAIL, FasL, or combinations thereof. Killing activity of hESC-/iPSC-derived NK cells may be assessed. Functional assays to assess differentiation into NK cells include direct cytolytic activity tumor cells (such as killing of K562 cells), Caspase-3/7 flow cytometry assay, immunological assays for cytotoxic granule or cytokine release, or antitumor activity in vivo xenograft models. See, e.g., Woll et al. Blood 113:6094-6101 (2009); Hermanson et al. in Hematopoietic differentiation of human pluripotent stem cells.69-19 (2015). [0128] In some embodiments, the NK cells are CD45+/CD56+ double-positive. In some embodiments, the immune cell is CD45+. In some embodiments, the immune cell is CD56+. In some embodiments the immune cell is CD45+, CD56+, or CD45+/CD56+. Further illustrative methods for making and using engineered cells are provided in U.S. Pat. Appl. Pub. Nos. US 2018/0298101 A1, US 2021/0230548 A1, and US 2021/0145883 A1; and Int’l. Pat. Appl. Pub. Nos. WO 2018/075664 A1 (corresponding to U.S. Pat. Appl. No. 17/481,404) and WO 2020/113029 A2 (corresponding to U.S. Pat. Appl. No.17/309,408), the disclosures of which are incorporated by reference herein in their entireties. [0129] The cell population of the disclosure may be a purified cell population. As used herein, a composition containing a “purified cell population” or “purified cell composition” means that at least 30%, 50%, 60%, typically at least 70%, and more preferably 80%, 90%, 95%, 98%, 99%, or more of the cells in the composition are of a similarly identified cell phenotype. [0130] The cell population may be an isolated cell population. As used herein, the term “isolated” means material that is substantially or essentially free from components that normally accompany it in its native state. In particular embodiments, the term “obtained” or “derived” is used synonymously with isolated. [0131] Introduction of a polynucleotide encoding a CAR may be achieved by transduction of either the NK cell or an iPSC or ESC precursor with a viral vector, such as a lentiviral vector or a retroviral vector. Suitable lentiviral vectors are known in the art and are described in, for example, Zufferey et al., (1997); Dull et al., (1998), US6013516A, and US5994136A. Suitable retroviral vectors are also known in the art. In variations, a non-viral vector, such as a lipid nanoparticle, a transfection reagent, or electroporation may be used to introduce a polynucleotide into a cell. [0132] In an aspect, the disclosure provides cells comprising the chimeric antigen receptor as described herein and/or a polynucleotide encoding the chimeric antigen receptor. The cell may be a pluripotent cell. In some embodiments, the pluripotent cell is an induced pluripotent stem cell (iPSC). In some embodiments, the iPSC is differentiated into an NK cell (i.e., an iPSC-derived NK cell). In some embodiments, the cell is an embryonic stem cell (ESC). In some embodiments, the ESC is differentiated into an NK cell (i.e., an ESC-derived NK cell). [0133] In some embodiments, the NK cell or pluripotent cell for differentiation into an NK cell (e.g., an iPSC-derived NK cell, ESC-derived NK cell) comprising the chimeric antigen receptor as described herein and/or a polynucleotide encoding the chimeric antigen receptor is genetically modified. In some embodiments, the cell comprises homozygous inactivating mutations of the cytokine-inducible SH2-containing protein (CISH) genes (e.g., CISH -/-). In some embodiments, the cell does not comprise homozygous inactivating mutations of the cytokine- inducible SH2-containing protein (CISH) genes (e.g., CISH+/+). In some embodiments, the cell comprises homozygous inactivating mutations of the transforming growth factor beta receptor 2 (TGFBR2) genes (e.g., TGFBR2 -/-). In some embodiments, the cell does not comprise homozygous inactivating mutations of the transforming growth factor beta receptor 2 (TGFBR2) genes (e.g., TGFBR2 +/+). In some embodiments, the cell comprises homozygous inactivating mutations of the beta-2-microglobulin (B2M) genes (e.g., B2M -/-). In some embodiments, the cell comprises homozygous inactivating mutations of the class II major histocompatibility complex transactivator (CIITA) genes (e.g., CIITA -/-). In some embodiments, the cell comprises homozygous inactivating mutations of the regulatory factor X (RFX) genes (e.g., RFX -/-). [0134] In some embodiments, the NK cell or pluripotent cell for differentiation into an NK cell (e.g., an iPSC-derived NK cell, ESC-derived NK cell) comprising the chimeric antigen receptor as described herein and/or a polynucleotide encoding the chimeric antigen receptor comprises homozygous inactivating mutations of two or more genes selected from the group consisting of CISH, TGFBR2, B2M, CIITA, and RFX genes. In some embodiments, the cell comprises homozygous inactivating mutations of the CISH, and TGFBR2 genes (e.g., CISH -/-; TGFBR2 -/-). In some embodiments, the cell comprises homozygous inactivating mutations of the CISH, TGFBR2, and B2M genes (e.g., CISH -/-; TGFBR2 -/-; B2M -/-). In some embodiments, the cell comprises homozygous inactivating mutations of the CISH, TGFBR2, and CIITA genes (e.g., CISH -/-; TGFBR2 -/-; CIITA -/-). In some embodiments, the cell comprises homozygous inactivating mutations of the CISH, TGFBR2, B2M, and CIITA genes (e.g., CISH -/-; TGFBR2 -/-; B2M -/- ; CIITA -/-). [0135] In some embodiments, the NK cell or pluripotent cell for differentiation into an NK cell (e.g., an iPSC-derived NK cell, ESC-derived NK cell) comprising the chimeric antigen receptor as described herein and/or a polynucleotide encoding the chimeric antigen receptor comprises expression and/or overexpression of a gene encoding a soluble IL-15 or membrane- bound IL-15. In some embodiments, the cell comprises expression and/or overexpression of a gene encoding CD16. In some embodiments, the cell comprises expression and/or overexpression of a gene encoding HLA-E. In some embodiments, the cell comprises expression and/or overexpression of a gene encoding a chemokine receptor (e.g., CXCR2). [0136] In some embodiments, the cell comprises inactivating mutations of the CISH and TGFBR2 genes (e.g., CISH -/-; TGFBR2 -/-), and overexpression of a gene encoding a soluble IL- 15 or membrane-bound IL-15. In some embodiments, the cell comprises inactivating mutations of the CISH and TGFBR2 genes (e.g., CISH -/-; TGFBR2 -/-), and overexpression of a gene encoding CD16. In some embodiments, the cell comprises expression and/or overexpression of a gene encoding CD16. In some embodiments, the cell comprises inactivating mutations of the CISH and TGFBR2 genes (e.g., CISH -/-; TGFBR2 -/-), overexpression of a gene encoding a soluble IL-15 or membrane-bound IL-15, and overexpression of a gene encoding CD16. In some embodiments, the cell comprises homozygous inactivating mutations of two or more genes selected from the group consisting of CISH, TGFBR2, B2M, CIITA, and RFX genes, and expression or overexpression of two proteins selected from the group consisting of IL-15 (soluble or membrane-bound), CD16, HLA-E, and a chemokine receptor. 7.5. Methods Of Use [0137] In an aspect, the disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering the CAR NK cell of the disclosure (such as an NK cell described in Section 7.4) or the pharmaceutical composition of the disclosure to the subject (such as a pharmaceutical composition described in Section 7.6). [0138] In an aspect, the disclosure provides a method of killing a target cell comprising contacting a population of target cells with a population of natural killer cells of the disclosure, wherein the chimeric antigen receptors of the natural killer cell comprise a ligand binding domain that specifically binds an antigen on the target cell, and wherein the natural killer cells induce specific killing of the target cells. [0139] In an aspect, the disclosure provides method of killing a target cell comprising contacting a population of target cells with a population of NK cells. [0140] Cell killing may be assessed by assays known in the art, including but not limited to those described in the examples. [0141] The cancer may result from proliferation of tumor cells that display an antigen specifically bound by the CAR or an NK cell expressing the CAR. The CARs provided herein include CARs having a ligand-binding domain that specifically binds CD19, CD20, and/or HER2. CD19 and CD20 can be expressed on tumor cells of various indolent and aggressive subtypes of B cell lymphomas and leukemias, including Non-Hodgkin's lymphoma (NHL), B-cell Chronic lymphocytic leukemia (CLL), and non-T acute lymphoblastic leukemia (ALL). Accordingly, the CARs described herein may be used to kill CD19+ and/or CD20+ tumor cells and/or treat cancer in subjects having Non-Hodgkin's lymphoma (NHL), B-cell Chronic lymphocytic leukemia (CLL), and non-T acute lymphoblastic leukemia (ALL). In some embodiments, the CARs described herein may be used to kill CD 19+ tumor cells. In some embodiments, the CARs described herein may be used to kill CD20+ tumor cells. In some embodiments, the CARs described herein may be used to treat cancer in subjects having Non-Hodgkin's lymphoma (NHL). In some embodiments, the CARs described herein may be used to treat cancer in subjects having B-cell Chronic lymphocytic leukemia (CLL). In some embodiments, the CARs described herein may be used to treat cancer in subjects having non-T acute lymphoblastic leukemia (ALL). HER2 expression has been found in several tumor types including, but not limited to, breast, esophageal, lung, cervical, endometrial, and ovarian cancer. Accordingly, the CARs described herein may be used to kill HER2+ tumor cells and/or treat cancer in subjects having breast, esophageal, lung, cervical, endometrial and/or ovarian cancer. In some embodiments, the CARs described herein may be used to kill HER2+ tumor cells. In some embodiments, the CARs described herein may be used to treat cancer in subjects having breast cancer. In some embodiments, the CARs described herein may be used to treat cancer in subjects having esophageal cancer. In some embodiments, the CARs described herein may be used to treat cancer in subjects having lung cancer. In some embodiments, the CARs described herein may be used to treat cancer in subjects having cervical cancer. In some embodiments, the CARs described herein may be used to treat cancer in subjects having endometrial cancer. In some embodiments, the CARs described herein may be used to treat cancer in subjects having ovarian cancer.
[0142] In an aspect, the disclosure provides methods of treating cancer in a subject in need thereof, comprising administering a population of NK cells, or pharmaceutical compositions comprising a population of NK cells.
[0143] The terms “subject,” “patient,” and “individual” are used interchangeably herein to refer to a vertebrate, such as a mammal, e.g., a human.
[0144] As used herein, the term “treatment” or “treating” embraces at least an amelioration of the symptoms associated with a disease or condition in the patient, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., a symptom associated with the condition being treated. As such, “treatment” or “treating” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from happening) or stopped (e.g., terminated) such that the patient no longer suffers from the condition, or at least the symptoms that characterize the condition.
[0145] In some embodiments, the method of treating cancer in a subject in need thereof comprises administering a therapeutically effective amount of CAR NK cells and/or pharmaceutical composition of the disclosure to the subject.
[0146] As used herein, “therapeutically effective” refers to an amount of cells or pharmaceutical composition that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with a disease, such as cancer.
[0147] In some embodiments, the CAR NK cells and pharmaceutical compositions comprising the same described herein are used as part of a combination therapy. In some embodiments, the CAR NK cells or pharmaceutical compositions of the instant disclosure are co-administered to the subject with tumor targeting antibodies.
[0148] In some embodiments, the CAR NK cells or pharmaceutical compositions of the instant disclosure exhibit increased cytotoxicity relative to cells or pharmaceutical compositions in which the cells have not been engineered in such a way as the instant disclosure. In some embodiments, the cells or pharmaceutical compositions of the instant disclosure exhibit increased effectiveness relative to equivalent cells or pharmaceutical compositions in cells have not been engineered in such a way as the instant disclosure.
[0149] The population of NK cells or pharmaceutical composition may be administered in an amount effective to kill target cells and/or in a therapeutically effective amount for treatment of the cancer.
[0150] In some embodiments, the disclosure provides a method of treating cancer with the chimeric receptor expressing cells provided herein or pharmaceutical compositions or formulations thereof. In some embodiments, the method comprises administering the chimeric receptor expressing cells provided herein, pharmaceutical compositions or formulations described herein to a subject in need thereof. The terms “administration” and “administering,” as used herein, refer to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and intramuscular, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.
[0151] In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced anti-tumor efficacy relative to a cell lacking the chimeric receptor. Methods for measuring anti-tumor efficacy in vitro and in vivo are known to those of skill in the art and include, for example, in vitro killing assays and implanting tumors into mouse models.
[0152] Cell killing may be assessed by assays known in the art, including but not limited to those described in the Examples provided in Section 9.
[0153] In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or about 1000% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 1% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 5% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 10% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 20% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 30% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 40% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 50% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 60% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 70% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 80% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 90% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 100% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by about 1000% compared to a cell that that lacks the chimeric receptor.
[0154]
[0155] In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 1% to 5%, between 5% to 10%, between 10% to 20%, between 20% to 30%, between 30% to 40%, between 40% to 50%, between 50% to 60%, between 60% to 70%, between 70% to 80%, between 80% to 90%, between 90% to 100%, or between 100% to 1000% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 1% to 5% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 5% to 10% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 10% to 20% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 20% to 30% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 30% to 40% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 40% to 50% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 50% to 60% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 60% to 70% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 70% to 80% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 80% to 90% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by between 90% to 100% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced cell killing by or between 100% to 1000% compared to a cell that that lacks the chimeric receptor.
[0156] In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or about 1000% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 1% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 5% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 10% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 20% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 30% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 40% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 50% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 60% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 70% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 80% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 90% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 100% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by about 1000% compared to a cell that that lacks the chimeric receptor.
[0157] In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 1% to 5%, between 5% to 10%, between 10% to 20%, between 20% to 30%, between 30% to 40%, between 40% to 50%, between 50% to 60%, between 60% to 70%, between 70% to 80%, between 80% to 90%, between 90% to 100%, or between 100% to 1000% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 1% to 5% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 5% to 10% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 10% to 20% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 20% to 30% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 30% to 40% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 40% to 50% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 50% to 60% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 60% to 70% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 70% to 80% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 80% to 90% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by between 90% to 100% compared to a cell that that lacks the chimeric receptor. In some embodiments, a cell expressing a chimeric receptor described herein exhibits enhanced specific killing by or between 100% to 1000% compared to a cell that that lacks the chimeric receptor.
7.6. Pharmaceutical Compositions
[0158] In an aspect, the disclosure provides a pharmaceutical composition comprising NK cells (such as an NK cell described in Section 7.4) having a CAR (such as a CAR described in Section 7.1) or cell population of the disclosure and one or more pharmaceutically acceptable excipients or diluents.
[0159] As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and/or non-human mammals.
[0160] As used herein, the term “pharmaceutically acceptable excipients or diluents” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and/or vehicle with which a cell of the disclosure is administered. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003).
[0161] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions.
7.7. Methods of Manufacture
[0162] In an aspect, the disclosure provides methods of making NK cells comprising providing a pluripotent cell, such as an induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC), and differentiating the pluripotent cell into an NK cell (such as in the Examples described below). The pluripotent cell may be genetically modified to comprise a polynucleotide encoding a CAR prior to or after differentiation. Known vectors may be used to deliver a polynucleotide, which may be stably maintained by the cell and/or integrated into the genome of the cell. In some embodiments, the polynucleotide is knocked-in to the genome of an NK cell or a pluripotent cell. In some embodiments, the polynucleotide is knocked into a safe harbor locus (e.g., AAVS1). In some embodiments, the polynucleotide is knocked into an essential gene (see, e.g., WO 2021/22615). [0163] In some embodiments, the pluripotent cell is genetically modified. In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the cytokine- inducible SH2-containing protein (CISH) genes (e.g., CISH -/-). In some embodiments, the pluripotent cell does not comprise homozygous inactivating mutations of the cytokine-inducible SH2-containing protein (CISH) genes (e.g., CISH+/+). In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the transforming growth factor beta receptor 2 (TGFBR2) genes (e.g., TGFBR2 -/-). In some embodiments, the pluripotent cell does not comprise homozygous inactivating mutations of the transforming growth factor beta receptor 2 (TGFBR2) genes (e.g., TGFBR2 +/+). In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the beta-2-microglobulin (B2M) genes (e.g., B2M -/-). In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the class II major histocompatibility complex transactivator (CIITA) genes (e.g., CIITA -/-). In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the regulatory factor X (RFX) genes (e.g., RFX -/-). [0164] In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of two or more genes selected from the group consisting of CISH, TGFBR2, B2M, CIITA, and RFX genes. In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the CISH, and TGFBR2 genes (e.g., CISH -/-; TGFBR2 -/-). In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the CISH, TGFBR2, and B2M genes (e.g., CISH -/-; TGFBR2 -/-; B2M -/-). In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the CISH, TGFBR2, and CIITA genes (e.g., CISH -/-; TGFBR2 -/-; CIITA -/-). In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of the CISH, TGFBR2, B2M,, and CIITA genes (e.g., CISH -/- ; TGFBR2 -/-; B2M -/- ; CIITA -/-). [0165] In some embodiments, the pluripotent cell comprises expression and/or overexpression of a gene encoding a soluble IL-15 or membrane-bound IL-15. In some embodiments, the pluripotent cell comprises expression and/or overexpression of a gene encoding CD16. In some embodiments, the pluripotent cell comprises expression and/or overexpression of a gene encoding HLA-E. In some embodiments, the pluripotent cell comprises expression and/or overexpression of a gene encoding a chemokine receptor (e.g., CXCR2). [0166] In some embodiments, the pluripotent cell comprises inactivating mutations of the CISH and TGFBR2 genes (e.g., CISH -/-; TGFBR2 -/-), and overexpression of a gene encoding a soluble IL-15 or membrane-bound IL-15. In some embodiments, the pluripotent cell comprises inactivating mutations of the CISH and TGFBR2 genes (e.g., CISH -/-; TGFBR2 -/-), and overexpression of a gene encoding CD16. In some embodiments, the pluripotent cell comprises expression and/or overexpression of a gene encoding CD16. In some embodiments, the pluripotent cell comprises inactivating mutations of the CISH and TGFBR2 genes (e.g., CISH -/-; TGFBR2 -/- ), overexpression of a gene encoding a soluble IL-15 or membrane-bound IL-15, and overexpression of a gene encoding CD16. In some embodiments, the pluripotent cell comprises homozygous inactivating mutations of two or more genes selected from the group consisting of CISH, TGFBR2, B2M, CIITA, and RFX genes, and expression or overexpression of two proteins selected from the group consisting of IL-15 (soluble or membrane-bound), CD16, HLA-E, and a chemokine receptor. 7.8. Kits [0167] In an aspect, the disclosure provides a kit comprising the CAR NK cells or the pharmaceutical composition of the disclosure (such as a pharmaceutical composition described in Section 7.6) and instructions for use. 8. EMBODIMENTS [0168] This invention provides the following non-limiting embodiments. A1. A chimeric antigen receptor (CAR) comprising a ligand-binding domain, a transmembrane domain, and a cytoplasmic portion, wherein the cytoplasmic portion comprises an amino acid sequence at least 90% identical to SEQ ID NO:1 and wherein the transmembrane domain is selected from the group consisting of a CD3, a CD4, a CD28, a TLR2, a CD64, a CD32a, a CD32c, a 2B4, a DNAM1, and a NTBA transmembrane domain, or functional variant thereof. A2. The CAR of embodiment A1, wherein the cytoplasmic portion comprises an amino acid sequence of SEQ ID NO: 1.
A3. The CAR of embodiment A1 or A2, wherein the cytoplasmic portion consists of an amino acid sequence at least 90% identical to SEQ ID NO:1.
A4. The CAR of embodiment A1 or A2, wherein the cytoplasmic portion consists of the amino acid sequence of SEQ ID NO: 1.
A5. The CAR of any one of embodiments A1 to A4, wherein the transmembrane domain is selected from the group consisting of the CD32a, the CD32c, the 2B4, and the NTBA transmembrane domain.
A6. The CAR of any one of embodiments A1 to A4, wherein the CD3 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:2.
A7. The CAR of any one of embodiments A1 to A4, wherein the CD4 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:3.
A8. The CAR of any one of embodiments A1 to A4, wherein the CD28 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4.
A9. The CAR of any one of embodiments A1 to A4, wherein the TLR2 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:5.
A10. The CAR of any one of embodiments A1 to A4, wherein the CD64 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:6. A1 l . The CAR of any one of embodiments A1 to A4, wherein the DNAM1 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:7.
A12. The CAR of any one of embodiments A1 to A5, wherein the CD32a transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:8.
A13. The CAR of any one of embodiments A1 to A5, wherein the CD32c transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:9.
A14. The CAR of any one of embodiments A1 to A5, wherein the 2B4 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 10.
A15. The CAR of any one of embodiments A1 to A5, wherein the NTBA transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:11.
A16. The CAR of any one of embodiments A1 to A15, wherein the extracellular protein comprises a ligand-binding domain.
A17. The CAR of embodiment A16, wherein the ligand-binding domain comprises an antibody-like domain.
A18. The CAR of embodiment A17, wherein the ligand-binding domain comprises a variable heavy (VH) domain and/or a variable light (VL) domain.
A19. The CAR of embodiment A17, wherein the ligand-binding domain comprises a singlechain variable fragment (scFv).
A20. The CAR of any one of embodiments A16 to A19, wherein the ligand-binding domain specifically binds a CD 19 antigen. A21. The CAR of embodiment A20, wherein the ligand-binding domain comprises the a CDR- H1 according to SEQ ID NO:86; a CDR-H2 according to SEQ ID NO:87; a CDR-H3 according to SEQ ID NO:88; a CDR-L1 according to SEQ ID NO:89; a CDR-L2 according to SEQ ID NO:32; and/or a CDR-L3 according to SEQ ID NO:33. A22. The CAR of embodiment A20, wherein the ligand-binding domain comprises a VH according to SEQ ID NO:38 and/or a VL according to SEQ ID NO:39, or functional variants having polypeptide sequences at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. A23. The CAR of embodiment A20, wherein the ligand-binding domain comprises an scFv according to SEQ ID NO:40, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. A24. The CAR of any one of embodiments A16 to A19, wherein the ligand-binding domain specifically binds a CD20 antigen. A25. The CAR of embodiment A24, wherein the ligand-binding domain comprises an scFv according to SEQ ID NO:41, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. A26. The CAR of any one of embodiments A16 to A19, wherein the ligand-binding domain specifically binds a HER2 antigen. A27. The CAR of embodiment A26, wherein the ligand-binding domain comprises an scFv according to SEQ ID NOs:42, 43, or 90, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto. A28. The CAR of any one of embodiments A1 to A27, wherein the CAR further comprises a hinge domain. A29. The CAR of embodiment A28, wherein the hinge domain selected from the group consisting of a CD8, a CD28, or an IgG4 hinge domain. A30. The CAR of embodiment A29, wherein the CD8 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 12.
A31. The CAR of embodiment A29, wherein the CD28 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 13 or SEQ ID NO: 14.
A32. The CAR of embodiment A29, wherein the IgG4 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 15.
A33. A CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 16.
A34. A CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 17.
A35. A CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 18.
A36. A CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 19.
A37. A cell comprising the CAR of any one of embodiments A1 to A36, wherein the cell is a pluripotent cell.
A38. The cell of embodiment A37, wherein the pluripotent cell is an induced pluripotent cell.
A39. A cell comprising the CAR of any one of embodiments A1 to A36, wherein the cell is a natural killer cell.
A40. The cell of embodiment A39, wherein the cell is an induced pluripotent stem cell-derived natural killer (iPSC-NK) cell. A41. The cell of any one of embodiments A37 to A40, wherein the cell comprises homozygous inactivating mutations in the cytokine-inducible SH2-containing protein (CISH) genes of the cell.
A42. A pharmaceutical composition comprising the cell of any one of embodiments A39 to A41, and a pharmaceutically acceptable solution.
A43. A method of killing a target cell comprising contacting a population of target cells with a population of natural killer cells according to any one of embodiments A39 to A41, wherein the chimeric antigen receptors of the natural killer cell comprise a ligand-binding domain that specifically binds on antigen on the target cell, and wherein the natural killer cells induce specific killing of the target cells.
A44. A method of treating cancer in a subject in need thereof, comprising administering the pharmaceutical compositions of embodiment A42 to the subject.
A45. Use of the natural killer cell of any one of embodiments A39 to A41, or the pharmaceutical composition of embodiment A42 for treatment of cancer.
A46. One or more polynucleotides encoding the CAR of any one of embodiments A1 to A36.
A47. One or more vectors comprising the one or more polynucleotides of embodiment A46.
A48. A cell comprising the one or more polynucleotides of embodiment A46, wherein the cell is a pluripotent cell.
A49. The cell of embodiment A48, wherein the pluripotent cell is an induced pluripotent cell.
A50. A cell comprising the one or more polynucleotides of embodiment A46, wherein the cell is a natural killer cell.
A51. The cell of embodiment A50, wherein the cell is an induced pluripotent stem cell-derived natural killer (iPSC-NK) cell.
A52. A method of making a natural killer cell, comprising providing a pluripotent cell with the one or more polynucleotides of embodiment A46 and differentiating the pluripotent cell into a natural killer cell. 9. EXAMPLES [0169] The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the disclosure should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. [0170] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the methods of the present disclosure and practice the claimed methods. The following working examples therefore specifically point out embodiments of the present disclosure and are not to be construed as limiting in any way the remainder of the disclosure. [0171] The materials and methods employed in these experiments are now described. Example 1: Screening of Chimeric Antigen Receptors Designs [0172] This example demonstrates that NK cells expressing a CAR having a cytoplasmic portion that includes a toll-like receptor 2 (TLR2) signaling domain, and a CD3 zeta (CD3ζ) signaling domain exhibited robust effector cell activity. [0173] Briefly, iPSC-derived NK cells were transduced as described below. Cell viability and CAR expression were measured on day 4, day 9/11 (pre-sorting), and day 17 (post-sorting). (Tables 6-8; FIG.2A-FIG.2L). [0174] Table 6: Cell viability and CAR expression on day 4 post-transduction
Figure imgf000088_0001
Figure imgf000089_0001
MFI: Mean Fluorescent Intensity [0175] Table 7: Cell viability and CAR expression on day 9/11 post-transduction.
Figure imgf000089_0002
MFI: Mean Fluorescent Intensity [0176] Table 8: Cell viability and CAR expression on day 4 post-transduction
Figure imgf000089_0003
Figure imgf000090_0001
[0177] On day 17 post-transduction, -sorting, and -expansion, the CAR expressing iNK cells were added to Raji 2.0 lymphoblast-like cells at a ratio of 5:1 (FIG.2A), 2.5:1 (FIG.2B), or 1.25:1 (FIG.2C), effector to target cells, and an impedance assay was performed as described below. The results demonstrated that CARs containing a NTBA, a 2B4, a CD28, an NKG2D, or a CD32a transmembrane domain were able to consistently exhibit improved NK cell mediated killing of the target cells, relative to untransduced cells. [0178] Similar results were observed when CAR expressing iNK cells were added to Raji lymphoblast-like cells expressing GFP Fluc and killing was measured by luciferase release. (FIG. 4, right columns; FIG.5B; and FIG.6A, FIG.6B, FIG.6D, and FIG.6F–FIG.6J, gray columns). In particular, CARs containing a NTBA, a 2B4, or an NKG2D transmembrane domain exhibited improved NK cell mediated killing of the target Raji cells at effector to target cell ratios ranging from 0.25:1 to 15:1, relative to untransduced cells (FIG.4, right columns; FIG.5B; and FIG.6I, FIG.6F, FIG.6B, and FIG.6A, gray columns). [0179] The functional cytolytic activity of CAR expressing iNK cells was further confirmed by adding the effector cells to SupB15 lymphoblast cells expressing GFP Fluc cells and measuring luciferase release. As show in FIG.5A, CARs containing the indicated transmembrane domains exhibited improved NK cell mediated killing of the target SupB15 cells at effector to target cell ratios ranging from 0.25:1 to 15:1, relative to untransduced cells (FIG.5A; and FIG.6A-FIG.6J, left columns). The results demonstrated that CARs containing a NTBA, a 2B4, a CD28, a DNAM1, a CD32a, a TLR2, or an NKG2D transmembrane domain were able to robustly exhibit improved NK cell mediated killing of the target cells, relative to untransduced cells, even at low effector to target cell ratios (FIG.4, left columns; FIG.5A; and FIG.6A-FIG.6J, left columns). [0180] Taken together, the results demonstrate that NK cells expressing a CAR having a cytoplasmic portion that includes a toll-like receptor 2 (TLR2) signaling domain, and a CD3 zeta (CD3ζ) signaling domain exhibited robust effector cell activity. Methods [0181] Construction of retroviral expression vectors and viral packaging: Polypeptide sequences of all candidate CAR components were obtained from Ensemble™ database (Howe et al. Nucleic Acids Res.2021, vol.49(1):884–891) and assembled into complete CAR designs using SnapGene™ molecular biology software. Nucleic acid sequences were determined using a publicly available codon optimization tool (Integrated DNA Technologies™). All CAR constructs included, as representative ligand binding domain, a CD19 scFv binder derived from the FMC63 monoclonal antibody (mAb) (See, e.g., U.S. Pat. No.9,701,758). CAR constructs were synthesized and cloned into a retroviral expression vector (pLXRN; Addgene Plasmid #99206) and maxi-preps were produced and sequence verified. Lentiviral particles were generated using the constructs described above, packaging plasmids (Rev, gag-pol, and VSVG; Cell Biolabs™), and the Lenti- X™ 293T cell line (Takara™). Viral supernatants were concentrated ~100X using a PEG-based concentration solution (Takara™) and viral titers were determined by flow cytometry of transduced Jurkat cells using a CD19 CAR detection reagent (CD19 CAR Detection Reagent human, Biotin; Miltenyi Biotec). [0182] Generation of iPSC-NK cells: iPSCs are collected and transferred to culture in serum- free media and formed into embryoid bodies by spin aggregation. Upon the appearance of CD34+ cells inside the Embryoid Body (EB) at day 6, EB are transferred into NK cell differentiation medium containing: a 2:1 mixture of Dulbecco modified Eagle medium/Ham F12), 2 mM L-, 1% penicillin/streptomycin, 25 mM b-mercaptoethanol, 20% heat-inactivated human serum AB, 5ng/mL sodium selenite, 50 mM ethanolamine, 20 mg/mL ascorbic acid, interleukin-3; for first week only, stem cell factor, interleukin-15, Fms-like tyrosine kinase 3 ligand, and interleukin-7. The EB is left in these conditions for 28 days receiving weekly media changes to produce NK cells (CD45+ CD56+ CD33- CD3- cells, as determined by flow cytometry). Then NK cells are harvested for expansion. [0183] Expansion: After differentiation, an immortalized leukemia cell line, K562 expressing mbIL21 and 4-1BB ligand (CD137), is used to stimulate NK cell expansion. NK cells are seeded with K562-41BBL-mbIL21 feeder cells and incubated at 1:2 ratio in Stromal Cell Growth Medium (SCGM) with 100U/mL IL-2. Non-adherent cells are removed and analyzed by flow cytometry to determine the purity of CD56+ NK cells. These cells are then stimulated with 2:1 aAPCs (irradiated at 10,000 Gy) to NK cells at 350,000 NK cells/mL of media containing CTS NK Xpander media with supplement and 10% human AB serum), supplemented with 50-100 U/mL IL-2. [0184] Transduction of iPSC-NK cells: Non-treated 24-well plates were coated with Retronectin (Takara). Equal amount of concentrated retrovirus was added to plates and centrifuged at 2000xg at 32°C for 2 hrs. iPSC-derived NK cells (3e5 cells) were added to each well in media containing IL-2 (50 U/ml) and centrifuged at 800xg at 32°C for 1 hr. Cells were incubated at 37°C for 48 hrs. Fresh media (with IL-2) was added, and cells were incubated for an additional 24 hrs. Four days post-transduction CAR expression was checked by flow cytometry using the CD19 CAR detection reagent. Following CAR expression check, cells were stimulated with 2:1 aAPCs (irradiated at 10,000 Gy) to NK cells at 350,000 NK cells/mL of media containing CTS NK Xpander media with supplement and 10% human AB serum), supplemented with 50-100 U/mL IL-2. On day 9 cells were FACS sorted using CD19 CAR detection reagents. Sorted CAR+ were then stimulated again 2:1 with sAPC. On day 17, cells were checked again for CAR expression prior to use in functional assays. [0185] Impedance Assay: RTCA E-plates were coated with CD40 tethering reagent (Agilent™) diluted in PBS overnight at 4°C. Plates were washed with PBS, target cells were added to each well, and impedance readings were allowed to equilibrate for 2-4 hrs. CAR+ iPSC-NK cells were added to each well at the indicated E:T ratio and impedance readings were measured every 15 min for 24 hrs. NK-specific killing was determined by normalizing impedance readings to “target cells alone” control wells and plotted as frequency of target killing. [0186] Luciferase Release Assay: To assess killing of SupB15 and Raji GFP Fluc targets by sorted effector cells expressing CD19 CARs using the Britelite plus, Ultra-high sensitivity Luminescence assay. SupB15 GFP Fluc and Raji GFP Fluc target cells (10,000 cells/well) were seeded in a 96 well U-bottom plate. In parallel, serial dilutions of effector cells were performed in a separate 96 well plate to achieve effector to target cell ratios ranging from 0.25:1 to 15:1. Effector cells were added to the target cells and centrifuged at 300xg for 5 min. The effector and target cells were incubated for 4 hours at 37 °C. Thereafter detector reagent was added and luciferase signal was measured. Example 2: In vivo Functional Testing of CARs having a CD19 Binding Domain [0187] iPSC derived NK cells expressing functional CAR constructs (determined by results from in vitro assays) are tested for in vivo efficacy using a CD19 expressing model of B cell malignancies. To initiate disease, 10-12 week-old NOD-SCID-gamma-/- (NSG) mice (Jackson Laboratory) are injected intravenously with luciferase expressing Raji cells (CD19 positive). After 1 day, the mice are administered iPSC-derived NK cells engineered to express (a) no CAR (negative control); (b) a 2nd generation T cell CAR (benchmark control); or (c) an NK CAR (test group). Subject mice are administered IL-2 every other day for the remainder of the experiment. Disease progression is monitored by survival, weight loss, and bioluminescent imaging of tumor burden (measured weekly). To assess the number of infused NK cells present during the course of the experiment, CD45+CD56+CD3- cells from peripheral blood are quantified weekly. * * * * [0188] While the invention has been described in connection with proposed specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims

CLAIMS What is claimed is:
1. A chimeric antigen receptor (CAR) comprising a ligand-binding domain, a transmembrane domain, and a cytoplasmic portion, wherein the cytoplasmic portion comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, and wherein the transmembrane domain is selected from the group consisting of a CD3, a CD4, a CD28, a TLR2, a CD64, a CD32a, a CD32c, a 2B4, a DNAM1, and a NTBA transmembrane domain, or functional variant thereof.
2. The CAR of claim 1, wherein the cytoplasmic portion comprises the amino acid sequence of SEQ ID NO: 1.
3. The CAR of claim 1 or 2, wherein the cytoplasmic portion consists of an amino acid sequence at least 90% identical to SEQ ID NO: 1.
4. The CAR of claim 1 or 2, wherein the cytoplasmic portion consists of the amino acid sequence of SEQ ID NO: 1.
5. The CAR of any one of claims 1 to 4, wherein the transmembrane domain is selected from the group consisting of the CD32a, the CD32c, the 2B4, and the NTBA transmembrane domain.
6. The CAR of any one of claims 1 to 4, wherein the CD3 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:2.
7. The CAR of any one of claims 1 to 4, wherein the CD4 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:3.
8. The CAR of any one of claims 1 to 4, wherein the CD28 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:4.
9. The CAR of any one of claims 1 to 4, wherein the TLR2 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:5.
10. The CAR of any one of claims 1 to 4, wherein the CD64 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:6.
11. The CAR of any one of claims 1 to 4, wherein the DNAM1 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:7.
12. The CAR of any one of claims 1 to 5, wherein the CD32a transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:8.
13. The CAR of any one of claims 1 to 5, wherein the CD32c transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO:9.
14. The CAR of any one of claims 1 to 5, wherein the 2B4 transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 10.
15. The CAR of any one of claims 1 to 5, wherein the NTBA transmembrane domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 11.
16. The CAR of any one of claims 1 to 15, wherein the ligand-binding domain comprises an antibody-like domain.
17. The CAR of claim 16, wherein the ligand-binding domain comprises an antibody domain.
18. The CAR of claim 16 or 17, wherein the ligand-binding domain comprises a variable heavy (VH) domain and/or a variable light (VL) domain.
19. The CAR of claim 16 or 17, wherein the ligand-binding domain comprises a single-chain variable fragment (scFv).
20. The CAR of any one of claims 16 to 19, wherein the ligand-binding domain specifically binds a CD 19 antigen.
21. The CAR of claim 20, wherein the ligand-binding domain comprises the a CDR-H1 according to SEQ ID NO:86; a CDR-H2 according to SEQ ID NO:87; a CDR-H3 according to SEQ ID NO:88; a CDR-L1 according to SEQ ID NO:89; a CDR-L2 according to SEQ ID NO:32; and/or a CDR-L3 according to SEQ ID NO:33.
22. The CAR of claim 20, wherein the ligand-binding domain comprises a VH according to SEQ ID NO:38 and/or a VL according to SEQ ID NO:39, or functional variants having polypeptide sequences at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto.
23. The CAR of claim 20, wherein the ligand-binding domain comprises an scFv according to SEQ ID NO:40, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto.
24. The CAR of any one of claims 16 to 19, wherein the ligand-binding domain specifically binds a CD20 antigen.
25. The CAR of claim 24, wherein the ligand-binding domain comprises an scFv according to SEQ ID NO:41, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto.
26. The CAR of any one of claims 16 to 19, wherein the ligand-binding domain specifically binds a HER2 antigen.
27. The CAR of claim 26, wherein the ligand-binding domain comprises an scFv according to SEQ ID NOs:42, 43, or 90, or a functional variant having a polypeptide sequence at least 55%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% identity thereto.
28. The CAR of any one of claims 1 to 27, wherein the CAR further comprises a hinge domain.
29. The CAR of claim 28, wherein the hinge domain selected from the group consisting of a CD8, a CD28, or an IgG4 hinge domain.
30. The CAR of claim 29, wherein the CD8 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 12.
31. The CAR of claim 29, wherein the CD28 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 13 or SEQ ID NO: 14.
32. The CAR of claim 29, wherein the IgG4 hinge domain comprises an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 15.
33. A CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 16.
34. A CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 17.
35. A CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 18.
36. A CAR comprising an amino acid sequence at least 55%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 19.
37. A cell comprising the CAR of any one of claims 1 to 36, wherein the cell is a pluripotent cell.
38. The cell of claim 37, wherein the pluripotent cell is an induced pluripotent cell.
39. A cell comprising the CAR of any one of claims 1 to 36, wherein the cell is a natural killer cell.
40. The cell of claim 39, wherein the cell is an induced pluripotent stem cell-derived natural killer (iPSC-NK) cell.
41. The cell of any one of claims 37 to 40, wherein the cell comprises homozygous inactivating mutations in the cytokine-inducible SH2-containing protein (CISH) genes of the cell.
42. A pharmaceutical composition comprising the cell of any one of claims 39 to 41, and a pharmaceutically acceptable solution.
43. A method of killing a target cell comprising contacting a population of target cells with a population of natural killer cells according to any one of claims 39 to 41, wherein the chimeric antigen receptors of the natural killer cell comprise a ligand-binding domain that specifically binds on antigen on the target cell, and wherein the natural killer cells induce specific killing of the target cells.
44. A method of treating cancer in a subject in need thereof, comprising administering the pharmaceutical compositions of claim 42 to the subject.
45. Use of the natural killer cell of any one of claims 39 to 41, or the pharmaceutical composition of claim 42 for treatment of cancer.
46. One or more polynucleotides encoding the CAR of any one of claims 1 to 36.
47. One or more vectors comprising the one or more polynucleotides of claim 46.
48. A cell comprising the one or more polynucleotides of claim 46, wherein the cell is a pluripotent cell.
49. The cell of claim 48, wherein the pluripotent cell is an induced pluripotent cell.
50. A cell comprising the one or more polynucleotides of claim 46, wherein the cell is a natural killer cell.
51. The cell of claim 50, wherein the cell is an induced pluripotent stem cell-derived natural killer (iPSC-NK) cell.
52. A method of making a natural killer cell, comprising providing a pluripotent cell with the one or more polynucleotides of claim 46 and differentiating the pluripotent cell into a natural killer cell.
PCT/US2024/049757 2023-10-04 2024-10-03 Chimeric antigen receptors comprising toll-like receptor 2 (tlr2) and cd3 zeta signaling domains and uses thereof Pending WO2025076196A2 (en)

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