WO2021223705A1 - Compositions et procédés pour modification de lymphocytes t - Google Patents

Compositions et procédés pour modification de lymphocytes t Download PDF

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WO2021223705A1
WO2021223705A1 PCT/CN2021/091879 CN2021091879W WO2021223705A1 WO 2021223705 A1 WO2021223705 A1 WO 2021223705A1 CN 2021091879 W CN2021091879 W CN 2021091879W WO 2021223705 A1 WO2021223705 A1 WO 2021223705A1
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immune cell
engineered immune
cell
antigen binding
domain
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PCT/CN2021/091879
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Hua Zhang
Lianjun SHEN
Huan SHI
Wei Cao
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Gracell Biotechnologies (Shanghai) Co., Ltd.
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Priority to CN202180033729.7A priority Critical patent/CN115777017A/zh
Publication of WO2021223705A1 publication Critical patent/WO2021223705A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • 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 A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/28—Expressing multiple CARs, TCRs or antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • CARs chimeric antigen receptors
  • CS1 also known as CRACC, SLAMF7 or CD319
  • SLAM Signaling Lymphocyte Activation Molecule family receptors. It can be expressed on NK cells, CD8+ T lymphocytes, mature dendritic cells and activated B cells.
  • CS1 is also overexpressed in multiple myeloma (MM) , which makes it an attractive target for immunotherapy such as CAR-T therapy.
  • MM myeloma
  • CD137 also known as 4-1BB or TNFRSF9
  • 4-1BB or TNFRSF9 is a member of the tumor necrosis factor (TNF) receptor family. It can be expressed on activated T cells, and to a larger extent on CD8+than on CD4+ T cells.
  • CD137 expression is found on dendritic cells, B cells, follicular dendritic cells, natural killer cells, granulocytes and cells of blood vessel walls at sites of inflammation.
  • CAR-T therapy uses autologous CAR-T cell infusion to prevent graft versus host disease (GVHD) .
  • GVHD graft versus host disease
  • some patients’ T cells are not sufficiently effective even after CAR redirection, which promotes the modification of allogeneic donor T cells.
  • compositions and methods for genetically modifying immune cells for cell therapy.
  • compositions and methods for CS1 targeted CAR-T cells and CD137 targeted CAR-T cells while circumventing using patient-specific T cell are also recognized herein.
  • an engineered immune cell comprising a chimeric antigen receptor (CAR) , wherein said CAR comprises: (i) a first antigen binding domain that specifically binds to CS1, (ii) a second antigen binding domain that specifically binds to CD7, and (iii) a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • said first antigen binding domain or said second antigen binding is a scFv.
  • said first antigen binding domain and said second antigen binding domain are arranged, from amino terminus to carboxyl terminus, in one of following patterns: (i) VL2-VH1-VL1-VH2; (ii) VH2-VL1-VH1-VL2; (iii) VL1-VH2-VL2-VH1; (iv) VH1-VL2-VH2-VL1; (v) VL2-VL1-VH1-VH2; (vi) VH2-VH1-VL1-VL2; (vii) VL1-VL2-VH2-VH1; or (viii) VH1-VH2-VL2-VL1; wherein VH1 is heavy chain variable domain of said first antigen binding domain, VL1 is light chain variable light domain of said first antigen binding domain, VH2 is heavy chain variable domain of said second antigen binding domain, and VL2 is light chain variable domain of said second antigen binding domain.
  • said first antigen binding domain and said second antigen binding domain are arranged, from amino terminus to carboxyl terminus, in one of following patterns: (i) VL2-VH2-VL1-VH1; (ii) VL2-VH2-VH1-VL1; (iii) VL1-VH1-VL2-VH2; (iv) VL1-VH1-VH2-VL2; (v) VH2-VL2-VL1-VH1; (vi) VH2-VL2-VH1-VL1; (vii) VH1-VL1-VL2-VH2; and (viii) VH1-VL1-VH2-VL2, wherein VH1 is heavy chain variable domain of said first antigen binding domain, VL1 is light chain variable light domain of said first antigen binding domain, VH2 is heavy chain variable domain of said second antigen binding domain, and VL2 is light chain variable domain of said second antigen binding domain.
  • the CAR further comprises a second transmembrane domain and a second intracellular signaling domain.
  • the first or second transmembrane domain comprises at least a portion of TCR alpha, TCR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or any combination thereof.
  • the first or second intracellular signaling domain comprises at least a portion of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d, or any combination thereof.
  • the first or second intracellular signaling domain further comprises a costimulatory domain.
  • the costimulatory domain is selected from the group consisting of CD127, CD27, CD28, 4-1BB (CD137) , OX40, CD30, CD40, PD-1, ICOS, MyD88, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
  • the first antigen binding domain and the second antigen binding domain are linked via a linker.
  • the linker is a cleavable linker.
  • the linker is a self-cleaving peptide.
  • the cleavable linker is selected from P2A, T2A, E2A, and F2A.
  • said engineered immune cell is a T cell, an NKT cell or an NK cell.
  • said T cell is an alpha beta T cell or a gamma delta T cell.
  • said engineered immune cell is derived from a stem cell.
  • said stem cell is a hematopoietic stem cell (HSC) or an induced pluripotent stem cell (iPSC) .
  • said engineered immune cell is an autologous cell or an allogeneic cell.
  • said engineered immune cell is obtained from a subject having a condition. In some embodiments, said engineered immune cell is obtained from a healthy donor.
  • an endogenous T cell receptor (TCR) of said engineered immune cell is inactivated.
  • a gene encoding a subunit of said endogenous TCR is inactivated such that said endogenous TCR is inactivated.
  • said subunit is selected from TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • an endogenous CS1 of said engineered immune cell is inactivated.
  • a gene encoding the endogenous CS1 of said engineered immune cell is inactivated.
  • an endogenous MHC molecule of said engineered immune cell is inactivated.
  • the endogenous MHC molecule comprises an MHC class I molecule and an MHC class II molecule.
  • the MHC class I molecule comprises HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.
  • a subunit of the endogenous MHC molecule in said engineered immune cell is inactivated such that the endogenous MHC molecule is inactive.
  • B2M subunit of the endogenous MHC molecule in said engineered immune cell is inactivated.
  • the engineered immune cell further comprises an enhancer moiety capable of enhancing one or more activities of said engineered immune cell.
  • said enhancer moiety is configured to constitutively enhance said one or more activities of said engineered immune cell.
  • said enhancer moiety is configured to constitutively upregulate one or more intracellular signaling pathways of said engineered immune cell.
  • said one or more intracellular signaling pathways are one or more cytokine signaling pathways.
  • said enhancer moiety is self-activating through self-oligomerizing. In some embodiments, said enhancer moiety is self-activating through self-dimerizing.
  • said enhancer moiety is a cytokine or a cytokine receptor.
  • said enhancer moiety is selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors for the same, functional fragments thereof, functional variants thereof, and combinations thereof.
  • said enhancer moiety functions as a trans-activating factor or a cis-activating factor.
  • the engineered immune cell further comprises an inducible cell death moiety capable of effecting death of said engineered immune cell upon contacting said chimeric polypeptide with a cell death activator.
  • said inducible cell death moiety is selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, Her2t, CD30, BCMA and EGFRt.
  • said inducible cell death moiety is EGFRt
  • said cell death activator is an antibody or an antigen binding fragment thereof that binds EGFRt.
  • said inducible cell death moiety is HSV-TK, and said cell death activator is GCV. In some embodiments, said inducible cell death moiety is iCasp9, and said cell death activator is AP1903. In some embodiments, said cell death activator comprises a nucleic acid, a polynucleotide, an amino acid, a polypeptide, lipid, a carbohydrate, a small molecule, an enzyme, a ribosome, a proteasome, a variant thereof, or any combination thereof. In some embodiments, said enhancer moiety is linked to said inducible cell death moiety.
  • said engineered immune cell exhibits enhanced viability while in presence of cells that are heterologous to said engineered immune cell.
  • the cells that are heterologous to said engineered immune cell comprises heterologous T cells and heterologous NK cells.
  • an isolated polynucleotide encoding the CARas described above.
  • a method of generating the engineered immune cell as described above comprising: (i) delivering a polynucleotide encoding the CAR as described above into an immune cell; and (ii) expressing the CAR in said immune cell, thereby generating said engineered immune cell.
  • a method of delivering an allogeneic cell therapy comprising administering to a subject in need thereof a population of engineered immune cells as described above.
  • an endogenous TCR of the engineered immune cell is functionally inactivated.
  • the cell reduces GvHD in the subject compared to an additional cell having a functionally active TCR.
  • the hematopoietic malignancy comprises leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) , chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia) , polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms) , multiple myeloma, Waldenstrom's macroglobul
  • acute leukemias such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,
  • kit comprising the engineered immune cells or the isolated polynucleotide as described above.
  • composition comprising the engineered immune cells or the isolated polynucleotide as described above.
  • an engineered immune cell comprising: (i) a chimeric antigen receptor (CAR) , wherein the CAR comprises a first antigen binding domain that specifically binds to CS1, a transmembrane domain and an intracellular signaling domain, (ii) an endogenous inactive T cell receptor (TCR) , (iii) an endogenous inactive MHC molecule, and
  • a gene encoding the endogenous CS1 in said engineered immune cell is inactivated.
  • a subunit of the endogenous TCR in said engineered immune cell is inactivated such that the endogenous TCR is inactive.
  • said subunit is selected from TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • a gene encoding the MHC molecule in said engineered immune cell is inactivated.
  • the MHC molecule comprises an MHC class I molecule and an MHC class II molecule.
  • the MHC class I molecule comprises HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.
  • a subunit of the endogenous MHC molecule in said engineered immune cell is inactivated such that the endogenous MHC molecule is inactive.
  • B2M subunit of the endogenous MHC molecule in said engineered immune cell is inactivated.
  • the CAR further comprises a second transmembrane domain and a second intracellular signaling domain.
  • the first or second transmembrane domain comprises at least a portion of TCR alpha, TCR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or any combination thereof.
  • the first or second intracellular signaling domain comprises at least a portion of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, or any combination thereof.
  • the first or second intracellular signaling domain further comprises a costimulatory domain.
  • the costimulatory domain is selected from the group consisting of CD127, CD27, CD28, 4-1BB (CD137) , OX40, CD30, CD40, PD-1, ICOS, MyD88, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.
  • the first antigen binding domain and the second antigen binding domain are linked via a linker.
  • the linker is a cleavable linker.
  • the linker is a self-cleaving peptide.
  • the cleavable linker is selected from P2A, T2A, E2A, and F2A.
  • the CAR further comprises a second antigen binding domain.
  • the second antigen domain binds to an antigen selected from the group consisting of CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GI
  • said first antigen binding domain or said second antigen binding is a scFv.
  • said first antigen binding domain and said second antigen binding domain are arranged, from amino terminus to carboxyl terminus, in any of following patterns: (i) VL2-VH1-VL1-VH2; (ii) VH2-VL1-VH1-VL2; (iii) VL1-VH2-VL2-VH1; (iv) VH1-VL2-VH2-VL1; (v) VL2-VL1-VH1-VH2; (vi) VH2-VH1-VL1-VL2; (vii) VL1-VL2-VH2-VH1; or (viii) VH1-VH2-VL2-VL1; wherein VH1 is heavy chain variable domain of said first antigen binding domain, VL1 is light chain variable light domain of said first antigen binding domain, VH2 is heavy chain variable domain of said second antigen binding domain, and VL2 is light chain variable domain of said second antigen binding domain.
  • said first antigen binding domain and said second antigen binding domain are arranged, from amino terminus to carboxyl terminus, in one of following patterns: (i) VL2-VH2-VL1-VH1; (ii) VL2-VH2-VH1-VL1; (iii) VL1-VH1-VL2-VH2; (iv) VL1-VH1-VH2-VL2; (v) VH2-VL2-VL1-VH1; (vi) VH2-VL2-VH1-VL1; (vii) VH1-VL1-VL2-VH2; and (viii) VH1-VL1-VH2-VL2, wherein VH1 is heavy chain variable domain of said first antigen binding domain, VL1 is light chain variable light domain of said first antigen binding domain, VH2 is heavy chain variable domain of said second antigen binding domain, and VL2 is light chain variable domain of said second antigen binding domain.
  • said first antigen binding domain and said second antigen binding domain are linked via a linker.
  • said linker comprises a self-cleaving peptide. In some embodiments, said linker does not comprise a self-cleaving peptide.
  • the CAR further comprises a third antigen binding domain.
  • the third antigen domain binds to an antigen selected from the group consisting of CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GI
  • the CAR further comprises a second transmembrane domain and a second intracellular signaling domain.
  • the first antigen binding domain and the second antigen binding domain are linked via a linker.
  • the linker is a cleavable linker.
  • the linker is a self-cleaving peptide.
  • the cleavable linker is selected from P2A, T2A, E2A, and F2A.
  • said engineered immune cell is a T cell, an NKT cell or an NK cell.
  • said T cell is an alpha beta T cell or a gamma delta T cell.
  • said engineered immune cell is derived from a stem cell.
  • said stem cell is a hematopoietic stem cell (HSC) or an induced pluripotent stem cell (iPSC) .
  • said engineered immune cell is an allogeneic cell.
  • said engineered immune cell is obtained from a subject having a condition. In some embodiments, said engineered immune cell is obtained from a healthy donor.
  • the engineered immune cell further comprises an enhancer moiety capable of enhancing one or more activities of said engineered immune cell.
  • said enhancer moiety is configured to constitutively enhance said one or more activities of said engineered immune cell.
  • said enhancer moiety is configured to constitutively upregulate one or more intracellular signaling pathways of said engineered immune cell.
  • said one or more intracellular signaling pathways are one or more cytokine signaling pathways.
  • said enhancer moiety is self-activating through self-oligomerizing. In some embodiments, said enhancer moiety is self-activating through self-dimerizing.
  • said enhancer moiety is a cytokine or a cytokine receptor.
  • said enhancer moiety is selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors for the same, functional fragments thereof, functional variants thereof, and combinations thereof.
  • said enhancer moiety functions as a trans-activating factor or a cis-activating factor.
  • the engineered immune cell further comprises an inducible cell death moiety capable of effecting death of said engineered immune cell upon contacting said chimeric polypeptide with a cell death activator.
  • said inducible cell death moiety is selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, Her2t, CD30, BCMA and EGFRt.
  • said inducible cell death moiety is EGFRt
  • said cell death activator is an antibody or an antigen binding fragment thereof that binds EGFRt.
  • said inducible cell death moiety is HSV-TK, and said cell death activator is GCV. In some embodiments, said inducible cell death moiety is iCasp9, and said cell death activator is AP1903. In some embodiments, said cell death activator comprises a nucleic acid, a polynucleotide, an amino acid, a polypeptide, lipid, a carbohydrate, a small molecule, an enzyme, a ribosome, a proteasome, a variant thereof, or any combination thereof. In some embodiments, said enhancer moiety is linked to said inducible cell death moiety.
  • said engineered immune cell exhibits enhanced viability while in presence of cells that are heterologous to said engineered immune cell.
  • he cells that are heterologous to said engineered immune cell comprises heterologous T cells and heterologous NK cells.
  • an engineered cell comprising a chimeric antigen receptor (CAR) , wherein said CAR comprises: (i) a first antigen binding domain that specifically binds to CS1, wherein the first antigen domain comprises a single domain antibody, (ii) a second antigen binding domain, and (iii) a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • said second antigen binding domain is a scFv.
  • said first antigen binding domain and said second antigen binding domain are arranged, from amino terminus to carboxyl terminus, in one of following patterns: (i) VHH1- VH2-VL2; (ii) VHH1-VL2-VH2; (iii) VH2-VL2-VHH1; (iv) VL2-VH2-VHH1; (v) VH2-VHH1-VL2; and (vi) VL2-VHH1-VH2, wherein VHH1 represents the single domain antibody of the first antigen binding domain, VH2 represents heavy chain variable domain of said second antigen binding domain, and VL2 represents light chain variable domain of said second antigen binding domain.
  • said second antigen binding domain comprises a single domain antibody.
  • said first antigen binding domain and said second antigen binding domain are arranged, from amino terminus to carboxyl terminus, in one of following patterns: (i) VHH1-VHH2; and (ii) VHH2-VHH1, wherein VHH1 represents the single domain antibody of the first antigen binding domain, VHH2 represents the single domain antibody of the second antigen binding domain.
  • the second antigen domain binds to an antigen selected from the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, T
  • said first antigen binding domain and said second antigen binding domain are linked via a linker.
  • said linker comprises a self-cleaving peptide.
  • said linker does not comprise a self-cleaving peptide.
  • said engineered immune cell is a T cell, an NKT cell or an NK cell.
  • said T cell is an alpha beta T cell or a gamma delta T cell.
  • said engineered immune cell is derived from a stem cell.
  • an engineered cell comprising a chimeric polypeptide, wherein the chimeric polypeptide comprises: (i) a first chimeric antigen receptor (CAR) , wherein the first CAR comprises: (a) a first antigen binding domain, (b) a second antigen binding domain, and (c) a first transmembrane domain and a first intracellular signaling domain, and (ii) a second chimeric antigen receptor (CAR) , wherein the second CAR comprises: (a) a third antigen binding domain, and (b) a second transmembrane domain and a second intracellular signaling domain; wherein at least one of the antigen binding domains specifically binds to CS1.
  • CAR chimeric antigen receptor
  • the first CAR and the second CAR are linked by a linker on the chimeric polypeptide.
  • the linker is a cleavable linker.
  • the linker is a self-cleaving peptide.
  • the cleavable linker is selected from P2A, T2A, E2A, and F2A.
  • first co-stimulatory domain and the second co-stimulatory domain are different. In some embodiments, the first co-stimulatory domain and the second co-stimulatory domain are the same.
  • said first or second transmembrane domain comprises at least a portion of TCR alpha, TCR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.
  • the first intracellular signaling domain and the second intracellular signaling domain are different.
  • the first intracellular signaling domain and the second intracellular signaling domain are the same.
  • said first or second intracellular signaling domain comprises at least a portion of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d.
  • the first or the second antigen binding domain is scFv.
  • said first antigen binding domain and said second antigen binding domain are arranged, from amino terminus to carboxyl terminus, in one of following patterns: (i) VL2-VH1-VL1-VH2; (ii) VH2-VL1-VH1-VL2; (iii) VL1-VH2-VL2-VH1; (iv) VH1-VL2-VH2-VL1; (v) VL2-VL1-VH1-VH2; (vi) VH2-VH1-VL1-VL2; (vii) VL1-VL2-VH2-VH1; and (viii) VH1-VH2-VL2-VL1; wherein VH1 represents heavy chain variable domain of said first antigen binding domain, VL1 represents light chain variable light domain of said first antigen binding domain, VH2 represents heavy chain variable domain of said second antigen binding
  • said first antigen binding domain and said second antigen binding domain are arranged, from amino terminus to carboxyl terminus, in one of following patterns: (i) VL2-VH2-VL1-VH1; (ii) VL2-VH2-VH1-VL1; (iii) VL1-VH1-VL2-VH2; (iv) VL1-VH1-VH2-VL2; (v) VH2-VL2-VL1-VH1; (vi) VH2-VL2-VH1-VL1; (vii) VH1-VL1-VL2-VH2; and (viii) VH1-VL1-VH2-VL2, wherein VH1 represents heavy chain variable domain of said first antigen binding domain, VL1 represents light chain variable light domain of said first antigen binding domain, VH2 represents heavy chain variable domain of said second antigen binding domain, and VL2 represents light chain variable domain of said second antigen binding domain.
  • the third antigen binding domain comprises a scFv or a single domain antibody.
  • the first antigen domain binds to an antigen selected from the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGF
  • the second antigen domain binds to an antigen selected from the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGF
  • the third antigen domain binds to an antigen selected from the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGF
  • an engineered immune cell comprising: (i) a chimeric antigen receptor (CAR) , wherein the CAR comprises a first antigen binding domain that specifically binds to CD137, a transmembrane domain and an intracellular signaling domain, and (ii) any two or more of an endogenous inactive T cell receptor (TCR) , an endogenous inactive MHC molecule, and an endogenous inactive CD137.
  • CAR chimeric antigen receptor
  • TCR endogenous inactive T cell receptor
  • the immune cell comprises an endogenous inactive T cell receptor (TCR) , and an endogenous inactive CD137. In some embodiments, the immune cell comprises an endogenous inactive MHC molecule and an endogenous inactive CD137.
  • TCR T cell receptor
  • CD137 endogenous inactive CD137
  • the immune cell comprises an endogenous inactive T cell receptor (TCR) and an endogenous inactive MHC molecule. In some embodiments, the immune cell comprises an endogenous inactive T cell receptor (TCR) , an endogenous inactive MHC molecule, and an endogenous inactive CD137. In some embodiments, the immune cell comprises a second antigen binding domain targeting a tumor antigen.
  • an engineered immune cell comprising: (i) a chimeric antigen receptor (CAR) , wherein the CAR comprises a first antigen binding domain that specifically binds to CS1, a transmembrane domain and an intracellular signaling domain, and (ii) any two or more of an endogenous inactive T cell receptor (TCR) , an endogenous inactive MHC molecule, and an endogenous inactive CS1.
  • CAR chimeric antigen receptor
  • TCR endogenous inactive T cell receptor
  • the immune cell comprises an endogenous inactive T cell receptor (TCR) , and an endogenous inactive CS1. In some embodiments, the immune cell comprises an endogenous inactive MHC molecule and an endogenous inactive CS1. In some embodiments, the immune cell comprises an endogenous inactive T cell receptor (TCR) and an endogenous inactive MHC. In some embodiments, the immune cell comprises an endogenous inactive T cell receptor (TCR) , an endogenous inactive MHC molecule, and an endogenous inactive CS1 molecule. In some embodiments, the immune cell comprises a second antigen binding domain targeting a tumor antigen.
  • an engineered immune cell comprising a chimeric antigen receptor (CAR) , wherein said CAR comprises: (i) a first antigen binding domain that specifically binds to CS1, (ii) a second antigen binding domain that specifically binds to CD137, (iii) a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • the immune cell comprises any two or more of an endogenous inactive T cell receptor (TCR) , an endogenous inactive MHC molecule, an endogenous inactive CD137, and an endogenous inactive CS1.
  • TCR endogenous inactive T cell receptor
  • CD137 endogenous inactive CD137
  • CS1 endogenous inactive CD137
  • the immune cell comprises an endogenous MHC molecule and an endogenous inactive CS1. In some embodiments, the immune cell comprises an endogenous inactive CD137 and an endogenous inactive CS1. In some embodiments, the immune cell comprises an endogenous inactive MHC molecule, an endogenous inactive CD137, and an endogenous inactive CS1. In some embodiments, the immune cell comprises an endogenous inactive T cell receptor (TCR) , an endogenous inactive CD137, and an endogenous inactive CS1. In some embodiments, the immune cell comprises an endogenous inactive T cell receptor (TCR) , an endogenous inactive MHC molecule, and an endogenous inactive CS1.
  • TCR endogenous inactive T cell receptor
  • the immune cell comprises an endogenous inactive T cell receptor (TCR) , an endogenous inactive MHC molecule, and an endogenous inactive CD137. In some embodiments, the immune cell comprises an endogenous inactive T cell receptor (TCR) , an endogenous inactive MHC molecule, an endogenous inactive CD137, and an endogenous inactive CS1.
  • Figure 1 illustrates exemplary designs of the CAR described herein targeting CS1 as an example and which can be changed to any antigen disclosed herein, where CAR S1-1 to CAR S1-6 illustrates the exemplary designs of the CS1 single CARs; CAR S2-1 to CAR S2-3, CAR S3-1 to CAR S3-3 and CAR S4-1 to CAR S4-3 illustrates the exemplary designs of the CS1 dual CARs in parallel, loop and tandem structures; and CAR S5-1 to CAR S5-4 and CAR S6-1 to CAR S6-4 illustrates the exemplary designs of the CS1 tri-CARs.
  • Figure 2A and 2B illustrate knockout efficiency of CS1 in CD4+ or CD8+ T cells.
  • FIG. 3 illustrates expression of anti-CS1 CAR on T cells.
  • Figure 4A and 4B illustrate binding capability of anti-CS1 CAR-T cells with CS1 antigen.
  • Figure 5A-5C illustrate cytotoxicity of anti-CS1 CAR-T cells on different target cells.
  • Figure 6A-6C illustrate cytokine release after co-incubation of anti-CS1 CAR-T cells with RPMI8226-LucG2#target cells.
  • FIG. 7 illustrates result of CAR-T tonic signaling analysis.
  • Figure 8 illustrates expression of anti-CS1 single CAR, dual CAR and triple CAR of the present application on T cells.
  • Figure 9A-9E illustrate knockout efficiency of TRAC, B2M, CD7 and CS1 in universal CAR-T cells of the present application.
  • Figure 10A-10D illustrate cytotoxicity of the universal anti-CS1 single CAR, dual CAR and triple CAR of the present application on target cells.
  • Figure 11A and 11B illustrate killing effect of the universal anti-CS1 single CAR, dual CAR and triple CAR of the present application on allogenic CTL or NK cells.
  • administering refers to physically introducing a product of the present disclosure into a subject using any of a variety of methods and delivery systems, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other routes of parenteral administration, for example by injection or infusion.
  • an antigen refers to a molecule or a fragment thereof capable of being bound by a selective binding agent.
  • an antigen can be a ligand that can be bound by a selective binding agent such as a receptor.
  • an antigen can be an antigenic molecule that can be bound by a selective binding agent such as an immunological protein (e.g., an antibody) .
  • An antigen can also refer to a molecule or fragment thereof capable of being used in an animal to produce antibodies capable of binding to that antigen.
  • an antigen may be bound to a substrate (e.g., a cell membrane) .
  • an antigen may not be bound to a substrate (e.g., a secreted molecule, such as a secreted polypeptide) .
  • antibody (Ab) include, but is not limited to, an immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an fragment thereof.
  • Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three constant domains CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region comprises a constant domain CL.
  • the VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FR) .
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • nucleotide generally refers to a base-sugar-phosphate combination.
  • a nucleotide can comprise a synthetic nucleotide.
  • a nucleotide can comprise a synthetic nucleotide analog.
  • Nucleotides can be monomeric units of a nucleic acid sequence (e.g. deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) ) .
  • nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP) , uridine triphosphate (UTP) , cytosine triphosphate (CTP) , guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof.
  • Such derivatives can include, for example, [ ⁇ S] dATP, 7-deaza-dGTP and 7-deaza-dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them.
  • nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.
  • ddNTPs dideoxyribonucleoside triphosphates
  • Illustrative examples of dideoxyribonucleoside triphosphates can include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.
  • a nucleotide can be unlabeled or detectably labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels.
  • Fluorescent labels of nucleotides can include but are not limited fluorescein, 5-carboxyfluorescein (FAM) , 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE) , rhodamine, 6-carboxyrhodamine (R6G) , N, N, N′, N′-tetramethyl-6-carboxyrhodamine (TAMRA) , 6-carboxy-X-rhodamine (ROX) , 4- (4′dimethylaminophenylazo) benzoic acid (DABCYL) , Cascade Blue, Oregon Green, Texas Red, Cyanine and 5- (2′-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) .
  • FAM 5-carboxyfluorescein
  • JE 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein
  • fluorescently labeled nucleotides can include [R6G] dUTP, [TAMRA] dUTP, [R110] dCTP, [R6G] dCTP, [TAMRA] dCTP, [JOE] ddATP, [R6G] ddATP, [FAM] ddCTP, [R110] ddCTP, [TAMRA] ddGTP, [ROX] ddTTP, [dR6G] ddATP, [dR110] ddCTP, [dTAMRA] ddGTP, and [dROX] ddTTP available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham
  • Chromosome Labeled Nucleotides BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg.
  • Nucleotides can also be labeled or marked by chemical modification.
  • a chemically-modified single nucleotide can be biotin-dNTP.
  • biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP) , biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP) , and biotin-dUTP (e.g. biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP) .
  • polynucleotide, oligonucleotide, ” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form.
  • a polynucleotide can be exogenous or endogenous to a cell.
  • a polynucleotide can exist in a cell-free environment.
  • a polynucleotide can be a gene or fragment thereof.
  • a polynucleotide can be DNA.
  • a polynucleotide can be RNA.
  • a polynucleotide can have any three dimensional structure, and can perform any function, known or unknown.
  • a polynucleotide can comprise one or more analogs (e.g. altered backbone, sugar, or nucleobase) . If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g.
  • rhodamine or fluorescein linked to the sugar thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine.
  • Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA (tRNA) , ribosomal RNA (rRNA) , short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA) , nucleic acid probes, and primers.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • gene refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript.
  • genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5’ and 3’ ends.
  • the term encompasses the transcribed sequences, including 5’ and 3’ untranslated regions (5’-UTR and 3’-UTR) , exons and introns.
  • the transcribed region will contain “open reading frames” that encode polypeptides.
  • a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region” ) necessary for encoding a polypeptide.
  • genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes.
  • rRNA ribosomal RNA genes
  • tRNA transfer RNA
  • the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters.
  • a gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism.
  • a gene can refer to an “exogenous gene” or a non-native gene.
  • a non-native gene can refer to a gene not normally found in the host organism but which is introduced into the host organism by gene transfer.
  • a non-native gene can also refer to a gene not in its natural location in the genome of an organism.
  • a non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence) .
  • endogenous refers to a nucleic acid molecule or polypeptide normally expressed in a cell or tissue.
  • exogenous refers to the nucleic acid molecule or polypeptide is not endogenously present in the cell or is present at a level sufficient to achieve the functional effects obtained upon overexpression.
  • exogenous includes any recombinant nucleic acid molecule or polypeptide expressed in a cell, e.g., a foreign, heterologous, and overexpressed nucleic acid molecule and polypeptide.
  • an autologous sample e.g., cells
  • an autologous sample can refer to a sample which is removed, processed, and then given back to the same subject (e.g., patient) at a later time.
  • Autologous with respect to a process, can be distinguished from an allogenic process in which the donor of a sample (e.g., cells) and the recipient of the sample are not the same subject.
  • allogeneic refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • T cell and NK cell consensus marker refers to a marker co-existing on T cells and NK cells, including but not limited to: CD2, CD7, CD38, CD45, CD48, CD50, CD52, CD56, CD69, CD100, CD122, CD132, CD161, CD159a, CD159c, CD314.
  • marker of T cells and/or NK cells refers to markers present in T cells or NK cells, respectively, or both T cells and NK cells, including but not limited to: CD2, CD3, CD4 , CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD160, CD161 CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, SLAMF7.
  • the term “functionally inactivate” or “functional inactivation” as used herein refers to that a functional gene or the product of the gene such as mRNA or protein is prevented or inhibited.
  • the inactivation may be achieved by deletion, addition or substitution of the gene or the promoter thereof, so that expression does not occur, or mutation of the coding sequence of the gene so that the gene product such as mRNA or protein is inactive.
  • the functional inactivation may be complete or partial. Inactivation of a gene can encompass all degrees of inactivation, including gene silencing, knockout, inhibition and disruption.
  • the functional inactivation is introduced by CRISPR-Cas9 system.
  • subject “individual, ” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal such as a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • treatment refers to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • a treatment can comprise administering a system or cell population disclosed herein.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more conditions (e.g., diseases or symptoms) under treatment.
  • a composition can be administered to a subject at risk of developing a particular condition, or to a subject reporting one or more of the physiological symptoms of a disease, even though the condition may not have yet been manifested.
  • CARs can comprise an extracellular antigen recognition region, for example, a scFv (single-chain variable fragment) or a single domain antibody (sdAb, such as a VHH antibody) , a transmembrane region, and an intracellular costimulatory signal region.
  • the extracellular domain of CARs can recognize a specific antigen and then transduce the signal through the intracellular domain, causing T cell activation and proliferation, cytolysis toxicity, and secretion of cytokines, thereby eliminating target cells.
  • the patient’s autologous T cells (or heterologous donors) can be first isolated, activated and genetically engineered to produce CAR-T cells, which can be then injected into the same patient. In this way, the probability of graft-versus-host disease may be reduced, and the antigen can be recognized by T cells in a non-MHC-restricted manner.
  • a CAR-T can treat all cancers that express the antigen.
  • the present disclosure provides compositions and methods to engineer a cell, e.g., an immune cell, such that it can target both disease-associated antigen (e.g., tumor-associated antigen or tumor cell marker such as CS1, BCMA, CD19) and immune cell antigen (e.g., CS1, CD7, CD137) through single, bispecific or multivalent CAR (s) .
  • disease-associated antigen e.g., tumor-associated antigen or tumor cell marker such as CS1, BCMA, CD19
  • immune cell antigen e.g., CS1, CD7, CD137
  • s single, bispecific or multivalent CAR
  • the present disclosure provides an engineered immune cell that can target a tumor cell marker and an immune cell antigen such as CS1.
  • the endogenous TCR can be inactivated (e.g., disrupted, inhibited, knocked out or silenced) .
  • the CAR-T of the present disclosure which targets the tumor cell marker and the immune cell antigen can eliminate positive tumor cells and clear host immune cell antigen positive T and NK cells, thereby avoiding host rejection (HVG) .
  • the endogenous MHC molecule can be inactivated (e.g., disrupted, inhibited, knocked out or silenced) to avoid the host rejection.
  • the endogenous expression of the immune cell antigen in the engineered immune cell can be inactivated (e.g., disrupted, inhibited, knocked out or silenced) to avoid fratricide.
  • the endogenous TCR of the engineered immune cell can be knocked out, and graft-versus-host disease (GVHD) can be prevented, thereby preparing a general-purpose or universal CAR-T (UCAR-T) cell.
  • the engineered immune cell can be derived from an autologous T cell or an allogeneic T cell.
  • the engineered immune cell can comprise a cell suicide element (e.g., inducible cell death moiety) , and the CAR-T can be inactivated/cleared at any time to reduce side effects.
  • the engineered immune cell can further comprise an enhancer moiety.
  • the enhancer moiety can regulate one or more activities of the engineered immune cell when the engineered immune cell is administered to a subject.
  • the enhancer moiety can be a cytokine (e.g., IL-5 or IL-7) or a cytokine receptor (e.g., IL-5R or IL-7R) .
  • the enhancer moiety can enhance a signaling pathway within the engineered immune cell, for example, STAT5 signaling pathway.
  • the engineered immune cell comprises a bispecific CAR targeting both CS1 and CD7 or CS1 and CD137.
  • the engineered immune cell show in this example can further comprise an inducible cell death moiety such as a truncated epidermal growth factor receptor (EGFRt or tEGFR, which can be used interchangeably herein; see U.S. Patent No. 9447194B2 and PCT Publication No. WO2018038945) .
  • EGFRt or tEGFR truncated epidermal growth factor receptor
  • the inducible cell death moiety or the enhancer moiety can be introduced in the immune cell via a separate expression vector.
  • the inducible cell death moiety and the enhancer moiety may be introduced into the immune cell via an expression vector comprising sequences encoding both moieties.
  • the inducible cell death moiety and the enhancer moiety are linked and are expressed as a chimeric polypeptide.
  • the application of the engineered immune cells provided herein in cell therapy can treat the disease (e.g., cancer) of a patient, be prepared in large-scale in advance to avoid GVHD and HvG, reduce treatment costs, inactivate CAR-T at any time if necessary, reduce side effects of immunotherapy, and ensure product safety.
  • the engineered cells provided herein can be referred to as universal CAR-T cells (UCAR-T cells) .
  • the cell e.g., immune cell or engineered immune cell
  • the CAR can include an extracellular domain, a transmembrane domain, and an intracellular signaling domain.
  • the extracellular domain can include a target-specific binding element (also known as an antigen binding domain) .
  • the intracellular domain can include a costimulatory signaling region and a zeta ( ⁇ ) chain portion.
  • a costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules other than antigens receptors or their ligands that may be needed for an efficient response of lymphocytes to antigen.
  • spacer domain generally means any oligo-or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain.
  • a spacer domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR.
  • the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • Transmembrane regions of particular use in the present disclosure may be derived from (e.g., comprise at least the transmembrane region (s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4.
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo-or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the cytoplasmic domain or otherwise the intracellular signaling domain of the CAR of the present disclosure can be responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • intracellular signaling domains for use in the CAR of the present disclosure include the cytoplasmic sequences of the TCR and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences) .
  • Primary cytoplasmic signaling sequences can regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.
  • ITAM containing primary cytoplasmic signaling sequences that are of particular use in the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that cytoplasmic signaling molecule in the CAR of the present disclosure comprises a cytoplasmic signaling sequence derived from CD3-zeta.
  • the cytoplasmic domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain (s) useful in the context of the CAR of the present disclosure.
  • the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region.
  • the costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that may be needed for an efficient response of lymphocytes to an antigen.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137) , OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • LFA-1 lymphocyte function-associated antigen-1
  • cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present disclosure may be linked to each other in a random or specified order.
  • a short oligo-or polypeptide linker preferably between 2 and 10 amino acids in length may form the linkage.
  • a glycine-serine doublet provides a particularly suitable linker.
  • the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28 and 4-1BB.
  • a CAR provided herein can comprise one or more antigen binding domains.
  • a CAR provided herein comprises an antigen binding domain that can target both an immune cell antigen (e.g., to inhibit killing activity of a T cell or NK cell) and a disease-associated antigen (e.g., a tumor-associated antigen) .
  • an antigen binding domain targeting both immune cell antigens and cancer antigens include, but not limited to, CD2, CD3, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD69, CD100, CD122, CD132, CD137, CD161, CD159a, CD159c, CD279, CD314, CD319 (CS1) and TCR.
  • a CAR provided herein comprises a single antigen binding domain.
  • the single antigen binding domain is a scFv or sdAb.
  • the CAR can comprise the structure of VHH-Hinge-TM-CD28/41BB-CD3 ⁇ .
  • CD28 can be an example of transmembrane domain and can be replaced with other transmembrane domains described herein.
  • 41BB can be an example of co-stimulatory domain and can be replaced with other co-stimulatory domains described herein.
  • VHH can be an sdAb capable of targeting an antigen.
  • the antigen can be any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1,
  • the CAR can comprise a single antigen binding domain, and the single antigen binding domain comprises multiple antigen binding units.
  • the antigen binding unit is an sdAb.
  • the CAR can comprise a structure of (VHH) n -Hinge-TM-CD28/41BB-CD3 ⁇ , CD28 can be an example of transmembrane domain and can be replaced with other transmembrane domains described herein.
  • 41BB can be an example of co-stimulatory domain and can be replaced with other co-stimulatory domains described herein.
  • VHH can be an antigen binding unit such as an sdAb, and n is an integer greater than 1.
  • n can be any integer of 2-20.
  • n can be any one of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
  • n is 2. In
  • the antigen can be any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1,
  • a CAR provided herein comprises two antigen binding domains such that one individual CAR is a bispecific CAR, targeting two different antigens.
  • the antigen binding domain may be selected from any antigen binding domain disclosed herein.
  • one of the antigen binding domains may be a scFv, and the other antigen binding domains may be a single domain antibody (sdAb) .
  • both antigen binding domains are scFv.
  • both antigen binding domains are sdAb.
  • the first antigen binding domain of the bispecific CAR targets an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, T
  • the second antigen binding domain of the bispecific CAR targets an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, T
  • one antigen binding domain can target CS1, and the other antigen binding domain can target CD7, CD137, CD19 or BCMA .
  • the two antigen binding domains of a bispecific CAR can have a tandem structure, a parallel structure or a loop structure.
  • a CAR can target CS1 and CD7.
  • the CAR can have a structure as formula I or I’: L-scFv1-I-scFv2-H-TM-C-CD3 ⁇ (I) and L-scFv2-I-scFv1-H-TM-C-CD3 ⁇ (I’) , wherein each “-” is independently a linker peptide or a peptide bond; L is optionally a signaling peptide sequence; I is a flexible linker; H is optionally a hinge region; TM is a transmembrane domain; C is a costimulatory domain; CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ ; one of scFv1 and scFv2 is an antigen binding domain targeting CS1, and the other one is an antigen binding domain targeting CD7.
  • a CAR can target CS1 and CD137.
  • the CAR can have a structure as formula I or I’: L-scFv1-I-scFv2-H-TM-C-CD3 ⁇ (I) and L-scFv2-I-scFv1-H-TM-C-CD3 ⁇ (I’) , wherein each “-” is independently a linker peptide or a peptide bond; L is optionally a signaling peptide sequence; I is a flexible linker; H is optionally a hinge region; TM is a transmembrane domain; C is a costimulatory domain; CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ ; one of scFv1 and scFv2 is an antigen binding domain targeting CS1, and the other one is an antigen binding domain targeting CD137.
  • the CAR can have a structure as formula II or II’: L-VHH1-I-VHH2-H-TM-C-CD3 ⁇ (II) , L-VHH2-I-VHH1-H-TM-C-CD3 ⁇ (II’) , wherein each “-” is independently a linker peptide or a peptide bond; L is optionally a signaling peptide sequence; I is a flexible linker; H is optionally a hinge region; TM is a transmembrane domain; C is a costimulatory domain; CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ ; VHH1 and VHH2 are single domain antibodies targeting to a first antigen and a second antigen, respectively.
  • the antigen can be selected from any antigen disclosed herein.
  • one of VHH1 and VHH2 is an sdAb targeting CS1, and the other is an sdAb targeting CD7.
  • one of VHH1 and VHH2 targets CS1, and the other one targets CD137.
  • the first antigen binding domain and said second antigen binding domain are arranged, from amino terminus to carboxyl terminus, in any of following patterns: (i) VHH1-VH2-VL2; (ii) VHH1-VL2-VH2; (iii) VH2-VL2-VHH1; (iv) VL2-VH2-VHH1; (v) VH2- VHH1-VL2; and (vi) VL2-VHH1-VH2, wherein VHH1 represents the single domain antibody of the first antigen binding domain, VH2 represents heavy chain variable domain of said second antigen binding domain, and VL2 represents light chain variable domain of said second antigen binding domain.
  • the CAR can have a structure represented by the following formula III or III': L3-VHH1-R-scFv2-H3-TM3-C3-CD3 ⁇ (IV) ; L3-scFv2-R-VHH1-H3-TM3-C3-CD3 ⁇ (III’) , wherein each "-" is independently a linker peptide or peptide bond; L3 is an optional signal peptide sequence; VHH1 is an antigen binding domain that targets CS1; R is a rigid or flexible joint; scFv2 is an antigen binding domain (e.g., an antibody single-chain variable region sequence) that targets CD7, CD137 or BCMA; H3 is an optional hinge region; TM3 is a transmembrane domain; C3 is a costimulatory domain; CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ .
  • formula III or III' L3-VHH1-R-scFv2-H3-TM3-
  • the CAR can have a structure as formula IV or IV’: L-VL-scFv-VH-H-TM-C-CD3 ⁇ (IV) , L-VH-scFv-VL-H-TM-C-CD3 ⁇ (IV’) , wherein each “-” is independently a linker peptide or a peptide bond; the elements L, H, TM, C and CD3 ⁇ as described above; scFv is an antigen binding domain targeting a CS1, VH is an anti-CD7 antibody heavy chain variable region, and VL is an anti-CD7 antibody light chain variable region; or scFv is an antigen binding domain targeting CD7, VH is CS1 antibody heavy chain variable region, and VL is CS1 antibody light chain variable region.
  • scFv is an antigen binding domain targeting a CS1
  • VH is an anti-CD137 antibody heavy chain variable region
  • VL is an anti-CD137 antibody light chain variable region
  • scFv is an antigen binding domain targeting CD137
  • VH is CS1 antibody heavy chain variable region
  • VL is CS1 antibody light chain variable region
  • a CAR can comprise a safety switch as described herein.
  • a CAR can have the structure of EGFRt-CD7 scFv-CS1 scFv-Hinge-TM-CD28/41BB-CD3 ⁇ , wherein EGFRt is a truncated EGFR, as a safety switch (e.g., inducible cell death moiety) , CD7 scFv is the scFv fragment of the heavy and light chain variable regions of an monoclonal antibody linked by a GS linker, and the CS1 scFv fragment of the heavy and light chain variable regions of an CS1 monoclonal antibody.
  • the structure of the CAR can further comprise a hinge, transmembrane regions, costimulatory signaling region of CD28 or 41BB, and/or CD3 ⁇ intracellular domain.
  • the nucleic acid construct of EGFRt-CD7 scFv-CS1 scFv-Hinge-TM-CD28/41BB-CD3 ⁇ can be inserted into a vector (e.g., a lentiviral vector) .
  • the vector can be packaged in 293T cells.
  • T cells can be sorted from PBMC, and after activation, TCR and PD-1 genes can be knocked out by CRISPR/CAS technology. T cells can then be infected with the vectors to express the CARs.
  • the prepared CAR-T cells can be used to detect the infection efficiency and gene editing efficiency of CAR by flowcytometry.
  • a CAR can comprise the structure of EGFRt-CD137 scFv-CS1 scFv-Hinge-TM-CD28/41BB-CD3 ⁇ , wherein EGFRt is a truncated EGFR, as a safety switch (e.g., inducible cell death moiety) , CD137 scFv is the scFv fragment of the heavy and light chain variable regions of an monoclonal antibody linked by a GS linker, and the CS1 scFv fragment is the scFv fragment of the heavy and light chain variable regions of an CS1 monoclonal antibody.
  • the structure of the CAR can further comprise a hinge, transmembrane regions, costimulatory signaling region of CD28 or 41BB, and/or CD3 ⁇ intracellular domain.
  • the nucleic acid construct of EGFRt-CD137 scFv-CS1 scFv-Hinge-TM-CD28/41BB-CD3 ⁇ can be inserted into a vector (e.g., a lentiviral vector) .
  • the vector can be packaged in 293T cells.
  • T cells can be sorted from PBMC, and after activation, TCR and PD-1 genes can be knocked out by CRISPR/CAS technology. T cells can then be infected with the vectors to express the CARs.
  • the prepared CAR-T cells can be used to detect the infection efficiency and gene editing efficiency of CAR by flowcytometry.
  • a CAR comprising two antigen binding domains arranged in a tandem form, as shown in CAR S4-1 to CAR S4-3 in Figure 1.
  • the first antigen binding domain or the second antigen binding domain is a scFv.
  • the first antigen binding domain and the second antigen binding domain is arranged, from amino terminus to carboxyl terminus, in any of following patterns: (i) VL2-VH2-VL1-VH1; (ii) VL2-VH2-VH1-VL1; (iii) VL1-VH1-VL2-VH2; (iv) VL1-VH1-VH2-VL2; (v) VH2-VL2-VL1-VH1; (vi) VH2-VL2-VH1-VL1; (vii) VH1-VL1-VL2-VH2; and (viii) VH1-VL1-VH2-VL2, wherein VH1 is heavy chain variable domain of the first antigen binding domain, VL1 is light chain variable light domain of the first antigen binding domain, VH2 is heavy chain variable domain of
  • the CAR can have a structure represented by the following formula V or V': L3-scFv1-R-scFv2-H3-TM3-C3-CD3 ⁇ (V) ; L3-scFv2-R-scFv1-H3-TM3-C3-CD3 ⁇ (V’) , wherein each "-" is independently a linker peptide or peptide bond; L3 is an optional signal peptide sequence; scFv1 is an antigen binding domain that targets CS1; R is a rigid or flexible joint; scFv2 is an antigen binding domain (e.g., an antibody single-chain variable region sequence) that targets CD7, CD137 or BCMA; H3 is an optional hinge region; TM3 is a transmembrane domain; C3 is a costimulatory domain; CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ .
  • V L3-scFv1-R-scFv2-H
  • the first antigen binding domain or the second antigen binding domain is an sdAb.
  • the CAR can have a structure represented by the following formula VI or VI': L3-VHH1-R-VHH2-H3-TM3-C3-CD3 ⁇ (VI) ; L3-VHH2-R-VHH1-H3-TM3-C3-CD3 ⁇ (VI’) , wherein each "-" is independently a linker peptide or peptide bond; L3 is an optional signal peptide sequence; VHH1 is an antigen binding domain that targets CS1; R is a rigid or flexible joint; VHH2 is an antigen binding domain (e.g., a single domain antibody) that targets CD7, or CD137 or BCMA; H3 is an optional hinge region; TM3 is a transmembrane domain; C3 is a costimulatory domain; CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ .
  • a CAR comprising two antigen binding domains arranged in a loop form as shown in CAR S3-1 to CAR S3-3 in Figure 1.
  • the first antigen binding domain and the second antigen binding domain is arranged, from amino terminus to carboxyl terminus, in any of following patterns: (i) VL2-VH1-VL1-VH2; (ii) VH2-VL1-VH1-VL2; (iii) VL1-VH2-VL2-VH1; (iv) VH1-VL2-VH2-VL1; (v) VL2-VL1-VH1-VH2; (vi) VH2-VH1-VL1-VL2; (vii) VL1-VL2-VH2-VH1; and (viii) VH1-VH2-VL2-VL1, wherein VH1 is heavy chain variable domain of the first antigen binding domain, VL1 is light chain variable light domain of the first antigen
  • the CAR can have the following formula VII, VII’, VII” or VII”’ structure: L8-VL1-VH2-I-VL2-VH1-H8-TM8-C8-CD3 ⁇ (VII) ; L8-VH1-VL2-I-VH2-VL1-H8-TM8-C8-CD3 ⁇ (VII’) ; L8-VL2-VH1-I-VL1-VH2-H8-TM8-C8-CD3 ⁇ (VII”) ; L8-VH2-VL1-I-VH1-VL2-H8-TM8-C8-CD3 ⁇ (VII”’) , wherein each "-" is independently a linker peptide or peptide bond; L8 is an optional signal peptide sequence; VH1 is an CS1 antibody heavy chain variable region, and VL1 is an CS1 antibody light chain variable region; VH2 is an CD7, CD137 or BCMA antibody heavy chain variable region;
  • a CAR comprising two antigen binding domains are arranged in a parallel form, as shown in CAR S2-1 to CAR S2-3 in Figure 1.
  • the parallel form can comprise a full construct of a first CAR having a first antigen binding domain linked to a full construct of a second CAR having a second antigen binding domain.
  • the first antigen binding domain or the second antigen binding domain is a scFv.
  • An example of parallel form can be tEGFR-CS1 scFv-CD28-CD3 ⁇ -CD7 scFv-41BB-CD3 ⁇ .
  • the tEGFR shown here can function as a safety switch, which can be replaced by other safety switches as described in the present disclosure.
  • CS1 scFv and CD7 scFv are two examples of antigen binding domains, which may be replaced with various antigen binding domains as described in the present disclosure.
  • CD28 can be an example of transmembrane domain and can be replaced with other transmembrane domains described herein.
  • 41BB can be an example of co-stimulatory domain and can be replaced with other co-stimulatory domains described herein.
  • a linker is used to link the first CAR and the second CAR.
  • the linker can be a cleavable linker.
  • the cleavable linker can be self-cleaving peptide such as 2A self-cleaving peptide.
  • tEGFR-CS1 scFv-CD28-CD3 ⁇ -CD137 scFv-41BB-CD3 ⁇ Another example of parallel form can be tEGFR-CS1 scFv-CD28-CD3 ⁇ -CD137 scFv-41BB-CD3 ⁇ .
  • the tEGFR shown here can function as a safety switch, which can be replaced by other safety switches as described in the present disclosure.
  • CS1 scFv and CD137 scFv are two examples of antigen binding domains, which may be replaced with various antigen binding domains as described in the present disclosure.
  • CD28 can be an example of transmembrane domain and can be replaced with other transmembrane domains described herein.
  • 41BB can be an example of co-stimulatory domain and can be replaced with other co-stimulatory domains described herein.
  • a linker is used to link the first CAR and the second CAR.
  • the linker can be a cleavable linker.
  • the cleavable linker can be self-cleaving peptide such as 2A self-cleaving peptide.
  • the first antigen binding domain or the second antigen binding domain is a sdAb.
  • An example of parallel form can be tEGFR-CS1 sdAb-CD28-CD3 ⁇ -CD7 sdAb-41BB-CD3 ⁇ .
  • the tEGFR shown here can function as a safety switch, which can be replaced by other safety switches as described in the present disclosure.
  • CS1 sdAb and CD7 sdAb are two examples of antigen binding domains, which may be replaced with various antigen binding domains as described in the present disclosure such as CD137 targeting sdAb.
  • CD28 can be an example of transmembrane domain and can be replaced with other transmembrane domains described herein.
  • 41BB can be an example of co-stimulatory domain and can be replaced with other co-stimulatory domains described herein.
  • a linker is used to link the first CAR and the second CAR.
  • the linker can be a cleavable linker.
  • the cleavable linker can be self-cleaving peptide such as 2A self-cleaving peptide.
  • tEGFR-CS1 sdAb-CD28-CD3 ⁇ -CD137 sdAb-41BB-CD3 ⁇ Another example of parallel form can be tEGFR-CS1 sdAb-CD28-CD3 ⁇ -CD137 sdAb-41BB-CD3 ⁇ .
  • the tEGFR shown here can function as a safety switch, which can be replaced by other safety switches as described in the present disclosure.
  • CS1 sdAb and CD137 sdAb are two examples of antigen binding domains, which may be replaced with various antigen binding domains as described in the present disclosure such as CD137 targeting sdAb.
  • CD28 can be an example of transmembrane domain and can be replaced with other transmembrane domains described herein.
  • 41BB can be an example of co-stimulatory domain and can be replaced with other co-stimulatory domains described herein.
  • a linker is used to link the first CAR and the second CAR.
  • the linker can be a cleavable linker.
  • the cleavable linker can be self-cleaving peptide such as 2A self-cleaving peptide.
  • a CAR provided herein is a multivalent CAR such as a tri-specific CAR (tri-CAR) comprising three antigen binding domains as shown in CAR S5-1 to S5-4, and CAR S6-1 to S6-4 in Figure 1.
  • tri-CAR tri-specific CAR
  • the first antigen binding domain of the tri-specific CAR targets an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA,
  • the second antigen binding domain of the tri-specific CAR targets an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA,
  • the third antigen binding domain of the tri-specific CAR targets an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O- acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA
  • the first antigen binding domain and the second antigen binding domain of the tri-CAR is arranged as the form of the dual CAR as described above (such as the tandem or the loop form) to form a bispecific unit, and the bispecific unit and a third antigen binding domain are further arranged in parallel form.
  • the first antigen binding domain, the second antigen binding domain and the third antigen binding domain of the tri-CAR is arranged, from amino terminus to carboxyl terminus, in any of following patterns: (i) VL2-VH1-VL1-VH2-CD28-CD3 ⁇ -VH3-VL3-41BB-CD3 ⁇ ; (ii) VH2-VL1-VH1-VL2-CD28-CD3 ⁇ -VH3-VL3-41BB-CD3 ⁇ ; (iii) VL1-VH2-VL2-VH1-CD28-CD3 ⁇ -VH3-VL3-41BB-CD3 ⁇ ; (iv) VH1-VL2-VH2-VL1-CD28-CD3 ⁇ -VH3-VL3-41BB-CD3 ⁇ ; (v) VL2-VL1-VH1-VH2-CD28-CD3 ⁇ -VH3-VL3-41BB-CD3 ⁇ ; (vi) VL2-VL
  • one antigen binding domain can target CS1, and the other two antigen binding domain can target any two selected from CD7, CD137, BCMA, and CD19.
  • the antigen binding domain of CS1 and an antigen binding domain of CD7, CD137, BCMA, or CD19 can have a tandem structure or a loop structure to form the bispecific unit, and the bispecific unit can form a tandem structure with a third antigen binding domain targeting CD7, CD137, BCMA, or CD19.
  • the antigen binding domains of any two of CD7, CD137, BCMA, or CD19 can have a tandem structure or a loop structure to form the bispecific unit, and the bispecific unit can form a tandem structure with the antigen binding domain of CS1.
  • the tri-specific CAR can be arranged, from amino terminus to carboxyl terminus, in any of following patterns: (i) CD7 VL-CS1 scFv-CD7 VH-CD28-CD3 ⁇ -C+BCMA scFv-41BB-CD3 ⁇ ; (ii) CD137 VL-CS1 scFv-CD137 VH-CD28-CD3 ⁇ -C+BCMA scFv-41BB-CD3 ⁇ ; (iii) BCMA VL-CS1 scFv-BCMA VH-CD28-CD3 ⁇ -C+CD19 scFv-41BB-CD3 ⁇ ; (iv) CD19 VL-BCMA scFv-CD19 VH-CD28-CD3 ⁇ -C+CS1 scFv-41BB-CD3 ⁇ ; (v) CS1 scFv-CD7 scFv-CD28-CD3 ⁇ -C+BCMA scFv-41BB-CD3
  • the nucleic acid can comprise a first sequence encoding one or more chimeric antigen receptors (CARs) , wherein the CAR can comprise a monovalent or multivalent (such as bivalent) antigen binding domain and wherein each CAR of the one or more CARs can further comprise a transmembrane domain and an intracellular signaling domain.
  • CARs chimeric antigen receptors
  • the antigen binding domain can target an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGF
  • the nucleic acid can comprise a first sequence encoding one or more chimeric antigen receptor (CAR) , wherein the CAR can comprise (i) a first antigen binding domain linked to (ii) a second antigen binding domain, and wherein each CAR of the one or more CARs can further comprise a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • the first antigen binding domain can target an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, T
  • the second antigen binding domain can target an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGF
  • the nucleic acid can comprise a first sequence encoding one or more chimeric antigen receptor (CAR) , wherein the CAR can comprise (i) a first antigen binding domain linked to (ii) a second antigen binding domain, (iii) a third antigen binding domain, and wherein each CAR of the one or more CARs can further comprise a transmembrane domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • the first antigen binding domain can target an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, T
  • the second antigen binding domain can target an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGF
  • the third antigen binding domain can target an antigen of any one selected the group consisting of CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGF
  • the nucleic acid molecule can further comprise a second sequence encoding an enhancer moiety, which enhancer moiety can enhance one or more activities of the CAR when expressed in a cell.
  • the enhancer moiety can be selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors for the same, functional fragments thereof, functional variants thereof, and combinations thereof.
  • the nucleic acid molecule can further comprise a second sequence encoding an inducible cell death moiety, which inducible cell death moiety, when expressed in a cell, can effect death of the cell upon contacting the inducible cell death moiety with a cell death activator.
  • the inducible cell death moiety can be selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, and EGFRt.
  • the nuclei acid molecule can further comprise a third sequence flanked by the first sequence and the second sequence, wherein the third sequence can encode a cleavable linker.
  • the cleavable linker can be a self-cleaving peptide.
  • the nucleic acid molecule can further comprise a regulatory sequence regulating expression of the first sequence and/or the second sequence.
  • kits comprising the nucleic acid molecule described herein.
  • the nucleic acid encoding the CAR described herein can be delivered into an immune cell for expression of the CAR to generate an engineered cell.
  • the present disclosure provides an engineered cell, such as an engineered immune cell.
  • the engineered immune cell can be prepared from a cell (e.g., an immune cell) isolated from a sample obtained from a subject.
  • the engineered immune cell can be prepared from a cell line cell.
  • the immune cell used to prepare the engineered immune cell can be a T cell, a B cell, a natural killer (NK) cell or a macrophage.
  • the immune cell used to prepare the engineered immune cell can be an innate lymphocyte (ILC) .
  • ILC innate lymphocyte
  • the immune cell used to prepare the engineered immune cell can be a stem cell.
  • the stem cell can be a hematopoietic stem cell (HSC) or an induced pluripotent stem cell (iPSC) .
  • HSC hematopoietic stem cell
  • iPSC induced pluripotent stem cell
  • the immune cell may comprise a T-cell receptor (TCR) .
  • TCR T-cell receptor
  • the TCR can be endogenous TCR of the immune cell.
  • the endogenous TCR can be inactivated.
  • a gene encoding a subunit of the TCR can be inactivated.
  • the immune cell can be an alpha beta T cells with impaired TCRs such that the immune cells can avoid GVHD.
  • the function of the endogenous TCR can be inhibited by an inhibitor such as TCR-derived peptides, peptides derived from amino acid sequences of fusion and other protein regions of various viruses, antibodies and small molecule inhibitors.
  • the viruses from which the TCR inhibiting peptides can be derived from include, but are not limited to, severe acute respiratory syndrome coronavirus (SARS-CoV) , herpesvirus saimiri (HVS) , human herpesvirus 6 (HHV-6) , Lassa virus (LASV) , lymphocytic choriomeningitis virus (LCMV) , Mopeia virus (MOPV) , Tacaribe virus (TACV) , Friend murine leukemia virus (MLV) ; human T lymphotropic virus type 1 (HTLV-1, ) ; herpesvirus ateles (HVA) ; Marburg virus (MARV) ; Sudan Ebola virus (SEBOV) ; and Zaire Ebola virus (ZEBOV) .
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • HVS herpesvirus saimiri
  • HHV-6 human herpesvirus 6
  • Lassa virus LL
  • the immune cells can be T cells containing TCRs that may not cause GVHD responses.
  • the immune cell can be an alpha beta T cell with TCRs that can recognize specific antigens such as viral specific antigen, tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs) .
  • TAAs tumor-associated antigens
  • TSAs tumor-specific antigens
  • the immune cell can be a gamma delta T cell or a natural killer T (NKT) cell.
  • the immune cell can be induced pluripotent stem cells produced from antigen-specific T cells (e.g., antigen-specific cytotoxic T cells) .
  • the immune cell can be cord-blood T cells.
  • the immune cell may comprise a cell surface marker.
  • the cell surface marker can be an immune cell antigen.
  • the gene encoding the immune cell antigen of the immune cell used for preparing the engineered immune cell can be inactivated.
  • immune cell antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD279, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, CD319 (CS1) , TCR ⁇ , TCR ⁇ and SLAMF7.
  • the immune cells can be isolated from a sample from a subject.
  • the subject can be a healthy donor.
  • the subject can have a condition (e.g., a disease such as cancer) .
  • the sample can be a bodily fluid or a tissue, including but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • a sample comprises NK cells, NKT cells, T-cells or T-cell progenitor cells.
  • the sample is an umbilical cord blood sample, a peripheral blood sample (e.g., a mononuclear cell fraction) or a sample from the subject comprising pluripotent cells.
  • a sample from the subject can be cultured to generate induced pluripotent stem (iPS) cells and these cells used to produce NK cells, NKT cells or T-cells.
  • Cell samples may be cultured directly from the subject or may be cryopreserved prior to use.
  • obtaining a cell sample comprises collecting a cell sample.
  • the sample is obtained by a third party.
  • a sample from a subject can be treated to purify or enrich the T-cells or T-cell progenitors in the sample.
  • the sample can be subjected to gradient purification, cell culture selection and/or cell sorting (e.g., via fluorescence-activated cell sorting (FACS) ) .
  • FACS fluorescence-activated cell sorting
  • the immune cell can be an NK cell.
  • the NK cells can be obtained from peripheral blood, cord-blood, or other sources described herein.
  • the NK cells can be derived from induced pluripotent stem cells.
  • a cell that can be utilized in a method provided herein can be positive or negative for a given factor.
  • a cell utilized in a method provided herein can be a CD3+ cell, CD3-cell, a CD5+ cell, CD5-cell, a CD7+ cell, CD7-cell, a CD14+ cell, CD14-cell, CD8+ cell, a CD8-cell, a CD103+ cell, CD103-cell, CD11b+ cell, CD11b-cell, a BDCA1+ cell, a BDCA1-cell, an L-selectin+ cell, an L-selectin-cell, a CD25+, a CD25-cell, a CD27+, a CD27-cell, a CD28+ cell, CD28-cell, a CD44+ cell, a CD44-cell, a CD56+ cell, a CD56-cell, a CD57+ cell, a CD57-cell, a CD62L
  • a cell may be positive or negative for any factor known in the art.
  • a cell may be positive for two or more factors.
  • a cell may be CD4+ and CD8+.
  • a cell may be negative for two or more factors.
  • a cell may be CD25-, CD44-, and CD69-.
  • a cell may be positive for one or more factors, and negative for one or more factors.
  • a cell may be CD4+ and CD8-.
  • a cellular marker provided herein can be utilized to select, enrich, or deplete a population of cells.
  • enriching comprises selecting a monocyte fraction. In some aspects, enriching comprises sorting a population of immune cells from a monocyte fraction. In some embodiments, the cells may be selected for having or not having one or more given factors (e.g., cells may be separated based on the presence or absence of one or more factors) . In some embodiments, the selected cells can also be transduced and/or expanded in vitro. The selected cells can be expanded in vitro prior to infusion. In some embodiments, selected cells can be transduced with a vector provided herein. It should be understood that cells used in any of the methods disclosed herein may be a mixture (e.g., two or more different cells) of any of the cells disclosed herein.
  • a method of the present disclosure may comprise cells, and the cells are a mixture of CD4+ cells and CD8+ cells.
  • a method of the present disclosure may comprise cells, and the cells are a mixture of CD4+ cells and cells.
  • a cell can be a stem memory TSCM cell comprised of CD45RO (-) , CCR7 (+) , CD45RA (+) , CD62L+ (L-selectin) , CD27+, CD28+ and IL-7R ⁇ +
  • stem memory cells can also express CD95, IL-2R ⁇ , CXCR3, and LFA-1, and show numerous functional attributes distinctive of stem memory cells.
  • Cells provided herein can also be central memory TCM cells comprising L-selectin and CCR7, where the central memory cells can secrete, for example, IL-2, but not IFN ⁇ or IL-4.
  • Cells can also be effector memory TEM cells comprising L-selectin or CCR7 and produce, for example, effector cytokines such as IFN ⁇ and IL-4.
  • a population of cells can be introduced to a subject.
  • a population of cells can be a combination of T cells and NK cells.
  • a population can be a combination of cells and effector cells.
  • a population of cells can be TILs.
  • the source immune cells can be T cells.
  • the T cells can be alpha beta T cells or gamma delta T cells.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • various T cell lines may be used.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll TM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS) .
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium may lead to magnified activation.
  • a washing step may be accomplished by methods such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions.
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL TM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (i.e., 3 ⁇ 28) -conjugated beads, such as M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In some embodiments, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells.
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process.
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • multiple rounds of selection can also be used. In certain embodiments, it may be useful to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • An example method can be cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • the concentration of cells and surface can be varied.
  • it may be desirable to significantly decrease the volume in which beads and cells are mixed together i.e., increase the concentration of cells
  • a concentration of 2 billion cells/ml is used.
  • a concentration of 1 billion cells/ml is used.
  • greater than 100 million cells/ml is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used.
  • concentrations can result in increased cell yield, cell activation, and cell expansion.
  • use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc. ) . Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • lower concentrations of cells may be used. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads) , interactions between the particles and cells is minimized.
  • This method can select for cells that express high amounts of desired antigens to be bound to the particles.
  • CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations.
  • the concentration of cells used is 5 ⁇ 10 6 /ml. In other embodiments, the concentration used can be from about 1 ⁇ 10 5 /ml to 1 ⁇ 10 6 /ml, and any integer value in between.
  • the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10°C. or at room temperature.
  • T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • one method involves using PBS containing 20%DMSO and 8%human serum albumin, or culture media containing 10%Dextran 40 and 5%Dextrose, 20%Human Serum Albumin and 7.5%DMSO, or 31.25%Plasmalyte-A, 31.25%Dextrose 5%, 0.45%NaCl, 10%Dextran 40 and 5% Dextrose, 20%Human Serum Albumin, and 7.5%DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C. or in liquid nitrogen.
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.
  • the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.
  • agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3
  • the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT) , cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT) , cyclophosphamide, or antibodies such as OKT3 or CAMPATH.
  • XRT external-beam radiation therapy
  • cyclophosphamide cyclophosphamide
  • antibodies such as OKT3 or CAMPATH.
  • the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
  • the engineered immune cell provided herein can exhibit enhanced activity toward tumor cells, but with reduced side effects such as GVHD, and/or host rejection of a graft (HVG) .
  • the engineered immune cell can target a disease-associated antigen (e.g., tumor-associated antigen, or tumor cell marker) and at the same time suppress host immune cells.
  • a disease-associated antigen e.g., tumor-associated antigen, or tumor cell marker
  • One or more endogenous genes e.g., a gene encoding a subunit of a TCR, a gene encoding a subunit of a MHC molecule, or a gene encoding a cell surface marker
  • the engineered immune cell comprises a single CAR.
  • the engineered immune cell comprises a first CAR and a second CAR, each targeting a different antigen. In some cases, the engineered immune cell comprises a CAR having a first antigen binding domain and a second antigen binding domain. In some cases, the engineered immune cell comprises a first CAR, a second CAR and a third CAR, each targeting a different antigen. In some cases, the engineered immune cell comprises a CAR having a first antigen binding domain, a second antigen binding domain and a third antigen binding domain.
  • the engineered immune cell can comprise one or more chimeric antigen receptors (CARs) comprising a binding moiety.
  • CARs chimeric antigen receptors
  • the binding moiety can comprise an antigen binding domain that can target both an immune cell antigen and a disease-associated antigen.
  • the CAR can further comprise a transmembrane domain and an intracellular signaling domain.
  • the antigen binding domain targets CS1.
  • the antigen binding domain targets CD137.
  • the engineered immune cell can also comprise an enhancer moiety capable of enhancing one or more activities of the engineered immune cell.
  • the endogenous antigen of the engineered immune cell can be inactivated in the engineered immune cell.
  • a gene encoding the endogenous antigen can be inactivated (e.g., silenced or knocked out) in the engineered immune cell.
  • a gene encoding endogenous CS1 can be inactivated (e.g., silenced or knocked out) in the engineered immune cell.
  • a gene encoding endogenous CD137 can be inactivated (e.g., silenced or knocked out) in the engineered immune cell.
  • the endogenous T cell receptor (TCR) of the engineered immune cell can be inactivated.
  • a gene encoding a subunit of the endogenous TCR of the engineered immune cell can be inactivated such that the endogenous TCR can be inactivated.
  • the gene encoding the subunit can be TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , or CD3 ⁇ .
  • the endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the endogenous MHC molecule comprises MHC class I molecule and MHC class II molecule.
  • a gene encoding MHC I molecule can be inactivated.
  • the gene encoding MHC I molecule includes but is not limited to HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.
  • the expression of one or more endogenous HLA genes of the engineered immune cell may be knocked out or partially knocked out.
  • any one of more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G of the engineered immune cell may be knocked out or partially knocked out.
  • an endogenous HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G can be knocked out or partially knocked out to reduce T cell killing activity.
  • any one of more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G of the engineered immune cell can remain intact.
  • a subunit of the endogenous MHC molecule in said engineered immune cell can be inactivated such that the endogenous MHC molecule is inactive.
  • B2M subunit of the endogenous MHC molecule in said engineered immune cell is inactivated.
  • B2M subunit of the endogenous MHC molecule in said engineered immune cell is knocked out or partially knocked out.
  • a killer/phagocyte inhibitor of the engineered immune cell can be overexpressed.
  • an endogenous HLA can be knocked out with co-expression of killer/phagocyte inhibitor (s) .
  • B2M can be knocked out with co-expression of killer/phagocyte inhibitors.
  • the killer/phagocyte inhibitor may suppress immune response toward the engineered immune cell.
  • the killer/phagocyte inhibitors include, but are not limited to, CD47, CD24, FASL, PDL1, or functional domains thereof.
  • any two or more of an endogenous antigen, an endogenous TCR, and an endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the endogenous antigen and the endogenous TCR of the engineered immune cell can be inactivated.
  • the endogenous TCR and the endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the endogenous antigen and the endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the antigen binding domain targets CS1, and the endogenous CS1 and the endogenous TCR of the engineered immune cell can be activated, the endogenous TCR and the endogenous B2M of the engineered immune cell can be inactivated, or the endogenous CS1 and an endogenous B2M of the engineered immune cell can be inactivated.
  • the antigen binding domain targets CD137, and the endogenous CD137 and the endogenous TCR of the engineered immune cell can be activated, the endogenous TCR and the endogenous B2M of the engineered immune cell can be inactivated, or the endogenous CD137 and the endogenous B2M of the engineered immune cell can be inactivated.
  • an endogenous antigen, an endogenous TCR, and an endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the antigen binding domain targets CS1, and the endogenous antigen CS1, an endogenous TCR, and the endogenous B2M of the engineered immune cell can be all inactivated.
  • the antigen binding domain targets CD137, and the endogenous antigen CD137, an endogenous TCR, and the endogenous B2M of the engineered immune cell can be all inactivated.
  • the CAR disclosed herein targets two or more of endogenous antigens of an immune cell, each of the endogenous antigens may be inactivated individually. In some cases, the endogenous antigens are all activated on the engineered immune cell disclosed herein.
  • the engineered immune cell can exhibit (i) enhanced degree of persistence by remaining viable in vitro while in presence of cells that are heterologous to the engineered immune cell, including but not limited to heterologous T cells, heterologous NK cells and the mixture of the heterologous T cells and heterologous NK cells, (ii) enhanced degree of expansion, or (iii) enhanced cytotoxicity against a target cell comprising the antigen, compared to an additional engineered immune cell comprising the one or more CARs without the inactivation of the TCR, MHC molecule and/or immune cell antigen.
  • cells that are heterologous to the engineered immune cell including but not limited to heterologous T cells, heterologous NK cells and the mixture of the heterologous T cells and heterologous NK cells, (ii) enhanced degree of expansion, or (iii) enhanced cytotoxicity against a target cell comprising the antigen, compared to an additional engineered immune cell comprising the one or more CARs without the inactivation of the TCR, MHC molecule and
  • the engineered immune cell can be characterized by exhibiting two or more of (i) enhanced degree of persistence by remaining viable in vitro while in presence of cells that are heterologous to the engineered immune cell, including but not limited to heterologous T cells, heterologous NK cells and the mixture of the heterologous T cells and heterologous NK cells, (ii) enhanced degree of expansion, and (iii) enhanced cytotoxicity.
  • the binding moiety can comprise a first antigen binding domain capable of binding to an immune cell antigen and a second antigen binding domain capable of binding to a disease-associated antigen.
  • Each CAR of the one or more CARs may further comprise a transmembrane domain and an intracellular signaling domain.
  • the engineered immune cell can also comprise an enhancer moiety capable of enhancing one or more activities of the engineered immune cell.
  • the endogenous immune cell antigen of the engineered immune cell be inactivated.
  • the endogenous T cell receptor (TCR) of the engineered immune cell can be inactivated.
  • the endogenous MHC molecule of the engineered immune cell can be inactivated.
  • any two or more of the endogenous immune cell antigen, the endogenous TCR, and the endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the endogenous immune cell antigen, the endogenous TCR, and the endogenous MHC molecule of the engineered immune cell can be all inactivated.
  • the engineered immune cell can exhibit (i) enhanced degree of persistence by remaining viable in vitro while in presence of cells that are heterologous to the engineered immune cell, including but not limited to heterologous T cells, heterologous NK cells and the mixture of the heterologous T cells and heterologous NK cells , (ii) enhanced degree of expansion, or (iii) enhanced cytotoxicity against a target cell comprising the immune cell antigen or the disease-associated antigen, compared to an additional engineered immune cell comprising the one or more CARs without the inactivation of the TCR, MHC molecule and/or immune cell antigen.
  • the engineered immune cell can be characterized by exhibiting two or more of (i) enhanced degree of persistence by remaining viable in vitro while in presence of cells that are heterologous to the engineered immune cell, including but not limited to heterologous T cells, heterologous NK cells and the mixture of the heterologous T cells and heterologous NK cells, (ii) enhanced degree of expansion, and (iii) enhanced cytotoxicity.
  • the engineered immune cell can comprise a multi-specific CAR.
  • the engineered immune cell comprises a bispecific CAR targeting an immune cell antigen and a disease-associated antigen.
  • the two antigen binding domains of the bispecific CAR can be arranged in any form as described in the present disclosure, for example, parallel form, loop form, and tandem form.
  • an engineered immune cell described herein can comprise a single chimeric antigen receptor (CAR) comprising (i) a first antigen binding domain that specifically binds CD7 and (ii) a second antigen binding domain capable of binding to CS1.
  • CAR chimeric antigen receptor
  • an engineered immune cell described herein can comprise a single chimeric antigen receptor (CAR) comprising (i) a first antigen binding domain that specifically binds CD137 and (ii) a second antigen binding domain capable of binding to CS1.
  • CAR chimeric antigen receptor
  • the endogenous T cell receptor (TCR) of the engineered immune cell can be inactivated.
  • the endogenous MHC molecule of the engineered immune cell can be inactivated.
  • a gene encoding endogenous CD7 and/or CD137 can be inactivated (e.g., silenced or knocked out) in the engineered immune cell.
  • any two or more of the endogenous immune cell antigen, the endogenous TCR, and the endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the endogenous immune cell antigen, the endogenous TCR, and the endogenous MHC molecule of the engineered immune cell can be all inactivated.
  • the first antigen binding domain or the second antigen binding domain can be an antibody or fragment thereof, for example, a scFv or a single domain antibody.
  • the disease-associated antigen described herein can be a tumor-associated antigen or a tumor-specific antigen.
  • tumor-associated antigens include, but are not limited to, BCMA, VEGFR2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, CD133, EGFR, GPC3; PMSA, CD 3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3 HER-2, HER3, ErbB4 /HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR , Flt4, CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A,
  • the tumor-associated antigens comprise CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R,
  • the immune cell antigen can be selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD279, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, CD319 (CS1) , TCR ⁇ , TCR ⁇ and SLAMF7.
  • the immune cell antigen is CD2, CD3, CD4, CD5, CD7, CD8, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD69, CD100, CD122, CD132, CD137, CD161, CD159a, CD159c, CD279, CD314, CD319 (CS1) , TCR ⁇ or TCR ⁇ .
  • the immune cell antigen is CD2, CD3, CD5, CD7, or CD137.
  • the immune cell antigen is CD7.
  • the immune cell antigen is CS1.
  • the immune cell antigen is CD137 (CD137) .
  • the enhancer moiety can be configured to constitutively enhance the one or more activities of the engineered immune cell.
  • the enhancer moiety can be configured to constitutively upregulate one or more intracellular signaling pathways of the engineered immune cell.
  • the one or more intracellular signaling pathways can be one or more cytokine signaling pathways.
  • the enhancer moiety can be sef-activating through self-oligomerizing.
  • the enhancer moiety can be self-activating through self-dimerizing.
  • the enhancer moiety can be a cytokine or a cytokine receptor.
  • the enhancer moiety can be selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors for the same, functional fragments thereof, functional variants thereof, and combinations thereof.
  • the engineered immune cell can further comprise an inducible cell death moiety, which inducible cell death moiety can effect suicide of the engineered immune cell upon contact with a cell death activator.
  • the inducible cell death moiety can be selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, and EGFRt.
  • the inducible cell death moiety is EGFRt
  • the cell death activator is an antibody or an antigen binding fragment thereof that binds EGFRt.
  • the inducible cell death moiety is HSV-TK, and the cell death activator is GCV.
  • the inducible cell death moiety is iCasp9
  • the cell death activator is AP1903.
  • the cell death activator can comprise a nucleic acid, a polynucleotide, an amino acid, a polypeptide, lipid, a carbohydrate, a small molecule, an enzyme, a ribosome, a proteasome, a variant thereof, or any combination thereof.
  • the engineered immune cell provided herein can comprise a chimeric polypeptide comprising (i) an enhancer moiety capable of enhancing one or more activities of the engineered immune cell, and (ii) an inducible cell death moiety capable of effecting death of the engineered immune cell upon contacting the chimeric polypeptide with a cell death activator, wherein the enhancer moiety is linked to the inducible cell death moiety.
  • the enhancer moiety and the inducible moiety may be linked by a linker.
  • the linker can be a cleavable linker, for example, a self-cleaving peptide.
  • the engineered immune cell can further comprise one or more chimeric polypeptide receptors (CPRs) comprising a binding moiety, wherein the binding moiety comprises (i) a first antigen binding domain, which first antigen binding domain suppresses or reduces a subject’s immune response toward the engineered immune cell when administered into the subject and (ii) a second antigen binding domain capable of binding to a disease-associated antigen.
  • An individual CPR of the one or more CPRs can comprise (i) the first antigen binding domain, (ii) the second antigen binding domain, or (iii) both the first antigen binding domain and the second antigen binding domain.
  • a CPR of the one or more CPRs can further comprise a transmembrane domain and an intracellular signaling domain.
  • the one or more CPRs in the engineered immune cell are one or more chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) .
  • the engineered immune cells comprise both CARs and engineered TCRs.
  • the engineered TCR can be a TCR fusion protein.
  • the TCR fusion protein can comprise a heterologous antigen binding domain fused to one or more subunits of a TCR complex.
  • the TCR fusion protein can comprise a TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain; and an antibody domain comprising an antigen binding domain, where the TCR subunit and the antibody domain are linked.
  • the TCR fusion protein can incorporate into a TCR complex when expressed in a T cell.
  • the TCR fusion protein can further comprise a TCR transmembrane domain.
  • the TCR extracellular domain, the TCR intracellular domain, or the TCR transmembrane domain can be derived from TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 epsilon, CD3 gamma, CD3 delta or CD3 zeta.
  • an endogenous TCR of the engineered immune cell comprising an engineered TCR is inactivated.
  • the engineered immune cell comprising inactivated endogenous TCR may not cause GVHD.
  • a gene encoding an endogenous TCR subunit can be inactivated.
  • a gene encoding an endogenous TCR subunit may be mutated such that an endogenous TCR may not be formed.
  • the engineered immune cell provided herein can comprise a chimeric polypeptide comprising (i) an enhancer moiety capable of enhancing one or more activities of the engineered immune cell, and (ii) an inducible cell death moiety capable of effecting death of the engineered immune cell upon contacting the chimeric polypeptide with a cell death activator.
  • the enhancer moiety is linked to the inducible cell death moiety.
  • the one or more chimeric antigen receptors (CARs) can comprise a binding moiety.
  • the binding moiety can comprise (i) a first antigen binding domain, which first antigen binding domain suppresses or reduces a subject’s immune response toward the engineered immune cell when administered into the subject and (ii) a second antigen binding domain capable of binding to a disease-associated antigen.
  • an individual CAR of the one or more CARs comprises (i) the first antigen binding domain or (ii) the second antigen binding domain.
  • an individual CAR of the one or more CARs comprises both the first antigen binding domain and the second antigen binding domain.
  • each CAR of the one or more CARs further comprises a transmembrane domain and an intracellular signaling domain.
  • the first antigen binding domain of the engineered immune cell can bind to an immune cell antigen.
  • the endogenous immune cell antigen of the engineered immune cell can be inactivated.
  • a gene encoding the endogenous immune cell antigen can be inactivated (e.g., silenced or knocked out) in the engineered immune cell.
  • a gene encoding endogenous CS1 or CD137 can be inactivated (e.g., silenced or knocked out) in the engineered immune cell.
  • the endogenous T cell receptor (TCR) of the engineered immune cell can be inactivated.
  • a gene encoding a subunit of the endogenous TCR of the engineered immune cell can be inactivated such that the endogenous TCR is inactivated.
  • the gene encoding the subunit can be TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , or CD3 ⁇ .
  • the endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the endogenous MHC molecule comprises MHC class I molecule and MHC class II molecule.
  • a gene encoding MHC I molecule is inactivated.
  • the gene encoding MHC I molecule comprises but is not limited to HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G.
  • the expression of one or more endogenous HLA genes of the engineered immune cell may be knocked out or partially knocked out.
  • any one of more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G of the engineered immune cell may be knocked out or partially knocked out.
  • an endogenous HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G can be knocked out or partially knocked out to reduce T cell killing activity.
  • any one of more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G of the engineered immune cell can remain intact.
  • a subunit of the endogenous MHC molecule in said engineered immune cell can be inactivated such that the endogenous MHC molecule is inactive.
  • B2M subunit of the endogenous MHC molecule in said engineered immune cell is inactivated.
  • B2M subunit of the endogenous MHC molecule in said engineered immune cell is knocked out or partially knocked out.
  • a killer/phagocyte inhibitor of the engineered immune cell can be overexpressed.
  • an endogenous HLA can be knocked out with co-expression of killer/phagocyte inhibitor (s) .
  • B2M can be knocked out with co-expression of killer/phagocyte inhibitors.
  • the killer/phagocyte inhibitor may suppress immune response toward the engineered immune cell.
  • the killer/phagocyte inhibitors include, but are not limited to, CD47, CD24, FASL, PDL1, or functional domains thereof.
  • any two or more of an endogenous immune cell antigen, an endogenous TCR, and an endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the endogenous immune cell antigen and the endogenous TCR of the engineered immune cell can be activated, an endogenous TCR and an endogenous MHC molecule of the engineered immune cell can be inactivated, or the endogenous immune cell antigen and an endogenous MHC molecule of the engineered immune cell can be inactivated.
  • the endogenous immune cell antigen, the endogenous TCR, and the endogenous MHC molecule of the engineered immune cell can be all inactivated.
  • endogenous T cell receptors of the engineered immune cell is inactivated.
  • TCRs T cell receptors
  • Various methods can be used to inactivate endogenous TCRs.
  • a gene encoding a subunit of the endogenous TCR can be inactivated such that the endogenous TCR is inactivated.
  • the gene encoding the subunit can be TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , or CD3 ⁇ .
  • the chimeric polypeptide may or may not comprise any self-cleaving peptide flanked by the enhancer moiety and the inducible cell death moiety.
  • the enhancer moiety can be configured to constitutively enhance the one or more activities of the engineered immune cell.
  • the enhancer moiety can be configured to constitutively upregulate one or more intracellular signaling pathways of the engineered immune cell.
  • the one or more intracellular signaling pathways can be one or more cytokine signaling pathways.
  • the enhancer moiety can be self-activating through self-oligomerizing.
  • the enhancer moiety can be self-activating through self-dimerizing.
  • the chimeric polypeptide described herein can be a secreted protein.
  • the chimeric polypeptide can be an intracellular protein.
  • the chimeric polypeptide can be a transmembrane protein.
  • the enhancer moiety or the inducible cell death moiety can be contained in an ectodomain of the transmembrane protein.
  • the enhancer moiety or the inducible cell death moiety is contained in an endodomain of the transmembrane protein.
  • the enhancer moiety can be contained in an endodomain of the transmembrane protein and the inducible cell death moiety can be contained in an ectodomain of the transmembrane protein.
  • the enhancer moiety can be contained in an ectodomain of the transmembrane protein, and the inducible cell death moiety can be contained in an endodomain of the transmembrane protein.
  • the enhancer moiety can be a cytokine or a cytokine receptor.
  • the enhancer moiety can be selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors for the same, functional fragments thereof, functional variants thereof, and combinations thereof.
  • the inducible cell death moiety can be selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, and EGFRt.
  • the inducible cell death moiety is EGFRt
  • the cell death activator is an antibody or an antigen binding fragment thereof that binds EGFRt.
  • the inducible cell death moiety is HSV-TK, and the cell death activator is GCV.
  • the inducible cell death moiety is iCasp9, and the cell death activator is AP1903.
  • the cell death activator may comprise a nucleic acid, a polynucleotide, an amino acid, a polypeptide, lipid, a carbohydrate, a small molecule, an enzyme, a ribosome, a proteasome, a variant thereof, or any combination thereof.
  • the engineered immune cell can be a CAR-T cell.
  • the CAR-T cell can express a CAR targeting CS1 or CD137.
  • the expression of endogenous CS1 or CD137 gene can be silenced in the CAR-T cell.
  • the CAR can be a single CAR targeting CS1 and CD7, or CS1 and CD137.
  • the CAR can comprise a first CAR targeting CS1 and a second CAR targeting CD7 or CD137.
  • the CAR-T cell can have one or more of the following characteristics: (a) expression of CS1, CD7 and/or CD137 gene is silenced in the CAR-T cell; (b) expression of TCR gene is silenced in the CAR-T cell; (c) the CAR-T cell expresses an exogenous cellular suicide element (e.g., inducible cell death moiety) ; d) expression of MHC gene is silenced in the CAR-T cell.
  • an exogenous cellular suicide element e.g., inducible cell death moiety
  • the engineered immune cell can express a CAR and/or an exogenous TCR, and the CAR and/or exogenous TCR target CS1, CD137, CS1 and CD7, or CS1 and CD137.
  • the engineered immune cell can comprise a cytokine-related signaling pathway that is enhanced.
  • the cytokine-related signaling pathway can comprise a related signaling pathway of a cytokine selected from a group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, or a combination thereof.
  • Enhancing the cytokine-related signaling pathway can comprise introducing a gene encoding a cytokine and/or a receptor thereof; up-regulating a gene encoding a cytokine and/or a receptor thereof; or exogenously adding a cytokine, a receptor of cytokine that is introduced, or a combination thereof.
  • the engineered immune cell can be a CAR-T cell having one or more characteristics selected from a group consisting of (a) gene expression an endogenous TCR being silenced; (b) expressing an element for cellular suicide; (c) normal expression of endogenous MHC molecule; (d) gene expression an endogenous MHC being silenced or partially silenced; (e) expression of CS1, CD7 and/or CD137 gene being silenced in the CAR-T cell.
  • the engineered immune cell can comprise a CAR or an exogenous TCR targeting a tumor cell marker.
  • the engineered immune cell can comprise a substance targeting a T cell or NK cell.
  • the engineered immune cell can comprise a CAR targeting a T cell and/or NK cell.
  • the engineered immune cell can comprise a bispecific CAR targeting both (i) a tumor cell marker and (ii) a T cell and/or NK cell marker.
  • the substance is an antibody.
  • the antibody target both T cell and NK cell can be TH-69, 3A1e, 3A1f, T3-3A1, RFT2, SDZ214-380 (SDZCHH380) , CD7-6B7, 124-1D1, 4H9, RPA-2.10, TS1/8, OKT11, AB75, 3E11, BH1, Lo-CD2a, OTI3B3, OTI3E3 OTI1F1, Elotuzumab, 1B9, 1G10, 24.1, 162.1, 4B4-1, BBK-2, 0. N. 185, G-1, EPR20238, OTI5H5, OTI7F9, ABM3D3.2E8, ABM2B2.1A5, 5F8, or JG1.6A.
  • the CAR-T cell provided herein can be a universal CAR-T cell.
  • the CAR-T cell can express a chimeric antigen receptor CAR that targets a tumor cell marker and the binding of the T cell receptor to PD-1 is inhibited.
  • the CAR-T cell can target CS1.
  • the endogenous TCR expression in the CAR-T cells provided herein can be knocked out by gene editing technology. Upon knocking out the endogenous TCRs of the CAR-T cells, the normal cells may not be recognized and killed by the CAR-T cells during the allogeneic infusion. The GVHD reaction may be inhibited.
  • the CAR-T can further comprise a suicide gene switch (e.g., an inducible cell death moiety) .
  • the CAR-T cells can be inactivated or removed by turning on the suicide gene switch (e.g., binding of an activator to the inducible cell death moiety) to reduce the side effects of the CAR-T cell therapy.
  • a CAR provided herein can have a structure of CS1 scFv-CD7 scFv-Hinge-TM-CD28/41BB-CD3 ⁇ , wherein the CD7 scFv fragment is a monoclonal 3A1e antibody, the heavy and light chain variable regions are joined by a GS linker, and the CS1 scFV fragment is the heavy and light chain variable region of tan anti-CS1 antibody linked by a GS linker.
  • the CAR can also include a hinge region and a transmembrane region in tandem, human CD28 and /or 41BB intracellular co-stimulatory elements, as well as human CD3 intracellular domain.
  • a gene fragment of a CAR construct CS1 scFv-CD7 scFv-Hinge-TM-CD28/41BB-CD3 ⁇ can be inserted into a lentiviral vector, and the recombinant vector can be packaged into viral particles in 293T cells.
  • T cells can be isolated from peripheral blood mononuclear cells, and after activation, some endogenous genes (e.g., CS1, TCR and PD-1 genes) can be knocked out by gene editing technologies such as CRISPR/CAS technology.
  • T cells can be infected by the viral particles containing the CAR construct describe herein to express the CAR.
  • the prepared CAR-T cells can be used to detect the infection efficiency and gene editing efficiency of CAR by flowcytometry.
  • the engineered immune cell may have one or more characteristics described herein: (a) the expression of the endogenous CS1 gene of the engineered immune cell is silenced; (b) the PD-1 gene expression of the engineered immune cell is silenced; (c) the TCR gene expression of the engineered immune cell is silenced; (d) the engineered immune cell expresses a cytokine or cytokine receptor complex and the pSTAT5 signaling level is up-regulated; (e) the engineered immune cell expresses an exogenous inducible cell death moiety; (f) the first CAR, and/or the second CAR in the engineered immune cell is co-expressed with the inducible cell death moiety; (g) the MHC gene expression of the engineered immune cell is silenced.
  • the engineered immune cell may comprise two different CARs, each having a different antigen binding domain target a different antigen.
  • the engineered immune cell may comprise a single CAR, which further comprises two antigen binding domains targeting two different antigens.
  • a first CAR, and/or a second CAR is linked to an inducible cell death moiety and/or an enhancer moiety by a self-cleaving element.
  • the enhancer moiety is a cytokine or cytokine complex.
  • cytokines or cytokine complexes examples include IL2, IL7, IL15, membrane-bound IL15 (mbIL15 or mb15) , and a constitutive activating cytokine receptor such as an IL7 receptor (C7R) .
  • mbIL and “mb” are used interchangeably to refer to a membrane-bound interleukin factor, for example, mbIL7 or mb7, and mbIL17 or mb17.
  • the engineered immune cell described herein may have the following characteristics: (a) the engineered immune cell expresses a CAR and/or an exogenous TCR, and the CAR and/or exogenous TCR targets tumor cell markers; and (b) the cytokine-associated signaling pathway is enhanced.
  • the engineered immune cell may be (i) chimeric antigen receptor T cells (CAR-T cells) ; (ii) chimeric antigen receptor NK cells (CAR -NK cells) ; or (iii) Exogenous T cell receptor (TCR) T cells (TCR-T cells) .
  • the engineered immune cell can be a CAR-T cell, preferably a universal CAR-T cell (UCAR-T cell) .
  • cytokine-related signaling pathway refers to a signaling pathway initiated by the cytokine binding to the corresponding receptor, converting the extracellular signal into an intracellular signal, which is then amplified, dispersed, and regulated by a signal cascade. A series of cellular responses can be produced.
  • the cytokine-related signaling pathway comprises a related signaling pathway of a cytokine selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, 25 or a combination thereof.
  • the engineered immune cell can comprise a bispecific CAR (or a dual CAR) .
  • the bispecific CAR can comprise both a first antigen binding domain and a second antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain can be linked via a linker.
  • the linker may not comprise a self-cleaving peptide.
  • the first antigen binding domain or the second antigen binding can be a scFv.
  • the first antigen binding domain and the second antigen binding domain can be arranged, from amino terminus to carboxyl terminus, in any of following patterns: (i) VL2-VH1-VL1-VH2; (ii) VH2-VL1-VH1-VL2; (iii) VL1-VH2-VL2-VH1; and (iv) VH1-VL2-VH2-VL1, wherein VH1 is heavy chain variable domain of the first antigen binding domain, VL1 is light chain variable light domain of the first antigen binding domain, VH2 is heavy chain variable domain of the second antigen binding domain, and VL2 is light chain variable domain of the second antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain can be arranged, from amino terminus to carboxyl terminus, in any of following patterns: (i) VL2-VH2-VL1-VH1; (ii) VL2-VH2-VH1-VL1; (iii) VL1-VH1-VL2-VH2; and (iv) VL1-VH1-VH2-VL1, wherein VH1 is heavy chain variable domain of the first antigen binding domain, VL1 is light chain variable light domain of the first antigen binding domain, VH2 is heavy chain variable domain of the second antigen binding domain, and VL2 is light chain variable domain of the second antigen binding domain.
  • the first antigen binding domain and the second antigen binding domain can bind to the immune cell antigen and the disease-associated antigen.
  • the engineered immune cell may not comprise a bispecific CAR.
  • an individual CAR of the engineered immune cell can comprise only the first antigen binding domain and an additional individual CAR of the engineered immune cell can comprise only the second antigen binding domain.
  • the immune cell antigen can be a surface protein or a secreted protein of an immune cell.
  • the immune cell can be an NK cell, a T cell, a monocyte, a macrophage or a granulocyte.
  • the immune cell antigen can be selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD279, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, CD319 (CS1) , TCR ⁇ , TCR ⁇ and SLAMF7.
  • the disease-associated antigen can be a tumor-associated antigen.
  • the tumor-associated antigen can be CS1 or other antigens described herein.
  • the first antigen binding domain can bind to an immune cell antigen selected from the group consisting of CD2, CD3, CD5, CS1, CD7 and CD137, and the second antigen binding domain can bind to CS1.
  • the first antigen binding domain can bind to CD7, and the second antigen binding domain can bind to CS1.
  • the first antigen binding domain can bind to CD137, and the second antigen binding domain can bind to CS1.
  • the expression of one or more endogenous human leukocyte antigen (HLA) genes of the engineered immune cell can remain intact.
  • HLA human leukocyte antigen
  • the expression of one or more of endogenous HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G genes of the engineered immune cell can remain intact.
  • the expression of one or more endogenous human leukocyte antigen (HLA) genes of the engineered immune cell can be inactivated.
  • the expression of one or more of endogenous HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G genes of the engineered immune cell can be inactivated.
  • the expression of one or more endogenous human leukocyte antigen (HLA) genes of the engineered immune cell can be downregulated.
  • the expression of one or more of endogenous HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G genes of the engineered immune cell can be downregulated.
  • the expression of one or more endogenous human leukocyte antigen (HLA) genes of the engineered immune cell can be knockout or partially knockout.
  • the engineered immune cell is a T cell, an NKT cell or an NK cell.
  • the engineered immune cell is derived from a stem cell.
  • the stem cell can be a hematopoietic stem cell (HSC) or an induced pluripotent stem cell (iPSC) .
  • a cell e.g., an engineered immune cell
  • a cell can comprise one or more chimeric antigen receptors (CARs) comprising a binding moiety, where the binding moiety can comprise an antigen binding domain capable of binding to an immune cell antigen.
  • CARs chimeric antigen receptors
  • Each CAR of the one or more CARs can further comprise a transmembrane domain and an intracellular signaling domain.
  • the cell can further comprise an enhancer moiety capable of enhancing one or more activities of the cell, where an endogenous T cell receptor (TCR) of the cell may be inactivated.
  • TCR endogenous T cell receptor
  • the enhancer moiety can enhance one or more activities of the cell.
  • the enhancer moiety can be configured to constitutively enhance the one or more activities of the cell.
  • the enhancer moiety can be configured to constitutively upregulate one or more intracellular signaling pathways of the cell.
  • the one or more intracellular signaling pathways can be one or more cytokine signaling pathways.
  • the enhancer moiety can be a cytokine or a cytokine receptor.
  • the enhancer moiety can be selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors for the same, functional fragments thereof, functional variants thereof, and combinations thereof.
  • the cell can further comprise an inducible cell death moiety capable of effecting death of the cell upon contacting the inducible cell death moiety with a cell death activator.
  • the inducible cell death moiety can be selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, Her2t, CD30, BCMA, and EGFRt.
  • the inducible cell death moiety can be EGFRt
  • the cell death activator can be an antibody or an antigen binding fragment thereof that binds EGFRt.
  • the inducible cell death moiety can be HSV-TK, and the cell death activator can be GCV.
  • the inducible cell death moiety can be iCasp9, and the cell death activator can be AP1903.
  • a gene encoding an endogenous surface marker of the cell can be inactivated, where the endogenous surface marker may be capable of binding to the first antigen binding domain when expressed.
  • the endogenous surface marker can be CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD279, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, CD319 (CS1) , TCR ⁇ , TCR ⁇ or SLAMF7.
  • the engineered immune cell can comprise a single antigen binding domain.
  • the antigen binding domain is a scFv or sdAb.
  • the antigen binding domain is a sdAb.
  • the antigen binding domain is multivalent comprising more than one antigen binding units such as sdAbs.
  • the antigen binding domain is bivalent comprising two antigen binding units such as two sdAbs.
  • the antigen binding domain targets an antigen of any one selected from CS1, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1,
  • the sdAb targets CS1. In some embodiments, the sdAb targets CD137. In some embodiments, the antigen binding domain is bivalent comprising two sdAb targeting CS. In some embodiments, the antigen binding domain is bivalent comprising two sdAb targeting CD137.
  • the engineered immune cell can comprise a first antigen binding domain and a second antigen binding domain.
  • a single or individual CAR of the engineered immune cell comprises both the first antigen binding domain and the second antigen binding domain.
  • two CARs of the engineered immune cell comprise the first antigen binding domain and the second antigen binding domain with each CAR contains only one antigen binding domain.
  • two engineered immune cells comprise the first antigen binding domain and the second antigen binding domain with each engineered immune cell comprises only one type of antigen binding domain.
  • the first antigen binding domain can target an immune cell antigen and the second antigen binding domain can target a disease-associated antigen.
  • the antigen binding domain can be a Fab, F (ab’) 2 , single domain antibody, single chain Fv (scFv) , centyrin, darpin, or other polypeptides with antigen binding specificities.
  • the antigen binding domain can target an immune cell antigen.
  • immune cell antigen include, but are not limited to, CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD279, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, CD319 (CS1) , TCR ⁇ , TCR ⁇ and SLAMF7.
  • the immune cell antigen is a cell marker expressed on both T cells and NK cells, including, but not limited to, CD2, CD7, CD38, CD45, CD48, CD50, CD52, CD56, CD69, CD100, CD122, CD132, CD134 (OX40) , CD137 (4-1BB) , CD178 (ICOS) , CD161, CD159a, CD159c and CD314 and SLAMF7 (CS1) .
  • the immune cell antigen is CS1, CD7, or CD137.
  • the antigen binding domain can target a disease-associated antigen, for example, tumor-associated antigen.
  • tumor-associated antigens include, but are not limited to, BCMA, VEGFR2, CD19, CD20, CD30, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD80, CD86, CD81, CD123, cd171, CD276, B7H4, CD133, EGFR, GPC3; PMSA, CD 3, CEACAM6, c-Met, EGFRvIII, ErbB2, ErbB3 HER-2, HER3, ErbB4 /HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR , Flt4, CD44V6, CEA, CA125, CD151, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TN
  • the tumor-associated antigens comprise CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R,
  • the antigen binding domain can target CD7.
  • CD7 is a transmembrane protein and is a member of the immunoglobulin superfamily.
  • CD7 proteins are expressed on the surface of mature T cells and NK cells as well as their precursor cells.
  • CD7 can bind to its ligand K12/SECTM1 and function as a co-stimulatory effecter on T cell activation.
  • CD7 knockout T cell precursors can develop into normal T cells with only a slight effect on T cell effector function.
  • T-ALL T-cell acute lymphoblastic leukemia
  • CD7 can also be expressed in NK lymphoma, T-cell lymphoma/leukemia, chronic myelogenous leukemia, acute myeloid leukemia, and lymphocyte-rich thymoma.
  • the antigen binding domain can target CD137.
  • CD137 also known as 4-1BB, is a member of the TNF receptor superfamily.
  • CD137 protein can be an activation-induced co-stimulatory receptor, which can be widely expressed in activated T cells, NK cells, dendritic cells, granulocytes and other immune cell types and certain tumor cells. CD137 expression was found on activated T and NK cells with little expression in T cells and inactivated T and NK cells. In T cells, CD137 can initiate NF- ⁇ B pathway through TRAF2 and participate in T cell proliferation, cytokine secretion and anti-apoptosis. In clinical applications, CD137 can be used as a marker for reactive T cells.
  • CD137 signaling pathway by CD137 natural ligand or agonistic antibody can increase cytokine secretion and antitumor activity of cytotoxic lymphocytes.
  • Inhibition of reactive T cells with specific inhibitory CD137 antibodies can reduce autologous rejection in transplant rejection or reduce GVHD response caused by autoreactive T cells of an allogeneic origin.
  • CD137 expression can be regulated by TCR signaling and downstream signaling of cytokine IL-2/IL-15. Knockout of CD137 molecule may have no effect on the function of mature T cells in a non-activated state.
  • the endogenous CD137 of the engineered immune cell can be knocked out to avoid fratricide.
  • the endogenous TCRs of the engineered immune cell can be inactivated to prevent GVHD.
  • the antigen binding domain can target CS1.
  • CS1 CRACC, SLAMF7 or CD319
  • SLAM Signal-on-Artactin
  • NK cells CD8+ T lymphocytes
  • mature dendritic cells CD8+ T lymphocytes
  • activated B cells CS1 is a robust marker of normal plasma cells and malignant plasma cells in multiple myeloma.
  • the antigen binding domain can target CD19.
  • CD19 is a 95 kDa glycoprotein on the surface of B cells that begins to express from the early development of B cells until it differentiates into plasma cells.
  • CD19 is a member of the immunoglobulin (Ig) superfamily and is involved in the regulation of the signal transduction process of B cell receptors as one of the constituent elements of the B cell surface signal transduction complex.
  • Ig immunoglobulin
  • the number of B cells in peripheral lymphoid tissue can be significantly reduced, and the response to vaccines and mitogens is also reduced, accompanied by a decrease in serum Ig levels. It can be generally believed that the expression of CD19 is restricted to the B-cell lineage and not to the surface of pluripotent hematopoietic stem cells.
  • CD19 can also be expressed on the surface of most B cell lymphomas, mantle cell lymphomas, ALLs, CLLs, hairy cell leukemias, and some acute myeloid leukemia cells.
  • CD19 can be a target for immunotherapy in the treatment of leukemia/lymphoma.
  • CD19 may not be expressed on most normal cell surfaces other than B cells, including pluripotent hematopoietic stem cells. This feature can make CD19 a safe therapeutic target for autoimmune diseases because the risk of irreversible bone marrow toxicity damage can be minimized.
  • the antigen binding domain can target BCMA.
  • B-cell maturation antigen BCMA or BCM
  • BCM B-cell maturation antigen
  • TNFRSF17 tumor necrosis factor receptor superfamily member 17
  • BAFF B-cell activating factor
  • BCMA is preferentially expressed in mature B lymphocytes. It has been shown to specifically bind to the tumor necrosis factor (ligand) superfamily, member 13b (TNFSF13B/TALL-1/BAFF) , and to lead to NF-kappaB and MAPK8/JNK activation.
  • the antigen binding domain provided herein can have a structure shown as V H -V L or V L -V H , wherein V H is a heavy chain variable region of an antibody; V L is a light chain variable region of an antibody; "-" is a linker peptide (or flexible linker) or a peptide bond.
  • the antigen binding domain targets a tumor-associated antigen.
  • the antigen binding domain targets an immune cell antigen.
  • the antigen binding domain targets CS1.
  • the monoclonal antibody of CS1 is selected from the group consisting of OTI3B3, OTI3E3 OTI1F1, Elotuzumab, 1B9, 1G10, 24.1, 162.1, or a combination thereof.
  • the antigen binding domain targets CD137.
  • the monoclonal antibody of CD137 is selected from the group consisting of 4B4-1, BBK-2, 0. N. 185, G-1, EPR20238, OTI5H5, OTI7F9, ABM3D3.2E8, ABM2B2.1A5, 5F8, or JG1.6A.
  • the antigen binding domain targets CD19.
  • the antigen binding domain that targets CD19 comprises the heavy chain variable region and the light chain variable region of the monoclonal FMC63 antibody.
  • the sequence of the linker peptide or flexible linker comprises 2-6, preferably 3-4 consecutive (GGGGS) amino acid sequences.
  • the immune cell antigen targets CD7.
  • the monoclonal antibody of CD7 is selected from the group consisting of TH-69, 3A1e, 3A1f, T3-3A1, RFT2, CD7-6B7, 124-1D1, 4H9, SDZ214-380, or a combination thereof.
  • the engineered immune cell provided herein can comprise an enhancer moiety.
  • the enhancer moiety can regulate one or more activities of the engineered immune cell, for example, enhance or upregulate one or more signaling pathways to enhance or upregulate effector functions of the engineered immune cell.
  • the signaling pathways can be a cytokine-related signaling pathway.
  • the enhancer moiety can be a cytokine.
  • the enhancer moiety can be a cytokine receptor.
  • the cytokine-related signaling pathway can comprise a related signaling pathway of a cytokine.
  • cytokines include, but are not limited to, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21 and IL25.
  • the cytokine-related signaling pathway comprises a related signaling pathway of two or more cytokines, wherein the cytokines include: IL-2 and IL-7, IL-2 and IL-15. IL-7 and IL-15, IL15 and IL21.
  • the cellular response can include regulation of downstream gene expression, changes in intracellular enzyme activity, changes in cellular bone architecture, changes in DNA synthesis, promotion of gene transcription, regulation of immune cell differentiation, proliferation, and resistance to cell death.
  • the cytokine-related signaling pathway is enhanced comprising: introducing or up-regulating a gene encoding a cytokine and/or a receptor thereof, exogenously adding a cytokine, being introduced into a cytokine receptor, or a combination thereof.
  • up-regulating the gene encoding the cytokine and/or its receptor comprises up-regulating the level of transcription and/or translation of the encoding gene.
  • the enhanced cytokine-related signaling pathway can be achieved by one or more of the following methods: expressing a gene encoding the cytokine and/or its receptor in the immune cell, increasing the copy number of the gene encoding the cytokine and/or its receptor in the immune cell, engineering a regulatory sequence (e.g., a promoter) of the encoding gene to enhance transcription speed (e.g., transcriptional initiation rate) , modifying a translational regulatory region of a messenger RNA carrying the encoded gene to enhance translational strength, modifying the coding gene itself to enhance mRNA stability, protein stability and to release protein feedback inhibition.
  • a regulatory sequence e.g., a promoter
  • enhance transcription speed e.g., transcriptional initiation rate
  • modifying a translational regulatory region of a messenger RNA carrying the encoded gene modifying the coding gene itself to enhance mRNA stability, protein stability and to release protein feedback inhibition.
  • the cytokine-related signaling pathway can be enhanced by membrane expression of a cytokine and its receptor, secretion of a cytokine, enhancement of transcriptional regulation of a cytokine and/or its receptor, or a combination thereof.
  • the membrane-expressed cytokine and its receptors can include: IL-15 and its receptor (e.g., mbIL15 fusion protein) , IL-7 and its receptor (e.g., mbIL7 fusion protein) , IL-17 and its receptor (e.g., mbIL17 fusion protein) , IL-2 and its receptor (e.g., mbIL2 fusion protein) , IL-21 and its receptor (e.g., mbIL21 fusion protein) , constitute the activated IL-7 receptor (C7R) , or a combination thereof.
  • C7R activated IL-7 receptor
  • the enhancer moiety comprised in the engineered immune cell is a secretive cytokine.
  • the secretive cytokine can function with various mechanisms, for example, the secretive cytokine can be a trans-activating factor or a cis-activating factor.
  • the secretive cytokines can include IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, or a combination thereof.
  • the enhancer is a membrane bound protein such as mbIL15, mbIL7, mbIL21 and mbIL2.
  • the enhancer moiety is constitutively active cytokine receptor downstream signaling protein such as STAT5 and STAT3.
  • the enhancer moiety is a constitutively active cytokine receptor such as constitutively active IL-7 receptor (C7R) or derivatives thereof.
  • the constitutively active cytokine receptor can be an engineered protein (e.g., referred to as “E3” in the present disclosure) where the ecto domain of C7R is replaced by a safety switch, such as EGFRt or truncated form of human epidermal growth factor receptor 2 (Her2t; see U.S. Patent Application Publication No. 20170267742A1) or other peptides described in the present disclosure.
  • the constitutively active cytokine receptor can be an engineered protein (e.g., referred to as “E4” in the present disclosure) where the ectodomain of C7R is replaced by an immune cell inhibitor, such as CD47, CD24 or other peptides that inhibit killer or phagocytic immune cell function and protect therapeutic cells (e.g., the engineered immune cells described herein) .
  • the cytokine can be a chemokine such as CCL21 and CCL19.
  • chemokines that may be used include CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLl l, CXCL16, CXCL13, CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13, CCL25, CCL3, CCL4, CCL5, CCL7, CCL14, CCL15, CCL16, CCL23, CX3CL1, XCL1, XCL2, CCL1, CCL17, CCL22, CCL11, CCL24, CCL26, CXCLl, CXCL2, CXCL3 and CXCL7.
  • the enhancer moiety is a ligand of CCR7, which can function to enhance infiltration of T cells, NK cells or dendritic cells.
  • CCR7 ligand includes, but not limited to, CCL21 and CCL19.
  • the enhancer moiety comprises co-expression of chemokines CCL21 and CCL19 for therapeutic use to treat lymphomas or other solid tumors.
  • the engineered immune cell is used as a therapeutic agent to treat liquid tumors, and in such situations, the enhancer moiety can comprise any cytokine in any form as described herein. In some situations, the engineered immune cell is used as a therapeutic agent to treat solid tumors, and in such situations, the enhancer moiety can comprise any cytokine in any form and further comprise one or more chemokines.
  • two cytokines may be used to enhance the cytokine-related signaling pathway in the engineered immune cell, including IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, and IL15 and IL21.
  • the cytokine-related signaling pathway enhancement can comprise the expression of a polypeptide selected from the group consisting of a mbIL fusion protein, a constitutively active IL-7 receptor (C7R) , an interleukin, or a combination thereof.
  • the enhancer moiety described herein can be interleukin 15 (IL-15) or IL-15 receptor.
  • IL-15 is a 14-15kDs glycoprotein composed of 114 amino acids and belongs to the family of four helix bundle cytokines.
  • IL-15 is structurally homologous to interleukin 2 (IL-2) .
  • IL-15 receptor comprises a high affinity IL-15 receptor alpha chain, an IL2/15 receptor beta chain, and a common gamma chain. Therefore, IL-15 may have some functions similar to IL-2, such as stimulating T cell activation and proliferation, enhancing NK cell killing activity and promoting B cell production of immunoglobulin.
  • IL-15 may play a role in the differentiation, proliferation and activation of NK cells, NKT cells and intestinal epithelial cells.
  • IL-15 and IL-17 may play a role in the regulation of CD8+ memory T cells.
  • IL-15 may also play a role in the non-immune system, such as regulation of skeletal muscle anabolism.
  • the enhancer moiety described herein can be interleukin 7 (IL-7) .
  • IL-7 can promote the growth of pre-B cells, pro-B cells, B cells, and T cells. It can also promote growth and anti-apoptosis of B cells and T cells. IL-7 can play a role in the early differentiation and proliferation of thymus and the development and differentiation of dendritic cells. However, IL-7 may not have an enhanced effect on the killing activity of antigen-specific cytotoxic T lymphocytes. It can first transfer from the thymus to the peripheral blood, then induce thymocytes or peripheral blood lymphocytes to produce lymphokines, activate and enhance lymphokine-activated killer cell activity of LAK cells. CD8+ subpopulation can be the main effector cell of IL-7, and IL-7 Can also support memory CD8+ T cell expansion and survival.
  • IL-7 can promote bone marrow tissue production.
  • IL-7 not only can stimulate myeloid precursor cells and megakaryocytes to produce colony forming units and platelets, but also can restore the body from the immunosuppression of cyclophosphamide. At higher concentrations, it can also induce cytotoxicity that enhances macrophages, function as a synergistic factor for the production of CTL cells, NK cells, and activated monocytes, induce monocyte-macrophages to secrete various cytokines and promote the expression of inflammatory factors such as macrophage inflammatory protein alpha (MIP-alpha) , MIP- ⁇ , IL-8 and monocyte chemoattractant protein-1 (MCP-1) and the like.
  • MIP-alpha macrophage inflammatory protein alpha
  • MIP- ⁇ MIP- ⁇
  • IL-8 monocyte chemoattractant protein-1
  • IL-7 By activating a large number of inflammatory factors produced by inflammatory cells, IL-7 not only can regulate the interaction between the components of the inflammatory process, but also enhance the inflammatory cytokine receptors (CCR) such as CCR1, CCR2 and CCR5. In addition, IL-7 can play a role in inducing immune responses. IL-7 can induce type I immune responses and increase the production of IFN- ⁇ and IL2. IL-7 can synergize with IL12 to induce IFN- ⁇ and T cell proliferation. IL-7 and transforming growth factor beta (TGF ⁇ ) can play a regulatory role and can be part of the immune regulatory mechanism.
  • CCR inflammatory cytokine receptors
  • TGF ⁇ transforming growth factor beta
  • IL-7 not only can promote immune reconstitution of T cells, but also can induce up-regulation of T cell cycle and BCL-2 expression, which broadens the diversity and persistence of circulating T cell receptor pools and increases the number of CD4+ and CD8+ T cells. Moreover, for HIV antigens, expanded T cells can also secrete IL2 and IFN- ⁇ , and have good antiviral function. Therefore, IL-7 can reverse the defects of HIV-specific T lymphocytes in proliferation, cytokine secretion and cell function.
  • the enhance moiety can regulate (e.g., activate) signal transducer and activator of transcription 5 (STAT5) -mediated signaling pathway.
  • STAT5 can be widely present in the cytoplasm.
  • cytokines e.g., IL2, IL7, IL15 and IL21
  • IL2 cytokine receptors
  • IL2 cytokine receptors
  • IL15 IL15
  • IL21 binds to the cytokine receptors
  • the receptor-coupled JAK is activated, thereby phosphorylating the Tyr residue at the C-terminus of the STAT5 protein.
  • the phosphorylated STAT5 can form homologous or heterologous dimers through its SH2 region.
  • the homologous or heterodimer can be transferred to the nucleus and bind to the target gene, thereby regulating the expression of the target gene including the cell regulatory factor and the anti-apoptotic gene.
  • Activation of STAT5 can play a role in maintaining normal cell function and regulating cell proliferation and differentiation. Therefore, regulating the activity of STAT5 signaling pathway may regulate the survival and persistence of CAR-T cells described herein.
  • the enhancer moiety can be introduced into a cell (e.g., an immune cell or an engineered immune cell) by delivering a nucleic acid molecule encoding the enhancer moiety into the cell.
  • the nucleic acid molecule can be a vector.
  • the enhancer moiety can be a part of a fusion construct.
  • a fusion protein or corresponding nucleic acid construct can have a structure as presented by a formula selected from: S-2A-L1-scFv-H-TM-C-CD3 ⁇ -2A-L2-IL15-IL15Ra (A) ; S-2A-L1-scFv-H-TM-C-CD3 ⁇ -2A-L2-IL15-IL15Ra-2A-L3-IL7 (B) ; S-2A-L1-scFv-H-TM-C-CD3 ⁇ -2A-L2-C7R (C) ; S-2A-L1-scFv-H-TM-C-CD3 ⁇ -2A-L2-IL7-IL7Ra (D) ; wherein: each ‘’-” is independently a linker peptide or a peptide bond; S is a safety switch; 2A is an optional self-cleaving peptide; each of L1, L2 and L3 is independently null or a signal peptide sequence; C7R is
  • the enhancer moiety can be part of a chimeric polypeptide.
  • the enhancer moiety can be linked to an inducible cell death moiety.
  • the enhancer moiety can be linked to the inducible cell death moiety by a linker.
  • the linker may not be cleaved.
  • the linker may not comprise a self-cleaving peptide.
  • the enhancer moiety and the inducible cell death moiety can be expressed in a cell from a same nucleic acid molecule and can be cleaved to form two polypeptides.
  • the engineered immune cell described herein may comprise an inducible cell death moiety, also referred to as “suicide gene switch. ” “suicide switch, ” “safety switch, ” or “cell suicide element. ”
  • the inducible cell death moiety can be used to effectively remove of the engineered immune cells (e.g., CAR-T cells) in vivo under the action of exogenous factors (e.g., drugs) .
  • the inducible cell death moiety described herein may be rapaCasp9, iCasp9, CD20 (and its mimotope) , RQR8, Her2t, CD30, BCMA, EGFRt, HSV-TK, mTMPK and the like.
  • iCasp9, CD20 (and its mimotope) , RQR8, and HSV-TK may have the same ability to clear T cells, but rapaCasp9, iCasp9, RQR8, and CD20 (and their mimotope) may be faster in comparison with HSV-TK.
  • an inducible cell death moiety is capable of effecting death of said cell upon contacting said inducible cell death moiety with a cell death activator.
  • the inducible cell death moiety can be, for example, rapaCasp9, iCasp9, HSV-TK, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, or EGFRt.
  • the inducible cell death moiety is EGFRt
  • said cell death activator is an antibody or an antigen binding fragment thereof that binds EGFRt.
  • the inducible cell death moiety is HSV-TK, and said cell death activator is GCV.
  • the inducible cell death moiety is iCasp9, and said cell death activator is AP1903.
  • the inducible cell death moiety can be linked to an enhancer moiety and can be co-expressed in a cell as a chimeric polypeptide as described above.
  • GVHD graft versus host disease
  • T cells for the treatment of malignant and infectious diseases
  • cell therapy by infusion of T cells can be designed to re-establish immunity against pathogens and malignancies.
  • the amount of time required to produce the T cells with tumor-targeting properties with a sufficient number of T cells in vitro can be generally incompatible with the patient's therapeutic window.
  • autologous T cells from patients with advanced disease may have impaired function and are tolerant to the desired antigen.
  • patients can be administered with allogeneic T cells but need to be prevented from immune-mediated rejection by host NK cells and T cells by recognizing different major or minor histocompatibility antigens on the infused cells.
  • Infusion of T cells without the expression of TCR alpha and beta chains and/or MHC molecules may not cause GVHD and HVG.
  • the T cells edited with CRISPR/CAS9 to delete TCR alpha chain and/or MHC molecules can serve as a source of universal effector donor cells.
  • Beta-2-Microglobulin may prevent donor CAR-T cells from being attacked by the host T cells.
  • the donor CAR-T cells may be attacked by host NK cells and affect the survival of CAR-T cells. Therefore, the present disclosure provides engineered immune cells which target tumor cells and host T cells and/or NK cells.
  • the engineered immune cells described herein can scavenge host T cells and/or NK cells, and enhance the survival, persistence and expansion ability of CAR-T cells, thereby being more effective against tumor cells.
  • TALENs transcription activator-like (TAL) effector nucleases
  • ZFNs Zinc finger nucleases
  • CRISPR/Cas9 system is used to edit the genes of the immune cells.
  • CRISPR/Cas9 system can be used to knockout endogenous TCRs or cell surface markers (e.g., CS1, CD7, CD137) of the immune cells to generate the engineered immune cells for T cell therapy.
  • the CRISPR/Cas9 (clustered regular interspaced short palindromic repeats) /Cas (CRISPR-associated) system is a natural immune system unique to prokaryotes that is resistant to viruses or exogenous plasmids.
  • the Type II CRISPR/Cas system has been applied in many eukaryotic and prokaryotic organisms as a direct genome-directed genome editing tool.
  • the development of the CRISPR/Cas9 system has revolutionized the ability of people to edit DNA sequences and regulate the expression levels of target genes, providing a powerful tool for accurate genome editing of organisms.
  • the simplified CRISPR/Cas9 system can comprise Cas9 protein and gRNA.
  • the principle of action is that gRNA forms a Cas9-gRNA complex with Cas9 protein through its own Cas9 handle, and the base complementary pairing sequence of gRNA in the Cas9-gRNA complex is paired with the target sequence of the target gene by the principle of base complementary pairing.
  • Cas9 uses its own endonuclease activity to cleave the target DNA sequence.
  • the CRISPR/Cas9 system has several distinct advantages: ease of use, simplicity, low cost, programmability, and the ability to edit multiple genes simultaneously.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an engineered immune cell described herein and a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition is a liquid composition.
  • the pharmaceutical composition can be administered into a subject, for example, by injection.
  • the concentration of the engineered immune cells in the preparation can be at least about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or more cells/ml.
  • the concentration of the engineered immune cells in the preparation can be 1 x 10 3 -1 x 10 8 cells/ml, or 1 x 10 4 -1 x 10 7 cells/ml.
  • compositions of the present disclosure may comprise engineered immune cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide) ; and preservatives.
  • Compositions of the present disclosure may be formulated for intravenous administration.
  • compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented) .
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • an immunologically effective amount When “an immunologically effective amount” , “an anti-tumor effective amount” , “an tumor-inhibiting effective amount” , or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject) . It can generally be stated that a pharmaceutical composition comprising the engineered immune cells (e.g., CAR-T cells) described herein may be administered at a dosage of 10 4 to 10 9 cells/kg body weight, or in some cases, 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques. The optimal dosage and treatment regime for a particular patient can readily be determined by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • CAR-T cells the engineered immune cells
  • compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v. ) injection, or intraperitoneally.
  • the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection.
  • the T cell compositions of the present disclosure are preferably administered by i. v. injection.
  • the compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
  • the present disclosure provides therapeutic applications with engineered immune cells (e.g., T cells or NK cells) transduced with a lentiviral vector (LV) encoding an expression cassette described herein.
  • Transduced T cells or NK cells can target tumor cell markers (such as CS1, CD19, BCMA) and activated T cell and/or NK cell consensus markers (such as CS1, CD7, CD137, etc. ) .
  • the engineered immune cells can be used for allogeneic tumor treatment and can be prepared on a large scale.
  • the present disclosure also provides a method of stimulating a T cell mediated immune response to a target cell population or tissue of a subject (e.g., a mammal) comprising the step of administering to the subject an engineered immune cell (e.g., CAR-T cell) of the disclosure.
  • a subject e.g., a mammal
  • an engineered immune cell e.g., CAR-T cell
  • the present disclosure provides a type of cell therapy comprising directly administering engineered universal CAR-T cells of the present disclosure to a patient in need thereof.
  • the CAR-T cells of the present disclosure may have the endogenous TCR expression knocked out or silenced in the cells by gene editing technology. Inactivation of the endogenous TCRs and/or MHC (such as B2M) can prevent killing of normal cells by the TCRs during the allogeneic infusion. The GVHD reaction may be prevented.
  • the CAR-T cells targeting a tumor cell marker such as CS1, CD19, BCMA
  • a marker for activated T cells and/or NK cells such as CS1, CD7, CD137
  • HVG host versus graft response
  • the cell therapy provided herein can also improve the survival and anti-tumor effect of allogeneic CAR-T cells in the subject.
  • provided herein is a method of treating or diagnosing a disease in a subject, comprising administering the pharmaceutical composition described herein to said subject.
  • the engineered immune cell in said pharmaceutical composition can be derived from an allogeneic immune cell.
  • the engineered immune cell derived from said allogeneic immune cell may not induce graft versus host disease (GvHD) in said subject.
  • the engineered immune cell in said pharmaceutical composition can be derived from an autologous immune cell.
  • the endogenous TCR and/or MHC (such as B2M) of said engineered immune cell in said pharmaceutical composition may be functionally inactive.
  • the engineered immune cell can reduce GVHD in said subject compared to an additional immune cell having a functionally active TCR and/or MHC (such as B2M) .
  • the disease can be a cancer.
  • the cancer can be, for example, lymphoma or leukemia.
  • the CAR-T cells of the present disclosure can undergo robust in vivo cell expansion and can be extended.
  • the CAR-mediated immune response can be part of a step of adoptive immunotherapy in which CAR-modified T cells can induce an immune response specific for the antigen-binding domain in the CAR.
  • anti-CS1 CAR-T cells elicit a specific immune response against cells expressing CS1.
  • the engineered immune cells provided herein can be used to treat cancers.
  • Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors.
  • the cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors.
  • Types of cancers to be treated with the CARs of the present disclosure include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • sarcomas e.g., sarcomas, carcinomas, and melanomas.
  • Adult tumors/cancers and pediatric tumors/cancers are also included.
  • Hematologic cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) , chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia) , polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms) , multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas) .
  • solid tumors such as sarcomas and carcinomas
  • solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms
  • the antigen bind moiety portion of the CAR of the present disclosure is designed to treat a particular cancer.
  • the CAR designed to target CS1 can be used to treat cancers and disorders including but are not limited to multiple myeloma, Plasmacytoma, T cell lymphoma, Light chain amyloidosis (AL) , B cell related autoimmune disorders such as lupus, HLA donor-specific antibodies (DSAs) .
  • the CAR designed to target CD19 can be used to treat cancers and disorders including but are not limited to pre-B ALL (pediatric indication) , adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage post allogeneic bone marrow transplantation, and the like.
  • pre-B ALL pediatric indication
  • adult ALL mantle cell lymphoma
  • diffuse large B-cell lymphoma diffuse large B-cell lymphoma
  • salvage post allogeneic bone marrow transplantation and the like.
  • the CAR designed to target BCMA can be used to treat cancers and disorders including but are not limited to multiple myeloma, plasmacytoma, Hodgkins'lymphoma, follicular lymphomas, small non-cleaved cell lymphomas, endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma, marginal zone lymphoma, extranodal mucosa-associated lymphoid tissue lymphoma, nodal monocytoid B cell lymphoma, splenic lymphoma, mantle cell lymphoma, large cell lymphoma, diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B cell lymphoma, pulmonary B cell angiocentric lymphoma, and small lymphocytic lymphoma.
  • cancers and disorders including but are not limited to multiple myeloma, plasmacytoma, Hodgkins'lymphoma, follicular lymphomas
  • the present disclosure should not be construed to be limited to solely to the antigen targets and diseases disclosed herein. Rather, the present disclosure should be construed to include any antigenic target that is associated with a disease where a CAR can be used to treat the disease.
  • the cell therapy disclosed herein can be co-formulated with, and/or co-administered with, one or more additional therapeutic agents, e.g., one or more anti-cancer agents, cytotoxic or cytostatic agents, hormone treatment, vaccines, and/or other immunotherapies.
  • the engineered immune cells are administered in combination with other therapeutic treatment modalities, including surgery, radiation, cryosurgery, and/or thermotherapy.
  • Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
  • the methods and compositions described herein are administered in combination with one or more antibody molecules, chemotherapy, other anti-cancer therapy (e.g., targeted anti-cancer therapies, or oncolytic drugs) , cytotoxic agents, immune-based therapies (e.g., cytokines) , surgical and/or radiation procedures.
  • anti-cancer therapy e.g., targeted anti-cancer therapies, or oncolytic drugs
  • cytotoxic agents e.g., cytokines
  • immune-based therapies e.g., cytokines
  • Exemplary cytotoxic agents that can be administered in combination with include antimicrotubule agents, topoisomerase inhibitors, anti-metabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis, proteasome inhibitors, and radiation (e.g., local or whole body ir-radiation) .
  • antimicrotubule agents include antimicrotubule agents, topoisomerase inhibitors, anti-metabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalating agents, agents capable of interfering with a signal transduction pathway, agents that promote apoptosis, proteasome inhibitors, and radiation (e.g., local or whole body ir-radiation) .
  • the combination therapy is used in combination with a standard of cancer care chemotherapeutic agent including, but not limited to, anastrozole bicalu-tamide bleomycin sulfate busulfan busulfan injection capecitabine N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin carmustine chlorambucil cisplatin cladribine cyclophosphamide ( or ) , cytarabine, cytosine arabinoside cytarabine liposome injection dacarbazine dactinomy-cin (Actinomycin D, Cosmegan) , daunorubicin hydrochloride daunorubicin citrate liposome injection dexamethasone, docetaxel doxorubicin hydro-chloride etoposide fludarabine phosphate 5- fluorouracil flutamide tezacitibine, Gemcitabine (di)
  • alkylating agents include, without limitation, nitrogen mustards, ethylenimine deriva-tives, alkyl sulfonates, nitrosoureas and triazenes) : uracil mustard (Aminouracil Uracil nitrogen ) , chlormethine cyclophosphamide ( Revimmune TM ) , ifosfamide melphalan Chlorambucil pi-pobroman triethylenemelamine triethy-lenethiophosphoramine, Temozolomide thiotepa busulfan carmustine lomustine streptozocin and dacarbazine
  • Additional exemplary alkylating agents include, without limitation, Oxaliplatin Temozolomide ( and ) ; Dactinomycin (also known as actinomycin-D, ) ; Melphalan (also known as L-PAM, L-sarcolysin, and phenylal-anine mustard, ) ; Altret
  • anthracyclines include, e.g., doxorubicin ( and ) ; bleomycin daunorubicin (dauorubicin hydrochloride, daunomycin, and rubidomycin hydro-chloride, ) ; daunorubicin liposomal (daunorubicin citrate liposome, ) ; mitoxantrone (DHAD, ) ; epirubicin (Ellence TM ) ; idarubicin ( Idamycin ) ; mitomycin C geldanamycin; herbimycin; ravidomycin; and desacetyl-ravidomycin.
  • vinca alkaloids examples include, but are not limited to, vinorelbine tartrate Vincristine and Vindesine vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, and ) ; and vinorelbine
  • proteasome inhibitors examples include, but are not limited to, bortezomib carfilzomib (PX-171-007, (S) -4-Methyl-N- ( (S) -1- ( ( (S) -4-methyl-1- ( (R) -2-methyloxiran-2-yl) -1-oxopentan-2-yl) amino) -1-oxo-3-phenylpropan-2-yl) -2- ( (S) -2- (2-morpholinoacetamido) -4-phenylbutanamido) -pentanamide) ; marizomib (NPI-0052) ; ixazomib citrate (MLN-9708) ; delanzomib (CEP-18770) ; 0-Methyl-N- [ (2-methyl-5-thiazolyl) carbonyl] -L-seryl-O-methyl-N-
  • the cell therapy may be used in combination with a tyrosine kinase inhibitor (e.g., a receptor tyrosine kinase (RTK) inhibitor) .
  • a tyrosine kinase inhibitor include, but are not limited to, an epidermal growth factor (EGF) pathway inhibitor (e.g., an epidermal growth factor receptor (EGFR) inhibitor) , a vascular endothelial growth factor (VEGF) pathway inhibitor (e.g., a vascular endothelial growth factor receptor (VEGFR) inhibitor (e.g., a VEGFR-1 inhibitor, a VEGFR-2 inhibitor, a VEGFR-3 inhibitor) ) , a platelet derived growth factor (PDGF) pathway inhibitor (e.g., a platelet derived growth factor receptor (PDGFR) inhibitor (e.g., a PDGFR- ⁇ inhibitor) ) , a RAF-1 inhibitor, a KIT inhibitor and a RET inhibitor
  • the anti-cancer agent used in combination with the hedgehog inhibitor is selected from the group consisting of: axitinib (AG013736) , bosutinib (SKI-606) , cediranib (RE-CENTIN, AZD2171) , dasatinib ( BMS-354825) , erlotinib gefitinib imatinib ( CGP57148B, STI-571) , lapatinib lestaurtinib (CEP-701) , neratinib (HKI-272) , nilotinib semaxanib (semaxinib, SU5416) , sunitinib ( SU11248) , toceranib vandetanib ( ZD6474) , vatalanib (PTK787, PTK/ZK) , trastuzumab bevacizumab rituximab cetuximab panitum
  • hedgehog inhibitors include, but are not limited to, vismodegib (2-chloro-N- [4-chloro-3- (2-pyridinyl) phenyl] -4- (methylsulfonyl) -benzamide, GDC-0449) ; 1- (4-Chloro-3- (trifluoromethyl) phenyl) -3- ( (3- (4-fluorophenyl) -3, 4-dihydro-4-oxo-2-quinazolinyl) methyl) -urea (CAS 330796-24-2) ; N- [ (2S, 3R, 3′R, 3aS, 4′aR, 6S, 6′aR, 6′bS, 7aR, 12′aS, 12′bS) -2′ ,3′, 3a, 4, 4′, 4′a, 5, 5′, 6, 6′, 6′a, 6′b, 7, 7′, 7a, 8′, 10′, 12′, 12′a, 12′b-
  • Selected tyrosine kinase inhibitors are chosen from sunitinib, erlotinib, gefitinib, or sorafenib erlotinib hydrochloride linifanib (N-[4- (3-amino-1H-indazol-4-yl) phenyl] -N′- (2-fluoro-5-methylphenyl) urea, also known as ABT 869, avail-able from Genentech) ; sunitinib malate bosutinib (4- [ (2, 4-dichloro-5-methoxyphenyl) amino] -6-methoxy-7- [3- (4-methylpiperazin-1-yl) propoxy] quinoline-3-carbonitrile, also known as SKI-606, described in U.S.
  • the cell therapy can be used in combination with a Vascular Endothelial Growth Factor (VEGF) receptor inhibitors, including but not limited to, Bevacizumab axitinib Brivanib alaninate (BMS-582664, (S) - ( (R) -1- (4-(4-Fluoro-2-methyl-1H-indol-5-yloxy) -5-methylpyrrolo [2, 1-f] [1, 2, 4] triazin-6-yloxy) propan-2-yl) 2-aminopropanoate) ; Sorafenib Pazopanib Sunitinib malate Cediranib (AZD2171, CAS 288383-20-1) ; Vargatef (BIBF1120, CAS 928326-83-4) ; Foretinib (GSK1363089) ; Telatinib (BAY57-9352, CAS 332012-40-5) ; Apatinib (YN968
  • the cell therapy described herein can be used in combination with a PI3K inhibitor.
  • the PI3K inhibitor can be an inhibitor of delta and gamma isoforms of PI3K.
  • PI3K inhibitors include, but are not limited to, 4- [2- (1H-Indazol-4-yl) -6- [ [4- (methylsulfonyl) piperazin-1-yl] methyl] thieno [3, 2-d] pyrimidin-4-yl] morpholine; 2-Methyl-2- [4- [3-methyl-2-oxo-8- (quinolin-3-yl) -2, 3-dihydroimidazo [4, 5-c] quinolin-1-yl] phenyl] propionitrile; 4- (trifluoromethyl) -5- (2, 6-dimorpholinopyrimidin-4-yl) pyridin-2-amine; Tozasertib (VX680 or MK-0457, CAS 6390
  • the cell therapy described herein can be used in combination with a mTOR inhibitor, e.g., one or more mTOR inhibitors chosen from one or more of rapamycin, temsirolimus AZD8055, BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126, OSI-027, GSK1059615, KU-0063794, WYE-354, Palomid 529 (P529) , PF-04691502, or PKI-587.
  • a mTOR inhibitor e.g., one or more mTOR inhibitors chosen from one or more of rapamycin, temsirolimus AZD8055, BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126, OSI-027, GSK1059615, KU-0063794, WYE-354, Palomid 529 (P529) , PF-04691502, or PKI
  • ridaforolimus (formally known as deferolimus, (1R, 2R, 4S) -4- [ (2R) -2 [ (1R, 9S, 12S, 15R, 16E, 18R, 19R, 21R, 23S, 24E, 26E, 28Z, 30S, 32S, 35R) -1, 18-dihydroxy-19, 30-dimethoxy-15, 17, 21, 23, 29, 35-hexamethyl-2, 3, 10, 14, 20-pentaoxo-11, 36-dioxa-4-azatricyclo [30.3.1.04, 9] hexatriaconta-16, 24, 26, 28-tetraen-12-yl] propyl] -2-methoxycyclohexyl di-methylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No.
  • the cell therapy can be used in combination with a BRAF inhibitor, e.g., GSK2118436, RG7204, PLX4032, GDC-0879, PLX4720, and sorafenib tosylate (Bay 43-9006) .
  • a BRAF inhibitor e.g., GSK2118436, RG7204, PLX4032, GDC-0879, PLX4720, and sorafenib tosylate (Bay 43-9006) .
  • a BRAF inhibitor includes, but is not limited to, regorafenib (BAY73-4506, CAS 755037-03-7) ; tuvizanib (AV951, CAS 475108-18-0) ; vemurafenib ( PLX-4032, CAS 918504-65-1) ; encorafenib (also known as LGX818) ; 1-Methyl-5- [ [2- [5- (trifluoromethyl) -1H-imidazol-2-yl] -4-pyridinyl] oxy] -N- [4- (trifluoromethyl) phenyl-1H-benzimidazol-2-amine (RAF265, CAS 927880-90-8) ; 5- [1- (2-Hydroxyethyl) -3- (pyridin-4-yl) -1H-pyrazol-4-yl] -2, 3-dihydroinden-1-one oxime (
  • the cell therapy described herein can be used in combination with a MEK inhibitor.
  • MEK inhibitor can be used in combination including, but not limited to, selumetinib (5- [ (4-bromo-2-chlorophenyl) amino] -4-fluoro-N- (2-hydroxyethoxy) -1-methyl-1H-benzimidazole-6-carboxamide, also known as AZD6244 or ARRY 142886) ; ARRY-142886 trametinib dimethyl sulfoxide (GSK-1120212, CAS 1204531-25-80) ; G02442104 (also known as GSK1120212) , RDEA436; N- [3, 4-Difluoro-2- [ (2-fluoro-4-iodophenyl) amino] -6-methoxyphenyl] -1- [ (2R) -2, 3-dihydroxypropyl] -cyclopropanesulfonamide (also known as RDEA119 or BAY8697
  • GDC-0973 Metal Organic Chemical Vaporescence Activated Cell Sorting: [3, 4-difluoro-2- [ (2-fluoro-4-iodophenyl) amino] phenyl] [3-hydroxy-3- (25) -2-piperidinyl-1-azetidinyl] -) , G-38963; and G02443714 (also known as AS703206) , or a pharmaceutically acceptable salt or solvate thereof.
  • MEK inhibitors include, but are not limited to, benimetinib (6- (4-bromo-2-fluorophenylamino) -7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxyethyoxy) -amide, also known as MEK162, CAS 1073666-70-2) ; 2, 3-Bis [amino [ (2-aminophenyl) thio] methylene] -butanedinitrile (also known as U0126 and described in U.S. Pat. No.
  • the cell therapy described herein can be used in combination with a JAK2 inhibitor, e.g., CEP-701, INCB18424, CP-690550 (tasocitinib) .
  • Example JAK inhibitors include, but are not limited to, ruxolitinib tofacitinib (CP690550) ; axitinib (AG013736, CAS 319460-85-0) ; 5-Chloro-N2- [ (1S) -1- (5-fluoro-2-pyrimidinyl) ethyl] -N4- (5-methyl-1H-pyrazol-3-y) -12, 4-pyrimidinediamine (AZD1480, CAS 935666-88-9) ; (9E) -15- [2- (1-Pyrrolidinyl) ethoxy] -7, 12, 26-trioxa-19, 21, 24-triazatetracyclo [18.3.1.12, 5.114, 18] -hexa
  • the combination therapies disclosed herein include paclitaxel or a paclitaxel agent, e.g., protein-bound paclitaxel (e.g., ) .
  • paclitaxel agents include, but are not limited to, nanoparticle albumin-bound paclitaxel (ABRAX-ANE, marketed by Abraxis Bioscience) , docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin, marketed by Protarga) , polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX, marketed by Cell Therapeutic) , the tumor-activated prodrug (TAP) , ANG105 (Angiopep-2 bound to three molecules of paclitaxel, marketed by Im-munoGen) , paclitaxel-EC-1 (paclitaxel bound to the erbB2-
  • the present disclosure provides methods for generating an engineered cell.
  • the method can comprise (a) delivering a nucleic acid molecule expressing a chimeric polypeptide into a cell; and (b) expressing the nucleic acid molecule in the cell, thereby generating the engineered cell.
  • the chimeric polypeptide can be a chimeric antigen receptor as described herein.
  • the present disclosure also provides methods for administering an engineered cell as described herein.
  • the engineered cell can be an engineered immune cell.
  • the engineered immune cell can be a T cell.
  • the engineered immune cell can be derived from an autologous T cell.
  • the engineered immune cell can be derived from an allogeneic T cell.
  • an engineered immune cell comprising a chimeric polypeptide comprising (i) an enhancer moiety capable of enhancing one or more activities of the engineered immune cell, and (ii) an inducible cell death moiety capable of effecting death of the engineered immune cell upon contacting the chimeric polypeptide with a cell death activator.
  • the enhancer moiety can be linked to the inducible cell death moiety.
  • the engineered immune cell can further comprise one or more chimeric antigen receptors (CARs) comprising a binding moiety.
  • the binding moiety can comprise a first antigen binding domain, which first antigen binding domain suppresses or reduces a subject’s immune response toward the engineered immune cell when administered into the subject.
  • the binding moiety can further comprise a second antigen binding domain capable of binding to a disease-associated antigen.
  • An individual CAR of the one or more CARs can comprise (i) the first antigen binding domain, (ii) the second antigen binding domain, or (iii) both the first antigen binding domain and the second antigen binding domain.
  • Each CAR of the one or more CARs can further comprise a transmembrane domain and an intracellular signaling domain.
  • an engineered immune cell comprising one or more chimeric antigen receptors (CARs) comprising a binding moiety.
  • the binding moiety can comprise a first antigen binding domain capable of binding to an immune cell antigen and a second antigen binding domain capable of binding to a disease-associated antigen.
  • Each CAR of the one or more CARs can further comprise a transmembrane domain and an intracellular signaling domain.
  • the engineered immune cell can further comprise an enhancer moiety capable of enhancing one or more activities of the engineered immune cell.
  • an endogenous T cell receptor (TCR) of the engineered immune cell can be inactivated.
  • the engineered immune cell can exhibit (i) enhanced degree of persistence by remaining viable in vitro for at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, or more days while in presence of cells (e.g., cancer cells, immune cells, or both) that are heterologous to the engineered immune cell, (ii) enhanced degree of expansion by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 200-fold, 250-fold, 300-fold
  • the engineered immune cell can exhibit enhanced degree of expansion by at least about 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 350-fold, 400-fold, 450-fold, 500-fold, or more within 30 days.
  • the engineered immune cell can exhibit enhanced degree of expansion by at least about 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000-fold, 8,000-fold, 9,000-fold, 10,000-fold, 20,000-fold, 30,000-fold, 40,000-fold, 50,000-fold, 60,000-fold, 70,000-fold, 80,000-fold, 90,000-fold, 100,000-fold, 200,000-fold, 300,000-fold, 400,000-fold, 500,000-fold, 600,000-fold, 700,000-fold, 800,000-fold, 900,000-fold, 1,000,000-fold, or more within 60 days.
  • the viability or expansion can be measured in the presence of stimulation, for example, stimulation by a cancer antigen or a cancer cell.
  • the viability or expansion can be measured in the presence of multiple rounds or repeated stimulations.
  • a method of administering a cell comprising a functionally inactive T cell receptor (TCR) .
  • the cell can further comprise one or more chimeric antigen receptors (CARs) .
  • Each individual CAR of the one or more CARs can comprise a binding moiety.
  • the binding moiety can comprise (i) a first antigen binding domain, which first antigen binding domain suppresses or reduces a subject’s immune response toward the engineered immune cell when administered into the subject and (ii) a second antigen binding domain that binds to a disease-associated antigen.
  • Each CAR of the one or more CARs can further comprise a transmembrane domain and an intracellular signaling domain.
  • an engineered immune cell comprising an enhancer moiety capable of enhancing one or more activities of the engineered immune cell.
  • the engineered cell can further comprise a chimeric antigen receptor (CAR) comprising an antigen binding domain that specifically binds CD7.
  • CAR can further comprise a transmembrane domain and an intracellular signaling domain.
  • the endogenous CD7 in the engineered immune cell can be inactivated.
  • the engineered cell can comprise a CAR comprising an antigen binding domain that specifically binds an immune cell antigen.
  • the immune cell antigen can be any immune cell antigen described herein such as CD2, CD3, CD4, CD5, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD279, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, CD319 (CS1) , TCR ⁇ , TCR ⁇ and SLAMF7.
  • the endogenous immune cell antigen of the engineered cell which the antigen binding domain binds, can be inactivated in the engineered cell.
  • an engineered immune cell comprising a single chimeric antigen receptor (CAR)
  • the single CAR comprises: (i) a first antigen binding domain that specifically binds CD7 and (ii) a second antigen binding domain capable of binding to CS1.
  • the CAR can further comprise a transmembrane domain and an intracellular signaling domain.
  • a gene encoding endogenous CS1 and/or CD7 can be inactivated in the engineered immune cell.
  • an engineered immune cell comprising a single chimeric antigen receptor (CAR)
  • the single CAR comprises: (i) a first antigen binding domain that specifically binds CD137 and (ii) a second antigen binding domain capable of binding to CS1.
  • the CAR can further comprise a transmembrane domain and an intracellular signaling domain.
  • a gene encoding endogenous CS1 and/or CD137 can be inactivated in the engineered immune cell.
  • the present disclosure also provides methods of treating or diagnosing a disease in a subject.
  • the method comprises administering a pharmaceutical composition comprising an engineered immune cell into a subject.
  • the engineered immune cell in the pharmaceutical composition can be derived from an allogeneic immune cell.
  • the engineered immune cell derived from the allogeneic immune cell may not induce graft versus host disease (GVHD) in the subject.
  • the engineered immune cell in the pharmaceutical composition can be derived from an autologous immune cell.
  • an endogenous TCR of the engineered immune cell in the pharmaceutical composition is functionally inactive.
  • the engineered immune cell can reduce GVHD in the subject compared to an immune cell having a functionally active TCR.
  • an endogenous MHC molecule (such as B2M) of the engineered immune cell in the pharmaceutical composition is functionally inactive.
  • the engineered immune cell can reduce GVHD in the subject compared to an immune cell having a functionally active MHC molecule (such as B2M) .
  • the disease can be a cancer.
  • the cancer can be lymphoma or leukemia.
  • the present disclosure also provides a method of delivering an allogeneic cell therapy comprising administering to a subject in need thereof a population of engineered immune cells.
  • An individual engineered immune cell of the population can comprise one or more chimeric antigen receptors (CARs) comprising a binding moiety.
  • the binding moiety can comprise a first antigen binding domain capable of binding to an immune cell antigen.
  • the binding moiety can further comprise a second antigen binding domain capable of binding to a disease-associated antigen.
  • the first antigen binding domain can suppress or reduce a subject’s immune response toward the engineered immune cell when administered into the subject.
  • the engineered immune cell can further comprise an enhancer moiety capable of enhancing one or more activities of the engineered immune cell.
  • the endogenous T cell receptor (TCR) of the engineered immune cell can be inactivated.
  • TCR T cell receptor
  • a gene encoding a subunit of TCR can be inactivated.
  • the endogenous MHC molecule of the engineered immune cell can be inactivated.
  • a gene encoding a subunit of MHC such as B2M can be inactivated.
  • Various gene editing methods described herein can be used to inactivate endogenous TCRs of a T cell.
  • a method provided herein can include activation of a population of cells.
  • the cell used to prepare the engineered immune cell can be activated before preparing the engineered immune cell.
  • the engineered immune cell can be activated.
  • Activation as used herein can refer to a process whereby a cell transitions from a resting state to an active state. This process can comprise a response to an antigen, migration, and/or a phenotypic or genetic change to a functionally active state.
  • activation can refer to the stepwise process of T cell activation.
  • a T cell can require one or more signals to become activated. For example, a T cell can require at least two signals to become fully activated.
  • the first signal can occur after engagement of a TCR by the antigen-MHC complex, and the second signal can occur by engagement of co-stimulatory molecules.
  • Anti-CD3 antibody (or a functional variant thereof) can mimic the first signal and anti-CD28 antibody (or a functional variant thereof) can mimic the second signal in vitro.
  • a method provided herein can comprise activation of a population of cells. Activation can be performed by contacting a population of cells with a surface having attached thereto an agent that can stimulate a CD3 TCR complex associated signal and a ligand that can stimulate a co-stimulatory molecule on the surface of the cells.
  • T cell populations can be stimulated in vitro such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) sometimes in conjunction with a calcium ionophore.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule can be used.
  • a population of cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions that can stimulate proliferation of the T cells.
  • 4-1BB can be used to stimulate cells.
  • cells can be stimulated with 4-1BB and IL-21 or another cytokine.
  • an anti-CD3 antibody and an anti-CD28 antibody can be used.
  • the agents providing a signal may be in solution or conjugated to a solid phase surface. The ratio of particles to cells may depend on particle size relative to the target cell.
  • the cells such as T cells
  • the cells can be combined with agent-coated beads, where the beads and the cells can be subsequently separated, and optionally cultured.
  • Each bead can be coated with either anti-CD3 antibody or an anti-CD28 antibody, or in some cases, a combination of the two.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • Cell surface proteins may be conjugated by allowing paramagnetic beads to which anti-CD3 antibody and anti-CD28 antibody can be attached (3x28 beads) to contact the T cells.
  • the cells and beads are combined in a buffer, for example, phosphate buffered saline (PBS) (e.g., without divalent cations such as, calcium and magnesium) .
  • PBS phosphate buffered saline
  • Any cell concentration may be used.
  • the mixture may be cultured for or for about several hours (e.g., about 3 hours) to or to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for or for about 21 days or for up to or for up to about 21 days.
  • Conditions appropriate for T cell culture can include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 5, (Lonza) ) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum) , interleukin-2 (IL-2) , insulin, IFN-g , IL-4, IL-7, GM-CSF, IL-10, IL-21, IL-15, TGF beta, and TNF alpha or any other additives for the growth of cells.
  • Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
  • Media can include RPMI 1640, A1 M-V, DMEM, MEM, ⁇ -MEM, F-12, X-Vivo 1 , and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine (s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g., penicillin and streptomycin, can be included only in experimental cultures, possibly not in cultures of cells that are to be infused into a subject.
  • the target cells can be maintained under conditions necessary to support growth; for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5%CO 2 ) .
  • an appropriate temperature e.g., 37°C
  • atmosphere e.g., air plus 5%CO 2
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • a soluble monospecific tetrameric antibody against human CD3, CD28, CD2, or any combination thereof may be used.
  • activation can utilize an activation moiety, a costimulatory agent, and any combination thereof.
  • an activation moiety binds: a CD3/T cell receptor complex and/or provides costimulation.
  • an activation moiety is any one of anti-CD3 antibody and/or anti-CD28 antibody.
  • a solid phase is at least one of a bead, plate, and/or matrix. In some aspects, a solid phase is a bead.
  • the activation moiety may be not be conjugated a substrate, e.g., the activation moiety may be free-floating in a medium.
  • a population of cells can be activated or expanded by co-culturing with tissue or cells.
  • a cell can be an antigen presenting cell.
  • An artificial antigen presenting cells (aAPCs) can express ligands for T cell receptor and costimulatory molecules and can activate and expand T cells for transfer, while improving their potency and function in some cases.
  • An aAPC can be engineered to express any gene for T cell activation.
  • An aAPC can be engineered to express any gene for T cell expansion.
  • An aAPC can be a bead, a cell, a protein, an antibody, a cytokine, or any combination.
  • An aAPC can deliver signals to a cell population that may undergo genomic transplant.
  • an aAPC can deliver a signal 1, signal, 2, signal 3 or any combination.
  • a signal 1 can be an antigen recognition signal.
  • signal 1 can be ligation of a TCR by a peptide–MHC complex or binding of agonistic antibodies directed towards CD3 that can lead to activation of the CD3 signal-transduction complex.
  • Signal 2 can be a co-stimulatory signal.
  • a co-stimulatory signal can be anti-CD28, inducible co-stimulator (ICOS) , CD27, and 4-1BB (CD137) , which bind to ICOS-L, CD70, and 4-1BBL, respectively.
  • Signal 3 can be a cytokine signal.
  • a cytokine can be any cytokine.
  • a cytokine can be IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof.
  • an artificial antigen presenting cell may be used to activate and/or expand a cell population. In some cases, an artificial may not induce allospecificity. An aAPC may not express HLA in some cases.
  • An aAPC may be genetically modified to stably express genes that can be used to activation and/or stimulation.
  • a K562 cell may be used for activation.
  • a K562 cell may also be used for expansion.
  • a K562 cell can be a human erythroleukemic cell line.
  • a K562 cell may be engineered to express genes of interest.
  • K562 cells may not endogenously express HLA class I, II, or CD1d molecules but may express ICAM-1 (CD54) and LFA-3 (CD58) .
  • K562 may be engineered to deliver a signal 1 to T cells.
  • K562 cells may be engineered to express HLA class I.
  • K562 cells may be engineered to express additional molecules such as B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, anti-CD3, anti-CD3 mAb, anti-CD28, anti-CD28mAb, CD1d, anti-CD2, membrane-bound IL-15, membrane-bound IL-17, membrane-bound IL-21, membrane-bound IL-2, truncated CD19, or any combination.
  • an engineered K562 cell can expresses a membranous form of anti-CD3 mAb, clone OKT3, in addition to CD80 and CD83. In some cases, an engineered K562 cell can expresses a membranous form of anti-CD3 mAb, clone OKT3, membranous form of anti-CD28 mAb in addition to CD80 and CD83.
  • An aAPC can be a bead.
  • a spherical polystyrene bead can be coated with antibodies against CD3 and CD28 and be used for T cell activation.
  • a bead can be of any size. In some cases, a bead can be or can be about 3 and 6 micrometers. A bead can be or can be about 4.5 micrometers in size.
  • a bead can be utilized at any cell to bead ratio. For example, a 3 to 1 bead to cell ratio at 1 million cells per milliliter can be used.
  • An aAPC can also be a rigid spherical particle, a polystyrene latex microbeads, a magnetic nano-or micro-particles, a nanosized quantum dot, a 4, poly (lactic-co-glycolic acid) (PLGA) microsphere, a nonspherical particle, a 5, carbon nanotube bundle, a 6, ellipsoid PLGA microparticle, a 7, nanoworms, a fluidic lipid bilayer-containing system, an 8, 2D-supported lipid bilayer (2D-SLBs) , a 9, liposome, a 10, RAFTsomes/microdomain liposome, an 11, SLB particle, or any combination thereof.
  • PLGA poly (lactic-co-glycolic acid)
  • an aAPC can expand CD4 T cells.
  • an aAPC can be engineered to mimic an antigen processing and presentation pathway of HLA class II-restricted CD4 T cells.
  • a K562 can be engineered to express HLA-D, DP ⁇ , DP ⁇ chains, Ii, DM ⁇ , DM ⁇ , CD80, CD83, or any combination thereof.
  • engineered K562 cells can be pulsed with an HLA-restricted peptide in order to expand HLA-restricted antigen-specific CD4 T cells.
  • the use of aAPCs can be combined with exogenously introduced cytokines for T cell activation, expansion, or any combination. Cells can also be expanded in vivo, for example in the subject’s blood after administration of genomically transplanted cells into a subject.
  • a method provided herein can comprise transduction of a population of cells.
  • a method comprises introducing a polynucleotide encoding for a cellular receptor such as a chimeric antigen receptor and/or a T cell receptor.
  • a transfection of a cell can be performed.
  • a viral supernatant comprising a polynucleotide encoding for a cellular receptor such as a CAR and/or TCR is generated.
  • a viral vector can be a retroviral vector, a lentiviral vector and/or an adeno-associated viral vector.
  • Packaging cells can be used to form virus particles capable of infecting a host cell. Such cells can include 293 cells, (e.g., for packaging adenovirus) , and Psi2 cells or PA317 cells (e.g., for packaging retrovirus) .
  • Viral vectors can be generated by producing a cell line that packages a nucleic acid vector into a viral particle.
  • the vectors can contain the minimal viral sequences required for packaging and subsequent integration into a host.
  • the vectors can contain other viral sequences being replaced by an expression cassette for the polynucleotide (s) to be expressed.
  • the missing viral functions can be supplied in trans by the packaging cell line.
  • AAV vectors can comprise ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA can be packaged in a cell line, which can contain a helper plasmid encoding the other AAV genes, namely rep and cap, while lacking ITR sequences.
  • the cell line can also be infected with adenovirus as a helper.
  • the helper virus can promote replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells can be used, for example, as described in US20030087817, incorporated herein by reference.
  • a host cell can be transiently or non-transiently transfected with one or more vectors described herein.
  • a cell can be transfected as it naturally occurs in a subject.
  • a cell can be taken or derived from a subject and transfected.
  • a cell can be derived from cells taken from a subject, such as a cell line.
  • a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences.
  • Non-limiting examples of vectors for eukaryotic host cells include but are not limited to: pBs, pQE-9 (Qiagen) , phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene) ; pTrc99A, pKK223-3, pKK233-3, pDR54O, pRIT5 (Pharmacia) .
  • Eukaryotic pWL-neo, pSv2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPv, pMSG, pSVL (Pharmiacia) .
  • any other plasmids and vectors can be used as long as they are replicable and viable in a selected host.
  • Any vector and those commercially available (and variants or derivatives thereof) can be engineered to include one or more recombination sites for use in the methods.
  • Such vectors can be obtained from, for example, Vector Laboratories Inc., Invitrogen, Promega, Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc., Stratagene, PerkinElmer, Pharmingen, and Research Genetics.
  • vectors of interest include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen) , pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech) , pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.
  • eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen) , pEUK-C1, pPUR, pMAM, p
  • vectors include pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificial chromosomes) , BAC's (bacterial artificial chromosomes) , P1 (Escherichia coli phage) , pQE70, pQE60, pQE9 (quagan) , pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene) , pcDNA3 (Invitrogen) , pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia) , pSPORT1, pSPORT2, pCMVSPORT2.0 and pSYSPORT1 (Invitrogen
  • Additional vectors of interest can also include pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBa-cHis2, pcDNA3.1/His, pcDNA3.1 (-) /Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pA081S, pPICZ, pPICZA, pPICZB, pPICZC, pGAPZA, pGAPZB, pGAPZC, pBlue-Bac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND (SP1) , pVgRXR, pcDNA2.1, pYES2, pZEr01.1, pZErO-2.1, p
  • Transduction and/or transfection can be performed by any one of: non-viral transfection, biolistics, chemical transfection, electroporation, nucleofection, heat-shock transfection, lipofection, microinjection, or viral transfection.
  • a provided method comprises viral transduction, and the viral transduction comprises a lentivirus.
  • Viral particles can be used to deliver a viral vector comprising a polypeptide sequence coding for a cellular receptor into a cell ex vivo or in vivo.
  • a viral vector as disclosed herein may be measured as pfu (plaque forming units) .
  • the pfu of recombinant virus or viral vector of the compositions and methods of the disclosure may be about 10 8 to about 5 ⁇ 10 10 pfu.
  • recombinant viruses of this disclosure are at least about 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 1 ⁇ 10 10 , 2 ⁇ 10 10 , 3 ⁇ 10 10 , 4 ⁇ 10 10 , and 5 ⁇ 10 10 pfu.
  • recombinant viruses of this disclosure are at most about 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 1 ⁇ 10 10 , 2 ⁇ 10 10 , 3 ⁇ 10 10 , 4 ⁇ 10 10 , and 5 ⁇ 10 10 pfu.
  • the viral vector of the disclosure may be measured as vector genomes.
  • recombinant viruses of this disclosure are 1 ⁇ 10 10 to 3 ⁇ 10 12 vector genomes, or 1 ⁇ 10 9 to 3 ⁇ 10 13 vector genomes, or 1 ⁇ 10 8 to 3 ⁇ 10 14 vector genomes, or at least about 1 ⁇ 10 1 , 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11 , 1 ⁇ 10 12 , 1 ⁇ 10 13 , 1 ⁇ 10 14 , 1 ⁇ 10 15 , 1 ⁇ 10 16 , 1 ⁇ 10 17 , and 1 ⁇ 10 18 vector genomes, or are 1 ⁇ 10 8 to 3 ⁇ 10 14 vector genomes, or are at most about 1 ⁇ 10 1 , 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11
  • a viral vector provided herein can be measured using multiplicity of infection (MOI) .
  • MOI may refer to the ratio, or multiple of vector or viral genomes to the cells to which the nucleic may be delivered.
  • the MOI may be 1 ⁇ 10 6 .
  • the MOI may be 1 ⁇ 10 5 to 1 ⁇ 10 7 .
  • the MOI may be 1 ⁇ 10 4 to 1 ⁇ 10 8 .
  • recombinant viruses of the disclosure are at least about 1 ⁇ 10 1 , 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11 , 1 ⁇ 10 12 , 1 ⁇ 10 13 , 1 ⁇ 10 14 , 1 ⁇ 10 15 , 1 ⁇ 10 16 , 1 ⁇ 10 17 , and 1 ⁇ 10 18 MOI.
  • recombinant viruses of this disclosure are 1 ⁇ 10 8 to 3 ⁇ 10 14 MOI, or are at most about 1 ⁇ 10 1 , 1 ⁇ 10 2 , 1 ⁇ 10 3 , 1 ⁇ 10 4 , 1 ⁇ 10 5 , 1 ⁇ 10 6 , 1 ⁇ 10 7 , 1 ⁇ 10 8 , 1 ⁇ 10 9 , 1 ⁇ 10 10 , 1 ⁇ 10 11 , 1 ⁇ 10 12 , 1 ⁇ 10 13 , 1 ⁇ 10 14 , 1 ⁇ 10 15 , 1 ⁇ 10 16 , 1 ⁇ 10 17 , and 1 ⁇ 10 18 MOI.
  • a viral vector is introduced at a multiplicity of infection (MOI) from about 1x10 5 , 2 x10 5 , 3x10 5 , 4x10 5 , 5 x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8 x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , or up to about 9x10 9 genome copies/virus particles per cell.
  • MOI multiplicity of infection
  • a method can comprise adding an infective agent to a composition comprising a population of cells.
  • An infective agent can comprise polybrene.
  • an infective agent can enhance efficiency of viral infection.
  • An infective agent can enhance viral infectivity from about 100 to 1,000 fold.
  • Polybrene can be added to a composition at a concentration from about 5ug to 10ug per ml.
  • a method provided herein can comprise a non-viral approach of introducing a cellular receptor to a cell.
  • Non-viral approaches can include but are not limited to: CRISPR associated proteins (Cas proteins, e.g., Cas9) , Zinc finger nuclease (ZFN) , Transcription Activator-Like Effector Nuclease (TALEN) , Argonaute nucleases, and meganucleases.
  • Nucleases can be naturally existing nucleases, genetically modified, and/or recombinant.
  • Non-viral approaches can also be performed using a transposon-based system (e.g. PiggyBac, Sleeping beauty) .
  • a method provided herein can utilize a PiggyBac system to introduce an exogenous polypeptide to a cell.
  • a PiggyBac system comprises two components, a transposon and a transposase.
  • the PiggyBac transposase facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed in the genome.
  • the predicted frequency of ‘TTAA’ in the genome is approximately 1 in every 256 base-pairs of DNA sequence.
  • the PB transposase also enables the excision of the transposon in a completely seamless manner, leaving no sequences or mutations behind.
  • PiggyBac offers a large cargo-carrying capacity (over 200 kb has been demonstrated) with no known upper limit.
  • PB performance levels can be increased by codon-optimization strategies, mutations, deletions, additions, substitutions, and any combination thereof.
  • PB can have a larger cargo (approximately 9.1–14.3 kb) , a higher transposition activity, and its footprint-free characteristic can make it appealing as a gene editing tool.
  • PB can comprise a few features: high efficiency transposition; large cargo; steady long-term expression; the trans-gene is integrated as a single copy; tracking the target gene in vivo by a noninvasive mark instead of traditional method such as PCR; easy to determine the integration site, and combinations thereof.
  • a method provided herein can utilize a Sleeping Beauty (SB) System to introduce a polypeptide coding for a cellular receptor to a cell.
  • SB Sleeping Beauty
  • the SB ITRs 230 bp
  • DRs imperfect direct repeats
  • Binding affinity and spacing between the DR elements within ITR has involved in transpositional activities.
  • the SB transposase can be a 39 kDa protein that possess DNA binding polypeptide, a nuclear localization signal (NLS) and the catalytic domain, featured by a conserved amino acid motif (DDE) .
  • Modified SBs can contain mutations, deletions and additions within ITRs of the original SB transposon. Modified SBs can comprise: pT2, pT3, pT2B, pT4, SB100X, and combinations thereof.
  • Non-limited examples of modified SBs can be selected from: SB10, SB11 (3-fold higher than SB10) , SB12 (4-fold higher than SB10) , HSB1–HSB5 (up to 10-fold higher than SB10) , HSB13–HSB17 (HSB17 is 17-fold higher than SB10) , SB100X (100-fold higher than SB10) , SB150X (130-fold higher than SB10) , and any combination thereof.
  • SB100X is 100-fold hyperactive compared to the originally resurrected transposase (SB10) .
  • SB transposition excision leaves a footprint (3 bp) at the cargo site.
  • Transposon integration occurs into TA dinucleotides of the genome, and results in target site duplications, generated by the host repair machinery. In some cases, SB appears to possess a nearly unbiased, close-to-random integration profile. Transposon integration can be artificially targeted ( ⁇ 10%) to a predetermined genomic locus in wildtype systems, however in chimeric systems provided herein, SB transposon integration can be directed to a predetermined locus with efficiencies over 10%.
  • a non-viral approach may be taken to introduce an exogenous polynucleic acid to a population of cells.
  • a non-viral vector or nucleic acid may be delivered without the use of a virus and may be measured according to the quantity of nucleic acid.
  • any suitable amount of nucleic acid can be used with the compositions and methods of this disclosure.
  • nucleic acid may be at least about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ⁇ g, 10 ⁇ g, 100 ⁇ g, 200 ⁇ g, 300 ⁇ g, 400 ⁇ g, 500 ⁇ g, 600 ⁇ g, 700 ⁇ g, 800 ⁇ g, 900 ⁇ g, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g.
  • nucleic acid may be at most about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ⁇ g, 10 ⁇ g, 100 ⁇ g, 200 ⁇ g, 300 ⁇ g, 400 ⁇ g, 500 ⁇ g, 600 ⁇ g, 700 ⁇ g, 800 ⁇ g, 900 ⁇ g, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g.
  • a non-viral approach of introducing a CAR and/or TCR sequence to a cell can include electroporation.
  • Electroporation can be performed using, for example, the Transfection System (ThermoFisher Scientific) or the Nucleofector ( Biosystems) . Electroporation parameters may be adjusted to optimize transfection efficiency and/or cell viability. Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type has a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance and resistance) . Application of optimal field strength causes electropermeabilization through induction of transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some cases, the electroporation pulse voltage, the electroporation pulse width, number of pulses, cell density, and tip type may be adjusted to optimize transfection efficiency and/or cell viability.
  • E Field Strength
  • electroporation pulse voltage may be varied to optimize transfection efficiency and/or cell viability.
  • the electroporation voltage may be less than about 500 volts.
  • the electroporation voltage may be at least about 500 volts, at least about 600 volts, at least about 700 volts, at least about 800 volts, at least about 900 volts, at least about 1000 volts, at least about 1100 volts, at least about 1200 volts, at least about 1300 volts, at least about 1400 volts, at least about 1500 volts, at least about 1600 volts, at least about 1700 volts, at least about 1800 volts, at least about 1900 volts, at least about 2000 volts, at least about 2100 volts, at least about 2200 volts, at least about 2300 volts, at least about 2400 volts, at least about 2500 volts, at least about 2600 volts, at least about 2700 volts,
  • the electroporation pulse voltage required for optimal transfection efficiency and/or cell viability may be specific to the cell type. For example, an electroporation voltage of 1900 volts may optimal (e.g., provide the highest viability and/or transfection efficiency) for macrophage cells. In another example, an electroporation voltage of about 1350 volts may optimal (e.g., provide the highest viability and/or transfection efficiency) for Jurkat cells or primary human cells such as T cells. In some cases, a range of electroporation voltages may be optimal for a given cell type.
  • an electroporation voltage between about 1000 volts and about 1300 volts may optimal (e.g., provide the highest viability and/or transfection efficiency) for human 578T cells.
  • a primary cell can be a primary lymphocyte.
  • a population of primary cells can be a population of lymphocytes.
  • electroporation pulse width may be varied to optimize transfection efficiency and/or cell viability.
  • the electroporation pulse width may be less than about 5 milliseconds.
  • the electroporation width may be at least about 5 milliseconds, at least about 6 milliseconds, at least about 7 milliseconds, at least about 8 milliseconds, at least about 9 milliseconds, at least about 10 milliseconds, at least about 11 milliseconds, at least about 12 milliseconds, at least about 13 milliseconds, at least about 14 milliseconds, at least about 15 milliseconds, at least about 16 milliseconds, at least about 17 milliseconds, at least about 18 milliseconds, at least about 19 milliseconds, at least about 20 milliseconds, at least about 21 milliseconds, at least about 22 milliseconds, at least about 23 milliseconds, at least about 24 milliseconds, at least about 25 milliseconds, at least about 26 milli
  • the electroporation pulse width required for optimal transfection efficiency and/or cell viability may be specific to the cell type. For example, an electroporation pulse width of 30 milliseconds may optimal (e.g., provide the highest viability and/or transfection efficiency) for macrophage cells. In another example, an electroporation width of about 10 milliseconds may optimal (e.g., provide the highest viability and/or transfection efficiency) for Jurkat cells. In some cases, a range of electroporation widths may be optimal for a given cell type. For example, an electroporation width between about 20 milliseconds and about 30 milliseconds may optimal (e.g., provide the highest viability and/or transfection efficiency) for human 578T cells.
  • the number of electroporation pulses may be varied to optimize transfection efficiency and/or cell viability.
  • electroporation may comprise a single pulse.
  • electroporation may comprise more than one pulse.
  • electroporation may comprise 2 pulses, 3 pulses, 4 pulses, 5 pulses 6 pulses, 7 pulses, 8 pulses, 9 pulses, or 10 or more pulses.
  • the number of electroporation pulses required for optimal transfection efficiency and/or cell viability may be specific to the cell type. For example, electroporation with a single pulse may be optimal (e.g., provide the highest viability and/or transfection efficiency) for macrophage cells.
  • electroporation with a 3 pulses may be optimal (e.g., provide the highest viability and/or transfection efficiency) for primary cells.
  • a range of electroporation widths may be optimal for a given cell type.
  • electroporation with between about 1 to about 3 pulses may be optimal (e.g., provide the highest viability and/or transfection efficiency) for human cells.
  • the starting cell density for electroporation may be varied to optimize transfection efficiency and/or cell viability. In some cases, the starting cell density for electroporation may be less than about 1x10 5 cells. In some cases, the starting cell density for electroporation may be at least about 1x10 5 cells, at least about 2x10 5 cells, at least about 3x10 5 cells, at least about 4x10 5 cells, at least about 5x10 5 cells, at least about 6x10 5 cells, at least about 7x10 5 cells, at least about 8x10 5 cells, at least about 9x10 5 cells, at least about 1x10 6 cells, at least about 1.5x10 6 cells, at least about 2x10 6 cells, at least about 2.5x10 6 cells, at least about 3x10 6 cells, at least about 3.5x10 6 cells, at least about 4x10 6 cells, at least about 4.5x10 6 cells, at least about 5x10 6 cells, at least about 5.5x10 6 cells, at least about 6x10 6 cells, at least about 6.5
  • the starting cell density for electroporation required for optimal transfection efficiency and/or cell viability may be specific to the cell type. For example, a starting cell density for electroporation of 1.5x10 6 cells may optimal (e.g., provide the highest viability and/or transfection efficiency) for macrophage cells. In another example, a starting cell density for electroporation of 5x10 6 cells may optimal (e.g., provide the highest viability and/or transfection efficiency) for human cells. In some cases, a range of starting cell densities for electroporation may be optimal for a given cell type. For example, a starting cell density for electroporation between of 5.6x10 6 and 5 x10 7 cells may optimal (e.g., provide the highest viability and/or transfection efficiency) for human cells such as T cells.
  • a method for treating a lymphoid malignancy can comprise administering to a patient in need thereof a population of engineered immune cells.
  • An individual engineered immune cell of the population can comprise one or more chimeric antigen receptors (CARs) comprising a binding moiety, where the binding moiety can comprise an antigen binding domain capable of binding to an immune cell antigen, and where each CAR of the one or more CARs can further comprise a transmembrane domain and an intracellular signaling domain.
  • An individual engineered immune cell of the population can further comprise an enhancer moiety capable of enhancing one or more activities of the engineered immune cell.
  • An endogenous T cell receptor (TCR) of the engineered immune cell may be inactivated.
  • the number of affected cells in peripheral blood or the number of affected cells in bone marrow of the patient can be reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more within a period (e.g., 3 weeks) after a last dosing of the engineered immune cells.
  • the period after a last dosing of the engineered immune cell can be about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more.
  • the number of any one or more of autologous T cell, granulocyte, and NK cell in peripheral blood of the patient can start to increase within a period (e.g., 3 weeks) after a last dosing of the engineered immune cells.
  • the period after a last dosing of the engineered immune cell can be about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or more.
  • the enhancer moiety can enhance one or more activities of the engineered immune cell.
  • the enhancer moiety can be configured to constitutively enhance the one or more activities of the engineered immune cell.
  • the enhancer moiety can be configured to constitutively upregulate one or more intracellular signaling pathways of the engineered immune cell.
  • the one or more intracellular signaling pathways can be one or more cytokine signaling pathways.
  • the enhancer moiety can be a cytokine or a cytokine receptor.
  • the enhancer moiety can be selected from the group consisting of IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, PD-1, PD-L1, CD122, CSF1R, CTAL-4, TIM-3, CCL21, CCL19, TGFR beta, receptors for the same, functional fragments thereof, functional variants thereof, and combinations thereof.
  • the engineered immune cell can further comprise an inducible cell death moiety capable of effecting death of the cell upon contacting the inducible cell death moiety with a cell death activator.
  • the inducible cell death moiety can be selected from the group consisting of rapaCasp9, iCasp9, HSV-TK, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, Her2t, CD30, BCMA, and EGFRt.
  • the inducible cell death moiety can be EGFRt
  • the cell death activator can be an antibody or an antigen binding fragment thereof that binds EGFRt.
  • the inducible cell death moiety can be HSV-TK, and the cell death activator can be GCV.
  • the inducible cell death moiety can be iCasp9, and the cell death activator can be AP1903.
  • a gene encoding an endogenous surface marker of the cell can be inactivated.
  • the endogenous surface marker can be capable of binding to the first antigen binding domain when expressed.
  • the endogenous surface marker can be CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A) , CD159c (NKG2C) , NKG2E, CD279, CD314 (NKG2D) , CD305, CD335 (NKP46) , CD337, CD319 (CS1) , TCR ⁇ , TCR ⁇ or SLAMF7.
  • the number of any one or more of autologous T cell, granulocyte, and NK cell in peripheral blood of the patient may start to increase before the number of affected cells in peripheral blood or the number of affected cells in bone marrow is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more.
  • the number of any one or more of autologous T cell, granulocyte, and NK cell in peripheral blood may start to increase after the number of affected cells in peripheral blood or the number of affected cells in bone marrow is reduced by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more.
  • Alpacas were immunized with CS1 antigen and CS1-positive cells for five rounds to obtain plasma antibodies with titers of ⁇ 1: 128000.
  • anti-CS1 nanobodies were obtained by enrichment and ELISA screening.
  • VHH regions were amplified by using the enriched phages as templates, and the amplified sequences were subject to next generation sequencing. The obtained sequences were ranked according to their sequence frequencies (Frequency) .
  • the Top30 sequences are as shown in Table 1.
  • CS1 single CARs, dual CARs and triple CARs were designed, and the exemplary structures of the CS1 single CARs, dual CARs and triple CARs are as shown in Figure 1.
  • CS1 CAR Primary T cells were transfected with lentiviral vectors comprising CS1 CAR listed in Table 3 below at a MOI of 2-8, in which CAR-1 to CAR-10 are CS1 single CAR structures comprising CS1 VHH antibodies, and CAR-R1 to CAR-R3 are CS1 single CAR structures comprising CS1 scFv antibodies.
  • the cells were transferred to a flask and cultured at 37 °C, 5%CO 2 .3 days after the transfection, the number of cells and CAR positive cells were detected by using anti-GFP, anti-Beacon, CS1 antigen.
  • the positive ratio of CS1 CAR in T cells was higher than 50%, as shown in Figure 3.
  • Table 3 Exemplary sequences of CS1 CAR for preparing CS1 single CAR-T cells
  • CS1 CAR-T cells were stained with different concentrations of CS1 antigen and then the positive ratio was analyzed by Flow Cytometry. Affinity of CARs on T cells with CS1 was calculated based on the positive ratio of CAR-T cells at different concentrations of CS1 antigen, as shown in Figure 4A and 4B.
  • sgRNA sequences targeting the TRAC were designed, synthesized and exemplary sequences of the sgRNA includes: uucggaacccaaucacugacGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCC GUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU.
  • the knockout efficiency of the gRNA was analyzed by FACS using anti-CD3 antibody, as shown in Figure 9A.
  • sgRNA sequences targeting the CS1 were designed, synthesized and exemplary sequences of the sgRNA are as shown in Table 4.
  • Table 4 Exemplary sgRNA sequence for KO CS1 in T cells.
  • sgRNA targeting CS1 and Cas9 protein were mixed at a ratio of 4: 1 in PCR tube to obtain the RNP mixture.
  • the mixture was incubated at 37 °C for more than 15 minutes, and then gently mixed with 10 7 T cells obtained from three donors (#190023, #20005, and #190036) followed by transfection through electroporation. After transfection, the cells were incubated in 80 ⁇ l pre-warmed medium and cultured in an incubator.
  • the transfected T cells were sorted by CD4+ or CD8+, and subject to flow cytometry after antigen staining to detect the expression of CS1.
  • the knockout efficiency (%) of CS1 in different donors are shown in Figure 2A and 2B.
  • sgRNA sequences targeting the TRAC were designed, synthesized and exemplary sequences of the sgRNA includes:
  • the knockout efficiency of the sgRNA was analyzed by FACS using anti-B2M antibody as shown in Figure 9B.
  • Luciferase gene can be transfected into target cells. After adding fluorescein, luciferase could react with the fluorescein to produce fluorescence. By detecting the intensity of the fluorescence, the activity of luciferase can be measured, and the survival of the target cells can be detected so that to evaluate the cytotoxicity of the CS1 CAR-T cells.
  • luciferase gene was transferred into target cells including MM. 1s-Luc ⁇ NCI-H929 and RPMI8226 cells. Then cytotoxicity of CS1 CAR-T cells with CS1 KO on the target cells were analyzed by detecting intensity of the fluorescence, as shown in Figure 5A-5C.
  • Figure 5A shows that, CAR-1 to CDR-10 CS1 CAR-T cells significantly increased the percentage of lysis of MM. 1s-Luc target cells at different effector : target ratios including 10: 1, 3: 1, and 1: 1.
  • Figure 5B shows that CAR-1 to CDR-10 CS1 CAR-T cells significantly increased the percentage of lysis of NCI-H929 target cells at E: T ratios of 10: 1 and 3: 1.
  • Figure 5C shows that CAR-1 to CDR-10 CS1 CAR-T cells and CAR-R1 to CAR-R3 CS1 CAR-T cells significantly increased the cytotoxicity of RPMI8226-Luc cells at E: T ratios of 10: 1, 3: 1, and 1: 1.
  • anti-beacon antibody was used to distinguish CS1 CAR positive cells from CS1 CAR negative cells. Meanwhile, the expression level of CD25 in positive and negative cells was detected, and the ratio of CD25 positive cells in CS1 CAR-positive and CS1 CAR-negative populations was calculated, as shown in Figure 7.
  • the cells were transfected by lentivirus.
  • CS-1-1, CS-1-2, CS-1-5, CS-1-8, CS-1-9, and CS-1-R1 CAR-T cells the electroporated T were transfected with lentivirus comprising CS1 CAR construct as listed in Table 5.
  • CS1-BCMA, CS1-CD19-BCMA and CS1-CD7 CAR-T cells the electroporated T were transfected with lentivirus comprising CS1-1-1 CAR construct and lentivirus comprising BCMA, CD19-BCMA or CD7 CAR construct, respectively, as listed in Table 5.
  • the transfected T were then transferred to a flask and cultured at 37 °C, 5%CO 2 .3 days after the transfection, the total number of cells, CAR positive cells and knockout efficiency were detected by Flow Cytometry.
  • the knockout efficiency of TRAC, B2M, CD7 or CS1 is as shown in Figure 9A-9E.
  • CS1 single CAR, dual CAR and triple CAR were all successfully expressed on T cells.
  • QKO group referred to the T cell in which TRAC, B2M, CD7 or CS1 were knockout but not transfected by lentivirus.
  • T cell group referred to the T cells not treated by electroporation or transfection.
  • Luciferase gene can be transfected into target cells. After adding fluorescein, luciferase could react with the fluorescein to produce fluorescence. By detecting the intensity of the fluorescence, the activity of luciferase can be measured, and the survival of the target cells can be detected so that to evaluate the cytotoxicity of the CAR-T cells.
  • luciferase gene was transferred into target cells including MM. 1S, RPMI-8226, CCRF-CEM, and Nalm6 cells. Then killing effects of universal CS1 single CAR, dual CAR and triple CAR-T cells on the target cells were analyzed by detecting intensity of the fluorescence, as shown in Figure 10A-10D.
  • the universal CS1 single CAR, dual CAR and triple CAR-T cells significantly increased the percentage of lysis of MM. 1s-Luc target cells at different effector : target ratios including 10: 1, 3: 1, and 1: 1.
  • Figure 10B shows that the universal CS1 single CAR, dual CAR and triple CAR-T cells significantly increased the percentage of lysis of RPMI8226-Luc target cells at an effector : target ratios of 1: 1.
  • Figure 10C shows that the universal CS1+CD7 dual CAR CAR-T cells significantly increased the percentage of lysis of CCRF-CEM-luc target cells at different effector : target ratios including 10: 1, 3: 1, and 1: 1.
  • Figure 10D shows that the universal CS1+CD19+BCMA triple CAR-T cells significantly increased the percentage of lysis of Nalm6-Luc target cells at different effector : target ratios including 10: 1, 3: 1, and 1: 1.
  • CFSE was used to label CAR-T cells.
  • the labeled cells were mixed with allogeneic primary expanded T cells (CTL) or primary allogeneic NK cells at different E: T ratios, and cultured for 16 hours, then centrifuged at 300g for 5 minutes. The cells in pellet were further resuspended by 100 ⁇ l FACS buffer comprising 1: 2000 DAPI.
  • the number of remaining viable NK and CTL cells were analyzed by Flow cytometry, as shown in Figure 11A and 11B. As can be seem from Figure 11A and Figure 11B, the remaining viable CTL and viable NK cells were reduced after incubation with the universal CS1 single CAR, dual CAR and triple CAR-T cells.

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Abstract

La présente divulgation concerne une cellule immunitaire modifiée et une utilisation associée. La cellule immunitaire modifiée comprend un CAR ou un TCR modifié. Les cellules immunitaires modifiées, lorsqu'elles sont administrées à un sujet, peuvent inhiber les cellules immunitaires hôtes telles que les lymphocytes T et/ou les cellules NK et améliorer la survie et la persistance des cellules immunitaires modifiées in vivo, présentant ainsi une activité tumoricide plus efficace.
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WO2022214887A1 (fr) * 2021-04-08 2022-10-13 Phosphogam, Llc Procédés et compositions d'amélioration de la cytotoxicité des lymphocytes t gamma/delta
WO2023093763A1 (fr) * 2021-11-24 2023-06-01 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour les références croisées dans le cadre d'immunothérapies axées sur les cellules

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CN106029875A (zh) * 2014-02-14 2016-10-12 塞勒克提斯公司 为了靶向存在于免疫细胞和病理细胞两者上的抗原而工程化的免疫治疗细胞
CN109153731A (zh) * 2015-08-11 2019-01-04 南京传奇生物科技有限公司 靶向bcma的嵌合抗原受体及其使用方法
CN109562126A (zh) * 2016-06-24 2019-04-02 美商生物细胞基因治疗有限公司 嵌合抗原受体(car)、组合物及其使用方法

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CN106029875A (zh) * 2014-02-14 2016-10-12 塞勒克提斯公司 为了靶向存在于免疫细胞和病理细胞两者上的抗原而工程化的免疫治疗细胞
CN109153731A (zh) * 2015-08-11 2019-01-04 南京传奇生物科技有限公司 靶向bcma的嵌合抗原受体及其使用方法
CN109562126A (zh) * 2016-06-24 2019-04-02 美商生物细胞基因治疗有限公司 嵌合抗原受体(car)、组合物及其使用方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022214887A1 (fr) * 2021-04-08 2022-10-13 Phosphogam, Llc Procédés et compositions d'amélioration de la cytotoxicité des lymphocytes t gamma/delta
WO2023093763A1 (fr) * 2021-11-24 2023-06-01 Hangzhou Qihan Biotechnology Co., Ltd. Systèmes et procédés pour les références croisées dans le cadre d'immunothérapies axées sur les cellules

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