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WO2025162379A1 - Polypeptide and viral vector containing gene encoding polypeptide - Google Patents

Polypeptide and viral vector containing gene encoding polypeptide

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Publication number
WO2025162379A1
WO2025162379A1 PCT/CN2025/075188 CN2025075188W WO2025162379A1 WO 2025162379 A1 WO2025162379 A1 WO 2025162379A1 CN 2025075188 W CN2025075188 W CN 2025075188W WO 2025162379 A1 WO2025162379 A1 WO 2025162379A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
deletion
substitution
viral vector
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/075188
Other languages
French (fr)
Chinese (zh)
Inventor
黄可
李宇航
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Genocury Biotech Co Ltd
Original Assignee
Shenzhen Genocury Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Genocury Biotech Co Ltd filed Critical Shenzhen Genocury Biotech Co Ltd
Publication of WO2025162379A1 publication Critical patent/WO2025162379A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of cell therapy, and in particular to a polypeptide and a viral vector comprising the polypeptide gene.
  • LNPs lipid nanoparticles
  • VLPs virus-like particles
  • adenovirus adeno-associated virus
  • RVV retroviral vector
  • LVV lentiviral vector
  • LVV and RVV are widely used in in vitro and in vivo transduction of T cells to prepare CAR-T cells because they can integrate target genes such as CAR genes into the genome of host cells, allowing the target genes to be stably expressed in host cells.
  • the CAR molecule can be expressed on the cell membrane of the packaging cell under the action of the signal peptide, and buds out from the packaging cell as the packaged LVV or RVV buds out, becoming part of the envelope of the LVV or RVV.
  • the envelope of the LVV or RVV fuses with the host cell membrane, and the CAR molecules contained in its envelope also become part of the host cell membrane. This transfer of CAR molecules between the viral vector envelope and the host cell membrane is called "pseudotransduction".
  • the CAR molecule transferred from the viral envelope to the host cell membrane is only detectable for a short period of time and is then degraded by the host cell shortly thereafter.
  • LVV or RVV can also transduce target cells such as cancer cells through the antigen binding region of the CAR molecule contained in its envelope, seriously affecting the efficacy of CAR-T cell therapy.
  • a polypeptide comprising an antigen-binding region but unlike a CAR molecule, not distributed or distributed in very small amounts on the envelope of LVV or RVV, and an LVV or RVV comprising the polypeptide gene and capable of effectively targeting and stimulating non-activated T cells for activation, are the key to reducing false transduction in traditional in vitro CAR-T cell therapy, reducing the ability of LVV or RVV to transduce target cells such as cancer cells in and outside the patient's body, improving the targeting and transduction efficiency of transduced T cells, and thus improving the efficacy of cell therapy.
  • the present invention provides a viral vector in one aspect
  • the viral vector comprises a polynucleotide encoding a T cell receptor chimeric protein (TCP);
  • the surface of the viral vector contains T cell activation signal molecules
  • the TCP includes:
  • TSP TCR/CD3 complex subunit related peptide
  • the viral vector is a lentiviral vector (LVV) or a retroviral vector (RVV).
  • the TCR/CD3 complex subunit is selected from at least one of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • the TCR/CD3 complex subunit functional fragment comprises at least one of the extracellular region, transmembrane region, intracellular region, variable region and constant region of the TCR/CD3 complex subunit.
  • the TSP comprises CD3 ⁇ or a functional fragment thereof
  • the TSP comprises the transmembrane region and the intracellular region of CD3 ⁇ ;
  • the TSP comprises the transmembrane region and the intracellular region of CD3 ⁇ and the extracellular region of at least one of the following proteins: TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ ;
  • the TSP comprises the transmembrane region and intracellular region of CD3 ⁇ and the extracellular region of (a) CD3 ⁇ , (b) CD3 ⁇ or (c) CD3 ⁇ ;
  • the C-terminus of the extracellular region of CD3 ⁇ , the extracellular region of CD3 ⁇ or the extracellular region of CD3 ⁇ is located towards the N-terminus of the transmembrane region of CD3 ⁇ , and the C-terminus of the transmembrane region of CD3 ⁇ is located towards the N-terminus of the intracellular region of CD3 ⁇ .
  • the TSP comprises CD3 ⁇ or a functional fragment thereof
  • the TSP comprises the transmembrane region and the intracellular region of CD3 ⁇ ;
  • the TSP comprises the transmembrane region and the intracellular region of CD3 ⁇ and the extracellular region of at least one of the following proteins: TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ ;
  • the TSP comprises the transmembrane region and intracellular region of CD3 ⁇ ; and (a) the extracellular region of CD3 ⁇ , (b) the extracellular region of CD3 ⁇ , or (c) the extracellular region of CD3 ⁇ ;
  • the C-terminus of the extracellular region of CD3 ⁇ , the extracellular region of CD3 ⁇ or the extracellular region of CD3 ⁇ is located in the N-terminal direction of the transmembrane region of CD3 ⁇ , and the C-terminus of the transmembrane region of CD3 ⁇ is located in the N-terminal direction of the intracellular region of CD3 ⁇ .
  • the TSP comprises CD3 ⁇ or a functional fragment thereof
  • the TSP comprises the transmembrane region and the intracellular region of CD3 ⁇ ;
  • the TSP comprises the transmembrane region and the intracellular region of CD3 ⁇ and the extracellular region of at least one of the following proteins: TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ ;
  • the TSP comprises the transmembrane region and intracellular region of CD3 ⁇ ; and (a) the extracellular region of CD3 ⁇ , (b) the extracellular region of CD3 ⁇ , or (c) the extracellular region of CD3 ⁇ ;
  • the C-terminus of the extracellular region of CD3 ⁇ , CD3 ⁇ or CD3 ⁇ is located towards the N-terminus of the transmembrane region of CD3 ⁇ , and the C-terminus of the transmembrane region of CD3 ⁇ is located towards the N-terminus of the intracellular region of CD3 ⁇ .
  • the TSP comprises CD3 ⁇ or a functional fragment thereof
  • the TSP comprises the transmembrane region and the intracellular region of CD3 ⁇ ;
  • the TSP comprises the transmembrane region and the intracellular region of CD3 ⁇ and the extracellular region of at least one of the following proteins: TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ ;
  • the TSP comprises the transmembrane region and intracellular region of CD3 ⁇ ; and (a) the extracellular region of CD3 ⁇ , (b) the extracellular region of CD3 ⁇ , or (c) the extracellular region of CD3 ⁇ ;
  • the C-terminus of the extracellular region of CD3 ⁇ , CD3 ⁇ or CD3 ⁇ is located in the direction of the N-terminus of the transmembrane region of CD3 ⁇ , and the C-terminus of the transmembrane region of CD3 ⁇ is located in the direction of the N-terminus of the intracellular region of CD3 ⁇ .
  • the TSP comprises TCR ⁇ or a functional fragment thereof and TCR ⁇ or a functional fragment thereof;
  • the TSP comprises the constant region of TCR ⁇ and the constant region of TCR ⁇ .
  • the constant region of the TCR ⁇ is mutated, and the mutation includes a cysteine substitution in the TCR ⁇ constant region; the mutation can enhance disulfide bond-based interchain interactions;
  • the TCR ⁇ constant region is derived from a human or mouse TCR ⁇ constant region, the human TCR ⁇ constant region comprises the amino acid sequence shown in SEQ ID NO: 39, and the mouse TCR ⁇ constant region comprises the amino acid sequence shown in SEQ ID NO: 40;
  • the mutation includes replacing the 47th amino acid Threonine T of the human TCR ⁇ constant region with Cysteine C (human TCR ⁇ constant region variant 1) or replacing the 47th amino acid Threonine T of the mouse TCR ⁇ constant region with Cysteine C (mouse TCR ⁇ constant region variant 1);
  • the human TCR ⁇ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:41
  • the mouse TCR ⁇ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:42.
  • the TCR ⁇ constant region is mutated, and the mutation includes a cysteine substitution in the TCR ⁇ constant region; the mutation can enhance disulfide bond-based interchain interactions;
  • the TCR ⁇ constant region is derived from a human TCR ⁇ constant region, and the human TCR ⁇ constant region comprises the amino acid sequence shown in SEQ ID NO: 43 (hTRBC1) or SEQ ID NO: 44 (hTRBC2), and the mutation includes replacing the 56th amino acid serine S of the human TCR ⁇ constant region with cysteine C (human TCR ⁇ constant region variant 1), and the human TCR ⁇ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
  • the TCR ⁇ constant region undergoes a mutation, wherein the mutation comprises replacing at least one uncharged amino acid in the TCR ⁇ constant region with a hydrophobic amino acid; the mutation increases the hydrophobicity of the TCR ⁇ transmembrane region, offsetting the instability caused by the positive charge carried by the TCR ⁇ transmembrane region, thereby enabling the TCR ⁇ and its dimer formed with TCR ⁇ to be more stably expressed on the T cell membrane, thereby achieving better function;
  • the TCR ⁇ constant region is derived from a human TCR ⁇ constant region, the human TCR ⁇ constant region comprising the amino acid sequence as shown in SEQ ID NO: 39, and the mutation comprises replacement of at least one of amino acids 115, 118, and 119 of the human TCR ⁇ constant region with a hydrophobic amino acid;
  • the mutations include at least one of the following mutations in the human TCR ⁇ constant region: substitution of amino acid serine S at position 115 with leucine L, substitution of amino acid glycine G at position 118 with valine V, substitution of amino acid phenylalanine F at position 119 with leucine L;
  • the mutation includes replacing the 115th amino acid serine S of the human TCR ⁇ constant region with leucine L, replacing the 118th amino acid glycine G with valine V, and replacing the 119th amino acid phenylalanine F with leucine L (human TCR ⁇ constant region variant 2), and the human TCR ⁇ constant region variant 2 comprises the amino acid sequence shown in SEQ ID NO:47.
  • the TCR ⁇ constant region is derived from a human TCR ⁇ constant region, the human TCR ⁇ constant region comprising the amino acid sequence set forth in SEQ ID NO: 39; the human TCR ⁇ constant region undergoes a mutation, the mutation comprising substitution of amino acid threonine T at position 47 of the human TCR ⁇ constant region with cysteine C, amino acid serine S at position 115 with leucine L, amino acid glycine G at position 118 with valine V, and amino acid phenylalanine F at position 119 with leucine L (human TCR ⁇ constant region variant 3), the human TCR ⁇ constant region variant 3 comprising the amino acid sequence set forth in SEQ ID NO: 48;
  • the TCR ⁇ constant region and the TCR ⁇ constant region are derived from human TCR ⁇ constant region and human TCR ⁇ constant region;
  • the human TCR ⁇ constant region comprises the amino acid sequence shown in SEQ ID NO:39, and the human TCR ⁇ constant region comprises the amino acid sequence shown in SEQ ID NO:43 or SEQ ID NO:44;
  • the human TCR ⁇ constant region and the human TCR ⁇ constant region are mutated, and the mutation includes the replacement of the 47th amino acid of the human TCR ⁇ constant region with cysteine and the 115th amino acid serine S
  • the human TCR ⁇ constant region variant 3 comprises the amino acid sequence shown in SEQ ID NO: 48
  • the human TCR ⁇ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.
  • any of the aforementioned TSPs may further comprise the hinge region of the TCR/CD3 complex subunit.
  • the antigen binding region is operably linked to the N-terminus of any of the aforementioned TSPs via a linker peptide.
  • the connecting peptide includes a flexible connecting peptide.
  • the flexible connecting peptide is the connecting peptide 1, the (G 4 S) 3 connecting peptide (connecting peptide 2) or the connecting peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 63).
  • the flexible connecting peptide is the connecting peptide 1.
  • the antigen binding region binds to a disease-associated antigen.
  • the disease is selected from cancer and autoimmune diseases; and the cancer includes blood cancer and solid cancer.
  • the disease-associated antigen is selected from:
  • TSHR CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38 , CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138 , DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3 , tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR
  • the disease-associated antigen is selected from at least one of CD19, CD20, CD33, MSLN, CD79B, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2 and GUCY2C.
  • the disease-associated antigen is a human disease-associated antigen.
  • the antigen-binding region comprises an antibody or an antigen-binding fragment thereof and/or a ligand or a receptor-binding fragment thereof, and the antibody or antigen-binding fragment thereof is selected from at least one of an immunoglobulin (full-length antibody), a half antibody, Fab, Fab', F(ab') 2 , an Fv fragment, a single-chain variable region fragment (scFv), a disulfide-stabilized antibody (dsFv), an antibody heavy chain variable region (VH) or a light chain variable region (VL), an Fd fragment consisting of a VH and a CH1 domain, a linear antibody, and a single-domain antibody (nanoantibody).
  • an immunoglobulin full-length antibody
  • Fab fragment fragment
  • Fab' fragment fragment
  • F(ab') 2 an Fv fragment
  • scFv single-chain variable region fragment
  • dsFv disulfide-stabilized antibody
  • VH antibody heavy chain variable
  • any of the aforementioned TCR ⁇ constant regions or variants thereof can be operably linked to the VH region or the VL region; any of the aforementioned TCR ⁇ constant regions or variants thereof can be operably linked to the VH region or the VL region.
  • any of the aforementioned TCR ⁇ constant regions or variants thereof when any of the aforementioned TCR ⁇ constant regions or variants thereof are operably linked to the VH region, any of the aforementioned TCR ⁇ constant regions or variants thereof are operably linked to the VL region; when any of the aforementioned TCR ⁇ constant regions or variants thereof are operably linked to the VL region, any of the aforementioned TCR ⁇ constant regions or variants thereof are operably linked to the VH region.
  • the VH region and VL region are derived from the same antibody or antigen-binding fragment thereof, or ligand or receptor-binding fragment thereof.
  • the antigen binding region is selected from at least one of the following antigen binding regions:
  • an antigen-binding region that binds to human CD19 wherein the antigen-binding region is a scFv derived from FMC-63 (FMC63-scFv), the amino acid sequence of the VH region of the FMC63-scFv being as shown in SEQ ID NO: 18, and the amino acid sequence of the VL region of the FMC63-scFv being as shown in SEQ ID NO: 19;
  • an antigen-binding region that binds to human asialoglycoprotein receptor ASGPR
  • ASGPR asialoglycoprotein receptor
  • antigen-binding region is an anti-human ASGPR scFv (anti-ASGPR-scFv)
  • amino acid sequence of the VL region of the anti-ASGPR-scFv is shown in SEQ ID NO: 51
  • amino acid sequence of the anti-ASGPR-scFv is shown in SEQ ID NO: 52;
  • an antigen-binding region that binds to human CD79B wherein the antigen-binding region is derived from the scFv (SN8-scFv) of the anti-human CD79B antibody SN-8; the amino acid sequence of the VH region of the anti-SN8-scFv is shown in SEQ ID NO:61; the amino acid sequence of the VL region of the SN8-scFv is shown in SEQ ID NO:62.
  • any of the aforementioned T cell receptor chimeric proteins does not comprise a signal peptide.
  • any of the aforementioned T cell receptor chimeric proteins comprises a signal peptide.
  • the signal peptide is not particularly limited as long as it can mediate the membrane expression of TCP.
  • the signal peptide is selected from the following signal peptides: CD8 ⁇ signal peptide, CD28 signal peptide, IgG signal peptide, HLA-A signal peptide, CD3 ⁇ signal peptide, CD3 ⁇ signal peptide, CD3 ⁇ signal peptide and CD3 ⁇ signal peptide.
  • the signal peptide is a human CD8 ⁇ signal peptide.
  • the amino acid sequence of the human CD8 ⁇ signal peptide is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:12.
  • the T cell receptor chimeric protein further comprises a co-stimulatory signaling domain.
  • the costimulatory signaling domain is derived from the costimulatory signaling domain of at least one of the following proteins:
  • the costimulatory signaling domain is derived from the costimulatory signaling domain of human 4-1BB and/or CD28.
  • the amino acid sequence of the costimulatory signaling domain of human 4-1BB is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49.
  • any of the aforementioned T cell receptor chimeric proteins when expressed in T cells, (a) is incorporated into an endogenous TCR/CD3 complex or a TCR/CD3 complex subunit or a functional fragment thereof; or (b) functionally interacts with an endogenous TCR/CD3 complex or an endogenous TCR/CD3 complex subunit or a functional fragment thereof.
  • any of the aforementioned TCPs is not expressed on the membrane in cells other than T cells, or the efficiency of membrane expression of the TCP on cells other than T cells is lower than the efficiency of membrane expression of the TCP on T cells.
  • the T cell activation signaling molecule includes a T cell activation primary signaling molecule.
  • Non-activated T cells are T cells that are not proliferated, differentiated, in a resting state, do not recognize antigens, and have not been activated by T cell activation signaling molecules such as primary and secondary T cell activation signaling molecules, such as T cells in the G0 phase of the cell cycle, resting/quiescent T cells, or immature T cells.
  • T cell activation signaling molecules such as primary and secondary T cell activation signaling molecules, such as T cells in the G0 phase of the cell cycle, resting/quiescent T cells, or immature T cells.
  • Resting T cells also known as quiescent T cells or naive T cells, are T cells that are not mitotically active or have not been exposed to cognate antigens presented on antigen-presenting cells, such as macrophages or dendritic cells.
  • T cell activation primary signal molecules bind to T cell surface proteins and participate in T cell receptor (T Cell Receptor, "TCR”)-mediated T cell activation (TCR-mediated T Cell Activation).
  • TCR T Cell Receptor
  • the T cell activation primary signal molecule is involved in converting TCR into active PTK (protein tyrosine kinase), which can phosphorylate a series of substrates to generate a large number of downstream signals.
  • PTK protein tyrosine kinase
  • the T cell activation primary signal molecule binds to at least one of a TCR/CD3 complex subunit and a functional fragment of a TCR/CD3 complex subunit; the TCR/CD3 complex subunit is selected from at least one of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ and TCR ⁇ .
  • the TCR/CD3 complex subunit is a human TCR/CD3 complex subunit.
  • CD3 and TCR form a TCR/CD3 complex in T cells, which participates in the activation of helper T cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells).
  • the T cell activation primary signal molecule comprises an anti-CD3 antibody or an antigen-binding fragment thereof.
  • the anti-CD3 antibody or antigen-binding fragment thereof specifically binds to human CD3.
  • the anti-CD3 antibody is selected from at least one of OKT3, UCHT1, YTH12.5 and TR66.
  • the anti-CD3 antibody is SP34.
  • the anti-CD3 antibody or antigen-binding fragment thereof cannot bind to the TSP.
  • the anti-CD3 antibody is an anti-CD3 ⁇ antibody
  • the TSP is not CD3 ⁇ , or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.
  • the anti-CD3 ⁇ antibody or its antigen-binding fragment specifically binds to human CD3 ⁇ (Uniprot ID: P07766).
  • the anti-CD3 ⁇ antibody or antigen-binding fragment thereof is UCHT1 or an antigen-binding fragment thereof;
  • the antigen-binding fragment of UCHT1 is a scFv (UCHT1-scFv);
  • amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NO: 87-89, respectively, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NO: 90-92, respectively.
  • the anti-CD3 ⁇ antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof;
  • the antigen-binding fragment of OKT3 is scFv (OKT3-scFv);
  • amino acid sequences of the HCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NOs: 104-106, respectively, and the amino acid sequences of the LCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NOs: 107-109, respectively;
  • the amino acid sequence of the VH region of the OKT3-scFv is shown in SEQ ID NO: 119, and the amino acid sequence of the VL region of the OKT3-scFv is shown in SEQ ID NO: 120.
  • the anti-CD3 ⁇ antibody or antigen-binding fragment thereof is SP34 or an antigen-binding fragment thereof;
  • the antigen-binding fragment of SP34 is scFv (SP34-scFv);
  • amino acid sequences of the HCDR1-3 regions of the SP34-scFv are shown as SEQ ID NOs: 113-115, respectively, and the amino acid sequences of the LCDR1-3 regions of the SP34-scFv are shown as SEQ ID NOs: 116-118, respectively;
  • amino acid sequence of the VH region of the SP34-scFv is shown in SEQ ID NO: 111
  • amino acid sequence of the VL region of the SP34-scFv is shown in SEQ ID NO: 112.
  • the TSP is:
  • CD3 ⁇ or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;
  • the anti-CD3 antibody is an anti-CD3 ⁇ antibody
  • the TSP is not CD3 ⁇ , or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.
  • the TSP is:
  • CD3 ⁇ or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;
  • the anti-CD3 antibody is an anti-CD3 ⁇ antibody
  • the TSP is not CD3 ⁇ , or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.
  • the anti-CD3 ⁇ antibody or antigen-binding fragment thereof is TR66 or an antigen-binding fragment thereof;
  • the antigen-binding fragment of TR66 is scFv (TR66-scFv).
  • the TSP is:
  • CD3 ⁇ or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;
  • the anti-CD3 antibody is an anti-CD3 ⁇ antibody
  • the TSP is not CD3 ⁇ , or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.
  • the anti-CD3 ⁇ antibody or antigen-binding fragment thereof is YTH12.5 or an antigen-binding fragment thereof;
  • the antigen-binding fragment of YTH12.5 is scFv (YTH12.5-scFv).
  • the TSP is:
  • CD3 ⁇ or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;
  • the T cell activation primary signal molecule includes at least one of an anti-TCR ⁇ antibody or an antigen-binding fragment thereof, an anti-TCR ⁇ antibody or an antigen-binding fragment thereof, an anti-TCR ⁇ antibody or an antigen-binding fragment thereof, and an anti-TCR ⁇ antibody or an antigen-binding fragment thereof.
  • the TSP when the T cell activation primary signal molecule comprises an anti-TCR ⁇ antibody or an antigen-binding fragment thereof, the TSP is not TCR ⁇ , or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.
  • the TSP when the T cell activation primary signal molecule comprises an anti-TCR ⁇ antibody or an antigen-binding fragment thereof, the TSP is not TCR ⁇ , or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.
  • the TSP when the T cell activation primary signal molecule comprises an anti-TCR ⁇ antibody or an antigen-binding fragment thereof, the TSP is not TCR ⁇ , or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.
  • the TSP when the T cell activation primary signal molecule comprises an anti-TCR ⁇ antibody or an antigen-binding fragment thereof, the TSP is not TCR ⁇ , or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.
  • the T cell activation signal molecule further includes a T cell activation secondary signal molecule.
  • T cell activation secondary signal molecules also known as co-stimulatory signal molecules (Co-Stimulatory Signal)
  • Co-Stimulatory Signal bind to other T cell surface receptors to provide additional signals necessary to avoid anergy and effective T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).
  • the T cell activation secondary signaling molecule binds to CD28.
  • the CD28 is human CD28 (Uniprot ID: P10747).
  • CD28-mediated co-stimulation is stronger than other co-stimulatory receptors (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).
  • the T cell activation secondary signal molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof.
  • the CD28 ligand or its receptor binding fragment includes CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.
  • the CD80 and CD86 are human CD80 and CD86.
  • the amino acid sequence of human CD80 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:85.
  • the amino acid sequence of human CD86 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:86.
  • the T cell activation secondary signaling molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof;
  • the anti-CD28 antibody or antigen-binding fragment thereof is a scFv derived from 15E8 (15E8-scFv), and the amino acid sequence of the 15E8-scFv is shown in SEQ ID NO: 17; the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO: 93-95, respectively, and the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO: 96-98, respectively.
  • the anti-CD28 antibody or antigen-binding fragment thereof is selected from at least one of CD28.2, 10F3 and TGN1412.
  • the T cell activation secondary signal molecule while comprising at least one of an anti-CD28 antibody or an antigen-binding fragment thereof that can bind to CD28 and a CD28 ligand or a receptor-binding fragment thereof, also includes at least one ligand or receptor-binding fragment selected from ICOS (inducible costimulator, "ICOS") ligand (ICOSL) or a receptor-binding fragment thereof, 4-1BB ligand (4-1BBL) or a receptor-binding fragment thereof, and OX40 ligand (OX40L) or a receptor-binding fragment thereof.
  • ICOS inducible costimulator, "ICOS”
  • ICOS is inducibly expressed on activated T cells (Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, et al. ICOS is an inducible T-cell costimulator structurally and functionally related to CD28. Nature 1999; 397: 263-6. [PubMed: 9930702])(Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).
  • TNFR family members OX40 (CD134) and 4-1BB (CD137) provide co-stimulatory signals by binding to their ligands OX40L and 4-1BBL (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).
  • the T cell activation secondary signaling molecule can also bind to at least one of CD27, HVEM, LIGHT, CD40, DR3, GITR, CD30, TIM1, SLAM, CD2 and CD226.
  • the T cell activation secondary signaling molecule may also be selected from at least one of B7-H2, CD70, LIGHT, HVEM, CD40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155 and CD112.
  • the T cell activation signal molecule is directly or indirectly connected to a transmembrane polypeptide and displayed on the surface of the viral vector.
  • the transmembrane polypeptide is selected from the transmembrane regions of the following proteins:
  • the transmembrane polypeptide is the CD8 ⁇ transmembrane region.
  • the transmembrane polypeptide is the human CD8 ⁇ transmembrane region.
  • the human CD8 ⁇ transmembrane region has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:15.
  • the T cell activation signaling molecule is indirectly linked to the transmembrane polypeptide via a linker domain and is displayed on the surface of the viral vector;
  • the linker domain is selected from:
  • an immunoglobulin hinge region wherein the immunoglobulin hinge region is selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge region;
  • the connecting domain is the CD8 ⁇ hinge region.
  • the connecting domain is the human CD8 ⁇ hinge region.
  • the human CD8 ⁇ hinge region is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:14.
  • the surface of the viral vector comprises a glycoprotein
  • the glycoprotein is selected from the envelope glycoprotein of the vesicular stomatitis virus strain and its variants, the envelope glycoprotein of the baboon endogenous retrovirus BaEV and its variants, the envelope glycoprotein RD114 of the feline endogenous retrovirus and its variants, and the envelope glycoprotein GALV of the gibbon ape leukemia virus and its variants.
  • the transmembrane polypeptide is the glycoprotein, and the glycoprotein is directly or indirectly linked to the T cell activation signaling molecule;
  • the glycoprotein is indirectly connected to the T cell activation signal molecule through a polypeptide linker.
  • the T cell activation primary signaling molecule is directly or indirectly linked to the T cell activation secondary signaling molecule
  • the T cell activation primary signal molecule is indirectly linked to the T cell activation secondary signal molecule via a polypeptide linker.
  • the polypeptide linker is a flexible connecting peptide
  • the glycoprotein is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a ( G4S ) n linker peptide, and the anti-CD3 antibody or antigen-binding fragment thereof is indirectly linked to the anti - CD28 antibody or antigen-binding fragment thereof, (ii) the CD80 extracellular domain, (iii) or the CD86 extracellular domain via a (G4S)n linker peptide; and/or
  • the glycoprotein is indirectly linked to (i) the anti-CD28 antibody or antigen-binding fragment thereof, (ii) the CD80 extracellular domain, (iii) the CD86 extracellular domain via a ( G4S ) n connecting peptide; the anti-CD28 antibody or antigen-binding fragment thereof, the CD80 extracellular domain, or the CD86 extracellular domain is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a ( G4S ) n connecting peptide;
  • n 3;
  • the anti-CD3 antibody or antigen-binding fragment thereof is the UCHT1-scFv;
  • the anti-CD28 antibody or antigen-binding fragment thereof is the 15E8-scFv.
  • the glycoprotein is selected from at least one of the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof.
  • the envelope glycoprotein and variants thereof of the vesicular stomatitis virus strain include the following envelope glycoproteins and variants thereof: envelope glycoprotein and variants of the Indiana strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Cocal strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Maraba strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Morreton strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Alagoas strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the New Jersey strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Carajas strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Chandipura strain of the ve
  • VSV-G Vesicular Stomatitis Virus strains
  • LDL-R low-density lipoprotein receptor
  • the extracellular domain of the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus contains the amino acid sequence shown in SEQ ID NO:1; the extracellular domain of the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus contains the amino acid sequence shown in SEQ ID NO:2.
  • amino acid sequence of the full-length protein of wild-type VSV-G (including the VSV-G signal peptide) is shown in SEQ ID NO: 24;
  • MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of the wild-type VSV-G.
  • the amino acid sequence of the full-length wild-type Cocal-G protein (including the Cocal-G signal peptide) is shown in SEQ ID NO: 99;
  • MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of the wild-type Cocal-G.
  • Activated T cells express LDL-R; therefore, artificially synthesized, biosafe LVV or RVV usually uses wild-type VSV-G to construct its envelope glycoprotein (VSV-G type LVV or RVV) to transduce activated T cells.
  • VSV-G type LVV or RVV envelope glycoprotein
  • the glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus or a variant thereof;
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • any of the aforementioned glycoproteins undergoes a first mutation, which reduces or loses the ability of the glycoprotein to bind to a glycoprotein receptor relative to before the first mutation occurs.
  • LDL-R is widely expressed on the surface of multiple cells, such as activated T cells, hepatocytes, cardiomyocytes, and endothelial cells. Therefore, VSV-G type LVV or RVV can also transduce other cells by binding to LDL-R, but the targeting of transduced T cells is relatively low.
  • VSV-G By weakening the ability of VSV-G to bind to LDL-R and at the same time making its envelope contain primary and secondary signal molecules for T cell activation such as anti-CD3 antibodies and anti-CD28 antibodies, the ability of VSV-G type LVV or RVV to target, activate and transduce T cells can be effectively improved.
  • the glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus, or a variant thereof;
  • the glycoprotein receptor is the low-density lipoprotein receptor (LDL-R); the glycoprotein undergoes a first mutation, such that the ability of the glycoprotein to bind to the LDL-R is reduced or lost relative to before the first mutation occurs;
  • LDL-R low-density lipoprotein receptor
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:
  • substitution or deletion of amino acid at position 8 substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 209 of SEQ ID NO: 1 or SEQ ID NO: 2.
  • substitution or deletion of amino acid 350 substitution or deletion of amino acid 352, substitution or deletion of amino acid 353, substitution of amino acid 354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; and
  • substitution or deletion of amino acid at position 8 substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 185, substitution or deletion of amino acid at position 186, substitution or deletion of amino acid at position 187, substitution or deletion of amino acid at position 188, substitution or deletion of amino acid at position 189, substitution or deletion of amino acid at position 190, substitution or deletion of amino acid at position 191, substitution or deletion of amino acid at position 192, substitution or deletion of amino acid at position 193, substitution or deletion of amino acid at position 194, substitution or deletion of amino acid at position 195, substitution or deletion of amino acid at position 195, substitution or deletion of amino acid at position 195, substitution or deletion of
  • the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:
  • the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:
  • the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:
  • deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, and substitution of R354 are located at positions equivalent to SEQ ID NO: 1 or SEQ ID NO: 2;
  • the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:
  • the first mutation includes a mutation in which the amino acid sequence comprises the following amino acids:
  • the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:
  • the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:3; relative to SEQ ID NO:1, SEQ ID NO:3 comprises a K47 deletion.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:4; relative to SEQ ID NO:1, SEQ ID NO:4 comprises R354Q.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:33 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:33; relative to SEQ ID NO:2, SEQ ID NO:33 comprises a K47 deletion.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:34 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:34; relative to SEQ ID NO:2, SEQ ID NO:34 comprises R354Q.
  • the glycoprotein having any of the aforementioned first mutations still retains the ability to fuse membranes and escape from endosomal/lysosomal regions.
  • the glycoprotein further undergoes a second mutation, which enhances the ability of the glycoprotein to antagonize inactivation by complement, or prevents inactivation by complement, compared to before the second mutation.
  • the glycoprotein having the second mutation is any of the aforementioned glycoproteins.
  • the complement system is composed of a series of proteins and is part of the innate immune system.
  • Complement (C) is present in the serum, tissue fluid, and cell membrane surfaces of normal humans and animals. Once activated, it possesses enzymatic activity and can undergo a complex cascade reaction. The complement system is initiated through a series of enzymes that cut each other, ultimately forming a membrane attack complex that resembles a hole on the target microorganism, causing the microorganism to rupture and die.
  • Complement components can be activated by antigen-antibody complexes or antibodies, and clear immune complexes through lysis, opsonization, phagocytosis, and mediation of inflammatory responses, demonstrating corresponding biological functions. Complement is widely involved in the body's defense response against microbial infection and immune regulation, and also mediates immunopathological damage responses. It is an effector system and effector method system with important biological functions in the body.
  • the regulatory complement components exist in soluble or membrane-bound forms, including properdin (P factor), C1 inhibitor (C1INH), factor I, factor H, C4 binding protein (C4BP), S protein, SP40/40, membrane cofactor protein (MCP), decay accelerating factor (DAF), homologous restriction factor (HRF) and membrane inhibitor of reactive lysis (MIRL).
  • P factor properdin
  • C1INH C1 inhibitor
  • C4BP C4 binding protein
  • S protein S protein
  • SP40/40 membrane cofactor protein
  • MCP membrane cofactor protein
  • DAF decay accelerating factor
  • HRF homologous restriction factor
  • MIRL membrane inhibitor of reactive lysis
  • pseudotyped LVV or RVV After entering the serum, pseudotyped LVV or RVV may be recognized and inactivated by complement, making it difficult to efficiently reach target cells and exert their effects; therefore, when used in vivo to prepare engineered T cells such as CAR-T or TCP-T cells, the efficiency of pseudotyped LVV or RVV in transducing non-activated T cells is low.
  • VSV-G By causing the second mutation in viral glycoproteins such as VSV-G, the ability of viral glycoproteins such as VSV-G to antagonize complement inactivation is improved, thereby making pseudotyped LVV or RVV more suitable for use in the in vivo preparation of engineered T cells such as CAR-T cells or TCP-T cells.
  • the glycoprotein that undergoes the second mutation is the envelope glycoprotein of the Indiana strain or Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the second mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:
  • the amino acid mutation includes at least one of amino acid deletion, insertion and substitution.
  • the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids:
  • the second mutation comprises an amino acid sequence as set forth in SEQ ID NO: 1 or at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO: 1, comprising at least one of the following position mutations:
  • the second mutation includes at least one of the following site mutations in the amino acid sequence:
  • T214N, T352A, K50T, and S146T are located equivalent to SEQ ID NO:1.
  • the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:
  • the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:
  • the second mutation comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the amino acid sequence as set forth in SEQ ID NO: 2, comprising at least one of the following position mutations:
  • the second mutation includes at least one of the following site mutations in the amino acid sequence:
  • the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:
  • the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:
  • the glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus or a variant thereof;
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO:1;
  • the glycoprotein undergoes any of the aforementioned first mutations, so that the ability of the glycoprotein to bind to LDL-R is reduced or lost relative to before the first mutation; the glycoprotein may also undergo any of the aforementioned second mutations, so that the ability of the glycoprotein to antagonize inactivation by complement is enhanced relative to before the second mutation, or is not inactivated by complement.
  • the glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus or a variant thereof;
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as set forth in SEQ ID NO:2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO:2;
  • the glycoprotein undergoes any of the aforementioned first mutations, so that the ability of the glycoprotein to bind to LDL-R is reduced or lost relative to before the first mutation; the glycoprotein may also undergo any of the aforementioned second mutations, so that the ability of the glycoprotein to antagonize inactivation by complement is enhanced relative to before the second mutation, or is not inactivated by complement.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 38.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:8; relative to SEQ ID NO:1, SEQ ID NO:8 comprises K47 deletion, T214N and T352A.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:9; relative to SEQ ID NO:1, SEQ ID NO:9 comprises K47 deletion, T214N, T352A, K50T and S146T.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 10; relative to SEQ ID NO: 1, SEQ ID NO: 10 comprises R354Q, T214N and T352A.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 11; relative to SEQ ID NO: 1, SEQ ID NO: 11 comprises R354Q, T214N, T352A, K50T and S146T.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:35 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:35; relative to SEQ ID NO:2, SEQ ID NO:35 comprises K47 deletion, K214N, T352A, K50T and S146T.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:36 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:36; relative to SEQ ID NO:2, SEQ ID NO:36 comprises K47 deletion, K214N and T352A.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:37 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:37; relative to SEQ ID NO:2, SEQ ID NO:37 comprises R354Q, K214N, T352A, K50T and S146T.
  • the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:38 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:38; relative to SEQ ID NO:2, SEQ ID NO:38 comprises R354Q, K214N and T352A.
  • the glycoprotein having any of the aforementioned second mutations still retains the ability to fuse membranes and escape from endosomal/lysosomal regions.
  • the glycoprotein having any of the aforementioned first and second mutations still retains the ability to fuse with membranes and escape from endosomal/lysosomes.
  • the efficiency of the T cell receptor chimeric protein in T cell extracellular expression is higher.
  • the TCP-T cells prepared after any of the aforementioned viral vectors contact with non-activated T cells have a higher killing efficiency.
  • control carrier 1 comprises at least one T cell targeting molecule on its surface
  • the T cell targeting molecule binds to CD5 or CD7;
  • the T cell targeting molecule binds to CD7;
  • the T cell targeting molecule is selected from at least one of an anti-CD7 antibody or an antigen-binding fragment thereof and a CD7 ligand or a receptor-binding fragment thereof; optionally, the anti-CD7 antibody or an antigen-binding fragment thereof is a scFv (TH69-scFv) derived from the monoclonal antibody TH-69; the amino acid sequence of the TH69-scFv is as shown in SEQ ID NO: 71; the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO: 72-74, respectively, and the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO: 75-77, respectively.
  • scFv derived from the monoclonal antibody TH-69
  • the amino acid sequence of the TH69-scFv is as shown in SEQ ID NO: 71
  • any of the aforementioned control vectors 1 is a vector having the same structure and/or characteristics as any of the aforementioned viral vectors except that it does not contain the protein encoding any of the aforementioned T cell activation signaling molecules.
  • the surface of any of the aforementioned viral vectors does not contain or contains only a small amount of the antigen binding region; the "small amount" is relative to the control vector 2 containing a polynucleotide encoding other polypeptides; the other polypeptides contain any of the aforementioned antigen binding regions but do not contain any of the aforementioned TSPs.
  • the viral vector does not undergo pseudo-transduction or pseudo-transduction is reduced compared to the control vector 2 comprising a polynucleotide encoding other polypeptides; the other polypeptide comprises any of the aforementioned antigen binding regions but does not comprise any of the aforementioned TSPs.
  • any of the aforementioned viral vectors (a) has a reduced ability to bind to target cell surface antigens or does not bind to target cell surface antigens; and/or (b) has a reduced ability to transduce target cells or does not transduce target cells; the target cell surface antigen can bind to the antigen binding region, and the other polypeptide comprises any of the aforementioned antigen binding regions but does not comprise any of the aforementioned TSPs.
  • the other polypeptide is linked to a signal peptide.
  • the other polypeptide comprises a chimeric antigen receptor, wherein the N-terminus of the chimeric antigen receptor is operably linked to the C-terminus of the signal peptide.
  • the signal peptide is selected from any one of the aforementioned signal peptides.
  • the chimeric antigen receptor is selected from at least one of first-generation, second-generation, third-generation and fourth-generation chimeric antigen receptors.
  • the structure of the chimeric antigen receptor from N-terminus to C-terminus includes, in sequence, an extracellular antigen binding region, a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signaling domain.
  • the target cell is selected from at least one of cancer cells and autoimmune disease-related cells.
  • the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Sjögren's syndrome, myasthenia gravis, celiac disease, type 1 diabetes, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis and scleroderma.
  • the target cells are cancer cells, including solid cancer cells and blood cancer cells.
  • the blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, high-grade B-cell lymphoma and multiple myeloma.
  • the cancer cells are blood cancer cells
  • the blood cancer is a CD19 + blood cancer
  • the CD19 + blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin's lymphoma and high-grade B-cell lymphoma.
  • the cancer cell is a blood cancer cell, and the blood cancer is selected from CD19 + blood cancer and CD33 + blood cancer;
  • the CD19 + blood cancer is selected from the group consisting of marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, and high-grade B-cell lymphoma;
  • the CD33 + blood cancer is selected from the group consisting of multiple myeloma (MM), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and acute monocytic leukemia (AMoL).
  • MM multiple myeloma
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • AMoL acute monocytic leukemia
  • the present invention further provides a TCP, which is any of the aforementioned TCPs provided by the present invention.
  • the present invention further provides a polynucleotide encoding any of the aforementioned TCPs provided by the present invention.
  • the polynucleotide is isolated.
  • the present invention also provides a method for transducing T cells, comprising contacting any one of the aforementioned viral vectors provided by the present invention with T cells.
  • the T cells are selected from at least one of activated T cells and non-activated T cells.
  • the contact occurs in vivo and/or in vitro in a subject; the subject is an individual who is administered T cells transduced by the method for transducing T cells and/or any of the aforementioned viral vectors provided by the present invention.
  • the present invention also provides an engineered T cell, which comprises the polynucleotide encoding any one of the aforementioned TCP molecules provided by the present invention and/or expresses any one of the aforementioned TCP molecules.
  • the engineered T cells are prepared by contacting T cells with any of the aforementioned viral vectors provided by the present invention.
  • the T cells are selected from at least one of activated T cells and non-activated T cells.
  • the contacting occurs in vivo and/or in vitro in a subject, and the subject is an individual who is administered the engineered T cells and/or any of the aforementioned viral vectors provided by the present invention.
  • the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implantation device administration.
  • the present invention also provides a composition comprising a pharmaceutically acceptable excipient or carrier and (a) any one of the aforementioned viral vectors provided by the present invention or (b) any one of the aforementioned engineered T cells.
  • the present invention also provides the use of any of the aforementioned TCPs, polynucleotides, viral vectors, engineered T cells or compositions provided by the present invention in the preparation of a drug for preventing and/or treating cancer.
  • the present invention also provides a method for treating cancer in a subject or killing cancer cells in a subject, comprising administering to the subject any one of the aforementioned viral vectors, engineered T cells or compositions provided by the present invention.
  • the cancer includes solid cancer and blood cancer.
  • the cancer is a blood cancer.
  • the blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, high-grade B-cell lymphoma and multiple myeloma.
  • the blood cancer is a CD19 + blood cancer.
  • the CD19 + blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma and high-grade B-cell lymphoma.
  • the blood cancer is selected from CD19 + blood cancer and CD33 + blood cancer;
  • the CD19 + blood cancer is selected from the group consisting of marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, and high-grade B-cell lymphoma;
  • the CD33 + blood cancer is selected from the group consisting of multiple myeloma (MM), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and acute monocytic leukemia (AMoL).
  • MM multiple myeloma
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • AMoL acute monocytic leukemia
  • the cancer cells express at least one antigen selected from CD19, CD20, CD33, MSLN, CD79B, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2 and GUCY2C.
  • the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implant device.
  • the inventors of the present invention discovered for the first time that when the primary signal molecule for T cell activation is an anti-CD3 antibody or an antigen-binding fragment thereof and cannot bind to the TSP contained in the TCP, for example, when the anti-CD3 antibody or its antigen-binding fragment is derived from the anti-CD3 ⁇ antibody UCHT1 (such as the UCHT1-scFv), the TSP is CD3 ⁇ rather than CD3 ⁇ , and the transduction efficiency of the viral vector is higher.
  • UCHT1 such as the UCHT1-scFv
  • T cells are one of several important white blood cells in the human immune system and play a crucial role in acquired immune responses.
  • One of the primary functions of T cells is immune-mediated cell death, a function primarily performed by two T cell subtypes: CD8 + T cells (cytotoxic T cells) and CD4 + T cells (helper T cells).
  • the T cells are CD4 + / CD8- , CD4- /CD8 + , CD4 + /CD8 + , CD4- / CD8- T cells, or combinations thereof.
  • CD4 + T cells produce IL-2, IFN, TNF, or a combination thereof after expressing TCP molecules and binding to target cells, such as CD19 + cancer cells.
  • CD8 + T cells lyse antigen-specific target cells after expressing TCP molecules and binding to target cells.
  • T Cell Receptor Chimeric Protein T Cell Receptor Chimeric Protein, "TCP” is a recombinant protein.
  • the TCP molecule includes various polypeptides and variants thereof that constitute the TCR/CD3 complex, such as TCR/CD3 complex subunits or their functional fragments and variants, and an antigen binding region that can specifically bind to at least one antigen; the TCP molecule is generally capable of binding to target cell surface antigens through the antigen binding region it contains.
  • TCR/CD3 complex subunit or its functional fragment TCR/CD3 complex subunit is selected from at least one of TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ ; and its functional fragment is the minimum unit of the TCR/CD3 complex subunit, including the extracellular, transmembrane, intracellular, variable and constant domains, which are sufficient to perform their respective functions.
  • the TCP molecules provided by the present invention can be (a) incorporated into endogenous TCR/CD3 complexes, endogenous TCR/CD3 complex subunits or functional fragments thereof in T cells; and/or (b) functionally interact with endogenous TCR/CD3 complexes, endogenous TCR/CD3 complex subunits or functional fragments thereof, for example, forming a TCR/CD3 complex or a functional fragment thereof with an endogenous CD3 subunit or a functional fragment thereof, thereby being expressed outside the membrane and anchored on the cell membrane of the T cell.
  • TCP/CD3 complex subunits such as TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ . Therefore, even under the action of signal peptides, the TCP molecule will not or relatively rarely be expressed in cells other than T cells, such as the common packaging cells HEK-293T cells. Therefore, it will not be transferred to the envelope of the packaged lentiviral vector or retroviral vector during the budding process.
  • CD19 is the most widely used target in CAR-T therapy and has been proven to be effective and safe in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma.
  • B-ALL B-cell acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • CD19 is widely and specifically expressed throughout the developmental stages of B cells until terminal differentiation into plasma cells. Therefore, CD19 has perfect coverage for B-cell malignancies, which has led to a very high complete remission rate (CRR) achieved by CAR-T-19 therapy (Wei, J., Han, X., Bo, J. et al. Target selection for CAR-T therapy. J Hematol Oncol 12, 62 (2019)).
  • CRR complete remission rate
  • CD33 is a sialoadhesive protein composed of a membrane-proximal immunoglobulin variable region (IgV) and a membrane-proximal immunoglobulin constant region (IgC) domain. It has become a viable target due to its near-universal expression on acute myeloid leukemia (AML) cells. Notably, CD33 is also present on precursor/mature myeloid cells and hematopoietic stem cells (HSCs) but is not essential for the development and function of human myeloid cells.
  • IgV membrane-proximal immunoglobulin variable region
  • IgC membrane-proximal immunoglobulin constant region
  • Flexible linkers are usually used when the connected domains need to move or interact to a certain extent (Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69.). Flexible linkers are usually composed of small, non-polar (such as Gly) or polar (such as Ser or Thr) amino acids (Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990; 211: 943–958.). These small amino acids provide flexibility while also allowing the movement of the connected functional domains.
  • the “antibody” herein includes a typical "four-chain antibody”, which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC);
  • the heavy chain refers to a polypeptide chain composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain from its N-terminus to its C-terminus; and, when the full-length antibody is of the IgE isotype, it optionally further includes a heavy chain constant region CH4 domain;
  • the light chain refers to a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) from its N-terminus to its C-terminus; the heavy chains and the light chains are linked by disulfide bonds to form a "Y"-shaped structure.
  • the CDR regions are identified according to the Kabat numbering scheme.
  • VHH domain and “single domain antibody” (sdAb) have the same meaning and are used interchangeably. They refer to the construction of a single domain antibody (sdAb) consisting solely of a single heavy chain variable region, obtained by cloning the variable region of a heavy chain antibody. SdAbs are minimal antigen-binding fragments with full functionality. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single heavy chain variable region.
  • CH1 light chain and heavy chain constant region 1
  • antibody herein also includes monoclonal antibodies or antigen-binding portions thereof.
  • Monoclonal antibodies or antigen-binding portions thereof may be non-human, chimeric, humanized or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).
  • the "antibodies” herein can be derived from any animal, including but not limited to humans and non-human animals, which can be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, monkeys (such as cynomolgus monkeys and rhesus monkeys), alpacas, sheep, rabbits, mice, rats or cartilaginous fish (such as sharks).
  • primates mammals, rodents and vertebrates, such as camelids, llamas, ostriches, monkeys (such as cynomolgus monkeys and rhesus monkeys), alpacas, sheep, rabbits, mice, rats or cartilaginous fish (such as sharks).
  • Antibody herein also includes the heavy chain variable region (VH) or light chain variable region (VL) of the antibody.
  • antigen-binding fragment refers to a fragment that does not have the entire structure of an intact antibody and only contains a portion or a partial variant of the intact antibody, wherein the portion or partial variant has the ability to bind to an antigen.
  • antibody or antigen-binding fragment thereof includes but is not limited to immunoglobulin (full-length antibody), half antibody, Fab, Fab', F(ab') 2 , Fv fragment, single-chain variable region fragment (scFv), disulfide bond-stabilized antibody (dsFv), the heavy chain variable region (VH) or light chain variable region (VL) of an antibody, an Fd fragment consisting of a VH and a CH1 domain, a linear antibody and a single-domain antibody (nanobody); the heavy chain (VH) of the scFv is connected to the light chain (VL) by a connecting peptide.
  • the connecting peptide can be selected from a flexible connecting peptide.
  • Ligand In receptor-ligand binding, a ligand is generally a molecule that binds to a site on a receptor to generate a signal, such binding typically resulting in a conformational change in the complex structure, thereby inducing the relevant physiological activity.
  • Receptor binding fragment refers to a fragment that lacks the full structure of a complete ligand and contains only a portion or partial variant of the complete ligand, which possesses the ability to bind to the receptor.
  • receptor binding fragment herein includes, but is not limited to, the extracellular domain, functional fragment, epitope, binding region, and variable region of the ligand.
  • Endocytosis refers to the process by which substances enter cells. During endocytosis, the substance to be taken in is surrounded by a region of the plasma membrane. The plasma membrane then buds into the cell to form a vesicle containing the taken in substance. Endocytosis can be divided into four categories: receptor-mediated endocytosis (also known as clathrin-mediated endocytosis), caveolae, pinocytosis, and phagocytosis (Marsh M, Endocytosis. Oxford University Press. p. vii., 2001).
  • Chimeric Antigen Receptor refers to an artificial cell surface receptor that has been modified to be expressed on immune effector cells such as lymphocytes and specifically bind to antigens, which at least contains (1) an extracellular antigen binding region, such as scFv or VHH; (2) a transmembrane region that anchors the CAR molecule into the immune effector cell, and (3) an intracellular signaling domain; the extracellular structure of the CAR may further include a hinge region, and the intracellular structure may further include one or more costimulatory molecules to form a costimulatory signaling domain.
  • CAR molecules can use the extracellular antigen binding region to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner.
  • chimeric refers to any nucleic acid molecule or protein that is non-endogenous and comprises a combination of sequences joined or linked together that are not naturally joined or linked together in nature.
  • a chimeric nucleic acid molecule can comprise nucleic acids encoding various domains from multiple different genes.
  • a chimeric nucleic acid molecule can comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature.
  • Antigen refers to a molecule capable of inducing an immune response.
  • the induced immune response may include the production of antibodies and/or the activation of specific immune competent cells.
  • Macromolecules including proteins, glycoproteins and glycolipids can be used as antigens.
  • Antigens can be derived from recombinant or genomic DNA. As contemplated herein, an antigen need not be (i) encoded solely by the full-length nucleotide sequence of a gene or (ii) fully encoded by a gene.
  • Antigens can be generated or synthesized, or the antigen can be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.
  • Reduction When referring to the ability of the glycoprotein or its variant to bind to its receptor “reduction”, the term “reduction” includes completely eliminating the ability of the glycoprotein or its variant to bind to its receptor, as well as significantly reducing the binding ability. In a specific embodiment, “significant reduction” refers to a reduction relative to the wild-type viral glycoprotein; “reduction” is selected from a reduction of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 4%, at least 3%, at least 2% and at least 1%.
  • Nucleic acid refers to any compound and/or substance including a polymer containing nucleotides, such as a polynucleotide.
  • nucleic acid refers to any compound and/or substance including a polymer containing nucleotides, such as a polynucleotide.
  • nucleic acid refers to any compound and/or substance including a polymer containing nucleotides, such as a polynucleotide.
  • nucleic acid refers to any compound and/or substance including a polymer containing nucleotides, such as a polynucleotide.
  • a purine or pyrimidine base i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)
  • a sugar i.e., deoxyribose or ribose
  • phosphate group i.e., deoxyribose or
  • nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is typically expressed as 5' to 3'.
  • nucleic acid encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • Nucleic acid can be linear or circular.
  • nucleic acid includes both a sense strand (coding strand) and an antisense strand (template strand), as well as single-stranded and double-stranded forms.
  • nucleic acids described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include nucleotide bases modified with derivatized sugars, phosphate backbone linkages, or chemically modified residues.
  • Nucleic acid vector means a vector that carries, contains or expresses any nucleic acid.
  • the nucleic acid vector may have specific functions such as expression, packaging, pseudotyping or transduction. If the nucleic acid vector is suitable for use as a cloning vector or shuttle vector, it may also have a manipulation function.
  • the structure of the vector may include any desired form that is feasible to manufacture and suitable for a particular use. Such forms include, for example, circular forms such as plasmids and phagemids, as well as linear or branched forms.
  • Nucleic acid vectors may be composed of, for example, DNA or RNA, as well as contain some or all nucleotide derivatives, analogs and mimetics. Such nucleic acid vectors may be obtained from natural sources, recombinantly produced or chemically synthesized.
  • Transgene Transgene, also known as payload gene (Payload gene).
  • the term “transgene” refers to a gene or polynucleotide encoding a protein of interest (e.g., any of the aforementioned TCPs, etc.), the expression of which is desired in host cells/target cells and has been transferred into cells by genetic engineering technology.
  • Transgenes can encode therapeutic proteins as well as proteins that serve as reporters, tags, markers, suicide proteins, etc.
  • Transgenes can come from natural sources, modifications of natural genes, or recombinant or synthetic molecules.
  • the transgene is a component of a viral vector.
  • Expression cassette refers to a unique component of a vector nucleic acid that comprises at least one transgene and regulatory sequences (e.g., promoter, 3'UTR) that control its expression in a host cell.
  • a tandem expression cassette refers to a component of a vector nucleic acid that comprises at least two transgenes that are under the control of a set of identical regulatory sequences for tandem expression of the at least two transgenes. In certain embodiments, the tandem expression cassette comprises at least two transgenes under the control of the same promoter.
  • the first transgene and the second transgene are separated by an internal ribosome entry site (IRES), a furin cleavage site, or a self-cleaving viral 2A peptide to allow co-expression of two proteins from a single mRNA.
  • IRS internal ribosome entry site
  • furin cleavage site a furin cleavage site
  • self-cleaving viral 2A peptide a self-cleaving viral 2A peptide to allow co-expression of two proteins from a single mRNA.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds.
  • Encoding refers to the inherent property of a specific polynucleotide sequence (such as DNA, cDNA and mRNA sequences) used as a template for synthesizing other polymers and macromolecules in biological processes, wherein the template has a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. Therefore, if the transcription and translation of the mRNA corresponding to the polynucleotide produces a protein in a cell or other biological system, the polynucleotide encodes the protein.
  • a specific polynucleotide sequence such as DNA, cDNA and mRNA sequences
  • nucleotide sequences encoding amino acid sequences include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.
  • Self-cleaving peptide or “self-cleaving peptide” or “2A peptide”: refers to a self-cleaving peptide that is configured to generate two or more proteins from a single open reading frame, including FT2A peptide, F2A peptide, E2A peptide, T2A peptide and P2A peptide, etc.
  • 2A peptides are 18 to 22 residues long viral oligopeptides that mediate the "cleavage" of polypeptides during translation in eukaryotic cells.
  • “2A peptide” can refer to peptides with different amino acid sequences.
  • the 2A peptides may be the same or different from each other.
  • Detailed methods for designing and using 2A peptides are provided by Szymczak-Workman et al. (2012) Cold Spring Harb. Protoc. 2012: 199-204.
  • Exogenous refers to any molecule that originates from outside an organism, including nucleic acids, proteins, peptides, or small molecule compounds.
  • endogenous refers to any molecule that originates from within an organism (i.e., produced naturally by the organism).
  • promoter is defined as a DNA sequence that is recognized by the cellular synthetic machinery or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence means a nucleic acid sequence required for expression of a gene product operably linked to the promoter/regulatory sequence.
  • the sequence may be a core promoter sequence, and in other cases, the sequence may include an enhancer sequence and other regulatory elements required for expression of the gene product.
  • the promoter/regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.
  • a “constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • an “inducible” promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.
  • tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is of the tissue type corresponding to the promoter.
  • viral envelope refers to the outermost layer of many viruses (Hurlbert, Ronald E., Fundamentals of Microbiology, 102. Chapter #11: Viruses. Archived from the original on 2008-11-10.).
  • the viral envelope protects the viral genetic material during its life cycle as it navigates through host cells. Not all viruses have a viral envelope. Many human pathogenic viruses are enclosed in a lipid bilayer and infect target cells by fusing their viral envelope with the cell membrane.
  • Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. This greater complexity allows the virus to regulate its life cycle, as it does during latent infection. Lentiviruses belong to a genus of retroviruses that can infect both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type I and HIV type II), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).
  • HIV human immunodeficiency virus, including HIV type I and HIV type II
  • equine infectious anemia virus feline immunodeficiency virus (FIV)
  • bovine immunodeficiency virus BIV
  • SIV simian immunodeficiency virus
  • Lentiviral vector is a vector derived from a lentivirus and contains one or more lentiviral packaging proteins and/or lentiviral proteins necessary for the expression of one or more genes carried by the vector. Lentiviral vectors are produced by multiple attenuation of virulence genes of lentiviruses such as HIV through gene editing and genetic engineering techniques. For example, deletion of the env, vif, vpr, vpu, and nef genes makes lentiviral vectors biosafe.
  • lentiviral vector is intended to mean a lentiviral particle that includes a viral envelope, has at least one characteristic of a lentivirus, and is capable of invading target cells without the ability to replicate itself.
  • lentiviral vectors include the so-called third-generation lentiviral packaging systems.
  • Third-generation lentiviral packaging systems typically consist of four plasmids: a transfer plasmid (containing the gene of interest, "GOI"), such as a transgene; a GagPol plasmid; a Rev plasmid; and an envelope plasmid (containing the viral glycoprotein gene, such as VSV-G or its variants or Cocal-G or its variants).
  • the "transfer plasmid” contains the lentiviral vector backbone genome and the transgene.
  • the transfer plasmid usually has one or more transgenes flanked by long terminal repeat (LTRs) sequences, which facilitate the integration of the transgene contained in the transfer plasmid into the host genome. LTRs are responsible for the reverse transcription and integration processes of the viral genome. Through these sequences, the lentivirus can integrate the transgene into the genome of the host cell.
  • LTRs long terminal repeat
  • the transfer plasmid is usually designed so that the resulting viral vector cannot replicate itself, for example, the transfer plasmid lacks the genetic elements necessary to produce an infectious lentiviral vector in the host cell.
  • the transfer plasmid can be designed to lack the 3'LTR, thereby making the virus "self-inactivating".
  • the TAT gene is eliminated from the third-generation pseudotype lentiviral vector packaging system by adding a chimeric 5'LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) to the transfer plasmid.
  • the transfer plasmid usually contains a ⁇ sequence (Psi sequence, also known as ⁇ packaging signal) located downstream of the 5'LTR.
  • the ⁇ sequence is responsible for packaging the transgenic RNA (Pack aged into) into the viral vector.
  • the ⁇ sequence ensures that only RNA containing the transgene is packaged into the viral vector.
  • the transfer plasmid may also optionally contain an internal ribosome entry site (Internal Ribosome Entry Site, "IRES") to allow simultaneous translation of two or more open reading frames (ORFs) on one mRNA, thereby achieving multi-gene expression.
  • IRS Internal Ribosome Entry Site
  • Some transfer plasmids may also contain a selection marker gene, such as an antibiotic resistance gene (such as PuroR, encoding puromycin resistance) or a fluorescent protein gene (such as GFP), for screening or tracking transduced cells.
  • a selection marker gene such as an antibiotic resistance gene (such as PuroR, encoding puromycin resistance) or a fluorescent protein gene (such as GFP), for screening or tracking transduced cells.
  • Third-generation lentiviral vector systems typically also include three packaging plasmids: a GagPol plasmid, a Rev plasmid, and an envelope plasmid.
  • the envelope plasmid typically carries a viral glycoprotein gene, with wild-type VSV-G or Cocal-G being one of the commonly used viral glycoproteins.
  • the viral glycoprotein gene is operably linked to a promoter, typically a CMV promoter, to initiate transcription of the viral glycoprotein gene.
  • Third-generation lentiviral vector systems also include two packaging plasmids, one containing genes encoding Gag and Pol proteins (GagPol packaging plasmid), and the other containing a gene encoding Rev protein (Rev plasmid) as a further safety feature, which is an improvement over the single packaging plasmid of the so-called second-generation packaging system.
  • the Gag gene encodes the Gag polyprotein precursor, which contains the lentiviral structural proteins and includes the matrix, capsid, and nucleocapsid.
  • the Pol gene encodes the Pol polyprotein precursor, which provides the lentiviral enzyme functions necessary for replication and includes protease, reverse transcriptase, and integrase.
  • the Rev gene encodes the Rev protein, which binds to the Rev response element (RRE) to allow nuclear export of unspliced and singly spliced HIV RNA during viral replication.
  • the Gag and Pol polyprotein precursors are cleaved during viral vector preparation.
  • the Rev protein binds to the Rev response element (RRE) sequence on the viral RNA and, by interacting with the host cell's nuclear export machinery, promotes the transport of incompletely spliced viral RNA from the nucleus to the cytoplasm.
  • These unspliced RNAs can be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral vectors.
  • the packaging plasmid includes but is not limited to pMD2.G, pRSV-rev, pMDLG-pRRE and pRRL-GOI.
  • Lentiviral vectors and lentiviral vector backbone genomes are known in the art, see Naldini, et al., (1996) Science 272:263-7; Zufferey et al., (1998) J. Virol. 72:9873-9880; Dull et al., (1998) J. Virol. 72:8463-8471, U.S. Pat. No. 6,013,516 and U.S. Pat. No. 5,994,136, each of which is incorporated herein by reference in its entirety.
  • pseudotyped retroviral vector packaging systems usually do not contain Rev plasmids. This is because the genomic RNA derived from retroviruses such as Moloney Murine Leukemia Virus (MMLV) can be naturally transported from the cell nucleus to the cytoplasm for translation and assembly, so there is no need to rely on specific nuclear export mechanisms such as Rev protein.
  • Pseudotyped retroviral vector packaging systems usually contain a transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transgene sequence contained in the transfer plasmid is sandwiched on both sides by long terminal repeat sequences (LTRs).
  • LTRs long terminal repeat sequences
  • LTR sequences promote the integration of the transfer plasmid sequence into the host genome. Typically, during viral transduction, the sequences between and including the LTRs will be integrated into the host genome.
  • the backbone genome of MMLV or murine stem cell virus (MSCV), including their respective LTRs, is typically used to construct transfer plasmids in pseudotyped retroviral vector packaging systems.
  • the GagPol packaging plasmid contains the Gag gene and the Pol gene; the envelope plasmid typically contains a polynucleotide encoding a viral glycoprotein, such as VSV-G or Cocal-G.
  • the envelope plasmid may also contain a nucleic acid encoding the T cell activation primary signaling molecule and/or the T cell activation secondary signaling molecule.
  • the production cells are transfected with a defined ratio of transfer plasmids, GagPol plasmids, envelope plasmids, and Rev plasmids.
  • the ratio of each plasmid is determined by mass, and there is no particular limitation as long as it can package a non-integrated lentiviral vector with biological activity.
  • the mass of each of the transfer plasmid and the GagPol plasmid is higher than the mass of each of the envelope plasmid and the Rev plasmid.
  • the defined ratio of the transfer plasmid, GagPol plasmid, envelope plasmid, and Rev plasmid is about 1:1:1:1 to about 9:4:2:2; in some embodiments of the present invention, the envelope plasmid may contain a nucleic acid encoding the T cell activation primary signaling molecule and/or the T cell activation secondary signaling molecule.
  • the envelope plasmid comprises a tandem expression cassette encoding any one of the aforementioned VSV-G or its variants or Cocal-G or its variants and the T cell activation primary signal molecule and/or T cell activation secondary signal molecule as disclosed herein.
  • the tandem expression cassette contained in the envelope plasmid comprises a polynucleotide encoding a first signal peptide, a polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule, a polynucleotide encoding an internal ribosome entry site (IRES), a furin cleavage site or one of the viral 2A peptides, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding VSV-G or its variants or Cocal-G or its variants.
  • IRS internal ribosome entry site
  • the polynucleotide encoding VSV-G or its variants or Cocal-G or its variants is located at the 5' end of the polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule. In other embodiments, the polynucleotide encoding VSV-G or its variants or Cocal-G or its variants is located at the 3' end of the polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule.
  • the polynucleotide encoding the T cell activation primary signaling molecule and/or the T cell activation secondary signaling molecule and the polynucleotide encoding VSV-G or its variant or Cocal-G or its variant are separated in a tandem cassette by a polynucleotide encoding an IRES, a furin cleavage site, or a viral 2A peptide, which allows co-expression of the two proteins from a single mRNA.
  • the viral 2A peptide is porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A), or variants thereof.
  • lentiviral/retroviral vector packaging systems relies on a "packaging cell line.”
  • a packaging cell line is a cell line that, when a transfer plasmid or one or more packaging plasmids are introduced into the cell, produces a non-replication-competent lentiviral or retroviral vector capable of infecting/transducing target cells.
  • An overview of available packaging lines is provided in J.M. Coffin, S.M. Hughes, et al., Cold Spring Harbour Laboratory Press, 1997, p. 447, which is incorporated herein by reference in its entirety.
  • various plasmids can be introduced into the packaging cell line using transfection methods including chemical-mediated transfection methods, physical-mediated transfection methods or biological-mediated transfection methods.
  • chemical-mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran or PEI (Polyethylenimine, polyethyleneimine transfection reagent), and physical-mediated transfection methods include transfection methods such as electroporation.
  • Production/host/packaging cells that can be used to prepare the viral vectors disclosed herein include human embryonic kidney (HEK) 293 cells and their derivatives.
  • the production cells can be adherent cell lines such as HEK293T production cells, or suspension cell lines such as HEK293T/17SF production cells.
  • the packaging cell/host cell is selected from CHO cells, BHK cells, MDCK cells, C3H-10T1/2 cells, FLY cells, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells and 211 cells;
  • the packaging cell/host cell is a HEK-293T cell.
  • Retrovirus and Retroviral Vector: Retrovirus and Retroviral Vector.
  • Retrovirus refers to an RNA virus with a single-stranded positive-sense RNA molecule. Retroviruses contain reverse transcriptase and integrase. After entering the target cell, the retrovirus uses its reverse transcriptase to transcribe its RNA molecule into a DNA molecule. Subsequently, the DNA molecule is integrated into the host cell genome using integrase. After integration into the host cell genome, the sequence from the retrovirus is called a provirus (e.g., a proviral sequence or a proviral sequence).
  • a provirus e.g., a proviral sequence or a proviral sequence
  • Retroviral vectors generally refer to pseudotyped retroviral vectors derived from retroviruses, illustratively from ⁇ -retroviruses. Unlike lentiviral vectors that can transduce dividing and non-dividing cells, retroviral vectors can only transduce dividing cells, and the exogenous transgenes they can carry are generally relatively small.
  • Lentiviral and retroviral vectors offer significant advantages for gene therapy by stably integrating exogenous cargo genes, such as shuttle genes, into the chromosomes of target cells, allowing for long-term expression of the delivered shuttle genes. Furthermore, they do not transfer viral genes, thus avoiding the problem of generating transduced cells that can be destroyed by cytotoxic T cells. Furthermore, they possess relatively large cloning capacities, sufficient for most anticipated clinical applications.
  • Envelope glycoprotein refers to the glycoprotein coated on the outer layer of the virus, which plays an important role in the adsorption and penetration of the virus into host cells, pathogenicity, downregulation of host surface protein expression, and increase in virus packaging and budding.
  • variant refers to a mutant having at least about 50% identity to the amino acid sequence of a non-mutant (wild type), and "at least about 50% identity” refers to about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of the non-mutant (wild type); or, a variant refers to a variant that is identical to the nucleic acid sequence encoding the non-mutant (wild type).
  • At least 50% identity means that the nucleic acid sequence encoding the variant is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the nucleic acid sequence encoding the non-mutant (wild type).
  • variants include mutants comprising conservative substitutions relative to non-mutants.
  • Constant substitutions are considered in the art to be substitutions of one amino acid with another amino acid having similar properties.
  • conservative substitutions are well known in the art (see, for example, WO97/09433, page 10, published on March 13, 1997; Lehninger, Biochemistry, 2nd edition; Worth Publishers, Inc. NY: NY (1975), pages 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), page 8).
  • “Pharmaceutically acceptable excipient or carrier” Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, preservatives, and/or adjuvants. Excipients are preferably nontoxic or substantially nontoxic to the recipient at the dosages and concentrations employed. Such excipients include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • compositions may contain substances for improving, maintaining, or preserving, for example, the pH, osmotic properties, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition.
  • the optimal pharmaceutical composition can be determined based on the intended route of administration, mode of delivery, and desired dosage.
  • compositions for in vivo administration are typically provided as sterile formulations. Sterilization is achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization, reconstitution, or dilution.
  • the pharmaceutical compositions of the present invention can be selected for parenteral delivery. Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, they can be prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.
  • a sterile access port such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.
  • the composition can be selected for inhalation or delivery through the digestive tract (such as orally).
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposomal carriers, bioerodible microparticles or porous beads, and depot injection) are also known to those skilled in the art.
  • the pharmaceutical composition is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or lyophilized powder.
  • the formulation can be stored in a ready-to-use form or in a form (e.g., lyophilized) that is redissolved before administration.
  • the present invention also provides a test kit for producing a single-dose administration unit.
  • the test kit of the present invention can each contain a first container with a dried protein and a second container with an aqueous formulation.
  • a test kit containing a single-chamber and multi-chamber prefilled syringe e.g., a liquid syringe and a lyophilizing syringe
  • Subject As used herein, “subject,” “patient,” and “individual” are used synonymously and include, but are not limited to, mammals, such as humans or non-human mammals, such as domestic animals, agricultural animals, or wild animals, as well as birds and aquatic animals.
  • a "patient” is a subject who suffers from a disease, disorder, or condition, or is at risk of developing a disease, disorder, or condition, or who is otherwise in need of any of the viral vectors, TCP-T cells, compositions, or treatment methods provided herein.
  • a “disease” is a state of health in a subject in which the subject is unable to maintain homeostasis and in which the subject's health continues to deteriorate if the disease does not improve.
  • a “disorder” or “adverse condition” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's health is less favorable than it would be in the absence of the disorder or adverse condition. Without treatment, a disorder or adverse condition does not necessarily result in a further deterioration in the subject's health.
  • cancer As used herein, the term “cancer” is defined as a disease characterized by the rapid, uncontrolled growth of abnormal cells. Abnormal cells may form solid tumors or constitute hematological malignancies. Cancer cells may spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, hematological cancers, such as B-lymphocyte malignancies and multiple myeloma; and solid cancers, such as breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, and lymphoma.
  • hematological cancers such as B-lymphocyte malignancies and multiple myeloma
  • solid cancers such as breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, and lymphoma.
  • Treatment refers to the use of a treatment method described herein to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor size, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth) in a subject.
  • the treatment method that effectively treats a patient may vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the treatment to elicit an anti-cancer response in the subject.
  • the therapeutically effective amount of the pharmaceutical composition containing any described engineered T cell and/or viral vector provided by the invention will be adopted and will depend on for example treatment degree and target.It will be appreciated by those skilled in the art that the appropriate dosage level for the treatment of will depend in part on the molecule sent, indication, administration route and patient condition (body weight, body surface or organ size) and/or situation (age and general health) and change.In some embodiments, clinician's titration dosage also changes administration route to obtain best therapeutic effect.
  • the frequency of administration will depend on the pharmacokinetic parameters of the engineered T cells or the viral vector in the formulation used. Clinicians typically administer the pharmaceutical composition until the dosage is achieved to achieve the desired effect.
  • the pharmaceutical composition can therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or administered as a continuous infusion via an implantable device or catheter.
  • administering The administration routes of the pharmaceutical composition are conventional in the art, such as oral, nasal, intravenous, subcutaneous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection, and can also be administered by sustained release system or by implantation device.
  • prevention refers to methods used to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a condition. As used herein, “prevention” and similar words also include lessening the intensity, effects, symptoms, and/or burden of a disease or condition prior to onset or recurrence.
  • “Stable integration” also known as “stable transfection, refers to the integration of exogenous polynucleotides into the host cell genome after introduction into the host cell, and their long-term stable expression in the host cell (Stable Gene Expression); in contrast, transient transfection and transient expression (Transient Expression).
  • Specific binding refers to the binding that occurs between paired molecular species (e.g., a receptor and a ligand, an antibody and an antigen). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, the specific binding between one or more species is direct. In some embodiments of the invention, the affinity of the specific binding is about 2 times greater than background binding (non-specific binding), about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.
  • sequence identity In general, “sequence identity” or “sequence homology” refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence.
  • Two or more sequences can be compared by determining their "percent identity.”
  • the percent identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100.
  • the advanced BLAST computer program available from the National Institutes of Health can also be used to compare sequence information to determine the percent identity.
  • the BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc.
  • the BLAST program defines identity as the number of aligned symbols (usually nucleotides or amino acids) that are identical divided by the total number of shorter symbols in the two sequences. The program can be used to determine percent identity over the entire length of the proteins being compared.
  • Signal peptide sometimes also called signal sequence, targeting signal, localization signal, localization sequence, transit peptide or leader peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), present at the N-terminus of most newly synthesized proteins that enter the secretory pathway (occasionally non-classically present at the C-terminus or internally) (Owji, et al., A comprehensive review of signal peptides: Structure, roles, and applications, European Jour nal of Cell Biology.97(6):422-441.(2018))(Blobel G,Dobberstein B,et al.,Transfer of proteins across membranes.I.Presence of proteolytically processe d and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma
  • Signal peptides are short peptides present at the N-terminus of newly synthesized proteins that are specific for the plasma membrane or secretory pathway.
  • Signal sequences typically contain a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a signal sequence cleavage site.
  • signal sequences cause newly synthesized proteins to translocate to the endoplasmic reticulum, where the protein is cleaved by a signal peptidase to produce the mature protein, which then enters its appropriate destination.
  • the diversity of signal sequence length and amino acid composition makes it difficult to accurately predict the cleavage site.
  • polypeptide sequences disclosed herein when referring to a signal sequence, polypeptide sequences in which no signal sequence or a partial signal sequence is present are also contemplated.
  • signal peptide The function of a signal peptide is to prompt the cell to transfer proteins, usually to the cell membrane.
  • signal peptides direct newly synthesized proteins to the SecYEG protein-conducting channel present in the plasma membrane.
  • a homologous system exists in eukaryotes, in which signal peptides direct newly synthesized proteins to the Sec6L channel, which has structural and sequence homology with SecYEG but is present in the endoplasmic reticulum (Rapoport TA, Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes, Nature. 450(7170):663-9(2007).).
  • SecYEG and Sec6L channels are often referred to as transporters, and transport through these channels is called translocation.
  • the transmembrane region may diffuse through the side gate in the translocon to be distributed to the surrounding membrane.
  • MOI Multiplicity of Infection
  • a polynucleotide is “operably linked” when it is in a functional relationship with another polynucleotide. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned so as to promote translation, the ribosome binding site is operably linked to a coding sequence.
  • operably linked means that the polynucleotides being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.
  • Transduction As used herein, the terms “transfection,” “transformation,” and “transduction” are used synonymously to refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell, packaging cell, or the like.
  • a “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include the primary subject cell and its progeny.
  • vectors such as viral vectors or isolated polynucleotides into mammalian cells are known in the art.
  • the described vectors can be transferred to immune effector cells by physical, chemical or biological methods.
  • vectors or isolated polynucleotides into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells containing vectors and/or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.).
  • the vector is introduced into the cell by electroporation.
  • the vector is introduced into the cell by PEI (Polyethylenimine, polyethyleneimine transfection reagent) transfection reagent.
  • Biological methods for introducing vectors or isolated polynucleotides into immune effector cells include the use of DNA and RNA vectors.
  • Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.
  • Chemical methods for introducing vectors or isolated polynucleotides into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system used as an in vitro delivery vehicle is a liposome.
  • Figure 1 is a flow cytometry result showing the expression efficiency of the CD19-CAR molecule or the CD19-TCP-E/G molecule in HEK-293T cells packaged with the control group lentiviral vector m-CAR and the targeted lentiviral vector m-TCP-E/G in Example 1;
  • FIG2 is a flow cytometry result showing the expression abundance of CD19 in the culture medium of Nalm-6 cells in each group to which the control group lentiviral vector m-CAR, the targeted lentiviral vectors m-TCP-E and m-TCP-G were added, respectively, in Example 1;
  • FIG3 is a graph showing the flow cytometry results of the expression efficiency of the CD19-TCP-E/G molecule in CD3 + T cells in PBMCs of the control group after the lentiviral vector m-7-E, the targeted lentiviral vectors m-TCP-E and m-TCP-G were respectively transduced into non-activated human PBMCs in Example 2;
  • FIG4 is a graph showing the proliferation curves of CD3 + T cells in PBMCs of each group after the targeted lentiviral vector m-TCP-G activated and stimulated the non-activated PBMCs of Donor 1 and Donor 2, respectively, in Example 3;
  • FIG5 is a flow cytometry result showing the killing efficiency of CD19-TCP-T cells prepared by transducing human non-activated T cells with the targeted lentiviral vector m-TCP-G in Example 4 against Nalm-6 cells in vitro;
  • Figure 6 shows in vivo imaging of three groups of model mice on Days 0, 5, and 7 in Example 5, to detect the killing efficiency of CD19-TCP-T cells prepared by transducing human PBMCs with the targeted lentiviral vector m-TCP-G against Nalm-6 cells in mice;
  • Figure 7 shows the flow cytometry results of the efficiency of CD3 + T cells expressing the CD19-TCP-G molecule in non-activated human PBMCs of each group, respectively, transduced by the control group lentiviral vector m-7-G, the targeted lentiviral vectors m-3-G, and m-TCP-G in Example 6;
  • FIG8 is a flow cytometry result showing the efficiency of CD3 + T cells expressing the CD19-TCP-G molecule after the targeted lentiviral vectors m-TCP-G and m-86-G were respectively transduced into non-activated human PBMCs in Example 7;
  • FIG9 is a flow cytometry result showing the efficiency of CD3 + T cells expressing the CD33-TCP-G molecule after the targeted lentiviral vector m-a33-G transduced human non-activated PBMCs in Example 8.
  • FIG9 is a flow cytometry result showing the efficiency of CD33-TCP-G molecule expression in CD3 + T cells after the targeted lentiviral vector m-a33-G transduced human non-activated PBMCs.
  • FIG10 is a flow cytometry result showing the killing efficiency of the CD33-TCP-T cells in killing CD33 + target MOLM-13 cells in vitro in Example 8.
  • Example 1 Construction of a lentiviral vector m-TCP-E/G targeting non-activated T cells
  • the polynucleotides encoding membrane-expressed anti-CD3 antibody ⁇ anti-CD28 antibody are as follows from the 5' end to the 3' end: a polynucleotide encoding a human CD8 ⁇ signal peptide, a polynucleotide encoding the UCHT1-scFv, a polynucleotide encoding a human CD8 ⁇ hinge region, a polynucleotide encoding a human CD8 ⁇ transmembrane region, a polynucleotide encoding an FT2A peptide, a polynucleotide encoding a human CD8 ⁇ signal peptide, a polynucleotide encoding the 15E8-scFv, a polynucleotide encoding a human CD8 ⁇ hinge region, and a polynucleotide
  • the membrane-expressed anti-CD3 antibody, UCHT1-scFv (membrane-expressed UCHT1-scFv) and the membrane-expressed anti-CD28 antibody, 15E8-scFv (membrane-expressed 15E8-scFv) are separately expressed on the cell membrane of the packaging cell, and as the lentiviral vector buds out, they are transferred to the envelope of the lentiviral vector, thereby enabling the lentiviral vector to have the ability to target and activate non-activated T cells.
  • a TCP molecule targeting CD19 (CD19-TCP) was constructed; the polynucleotides encoding the CD19-TCP molecule were, from the 5' end to the 3' end, the following: a polynucleotide encoding the human CD8 ⁇ signal peptide, a polynucleotide encoding the extracellular antigen binding region targeting human CD19, a polynucleotide encoding the connecting peptide 1, and a polynucleotide encoding human CD3 ⁇ (CD19-TCP-E) or human CD3 ⁇ (CD19-TCP-G);
  • the extracellular antigen binding region is the FMC63-scFv that can specifically bind to human CD19; the amino acid sequence of the VH region of the FMC63-scFv is shown in SEQ ID NO: 18, the amino acid sequence of the VL region of the FMC63-scFv is shown in SEQ ID NO: 19; the amino acid sequence of the FMC63-scFv is shown in SEQ ID NO: 64; the amino acid sequences of the HCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NO: 65-67, respectively, and the amino acid sequences of the LCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NO: 68-70, respectively;
  • the C-terminus of the extracellular antigen binding region is operably connected to the N-terminus of the human CD3 ⁇ or human CD3 ⁇ through the connecting peptide 1;
  • the amino acid sequence of the CD19-TCP-E is shown in SEQ ID NO: 100;
  • the amino acid sequence of the CD19-TCP-G is shown in SEQ ID NO:101.
  • An envelope plasmid (envelope plasmid 1) carrying a polynucleotide encoding a mutant VSV-G and a polynucleotide encoding the membrane-expressed anti-CD3 ⁇ anti-CD28 dual antibody, a pMDLg/pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a master plasmid/transfer plasmid (CD19-TCP-E/G master plasmid) carrying a polynucleotide encoding the CD19-TCP-E/G molecule; the envelope plasmid 1 and the master plasmid were synthesized by conventional molecular cloning methods;
  • the amino acid sequence of the mutant VSV-G extracellular domain is shown in SEQ ID NO:8; relative to SEQ ID NO:1, SEQ ID NO:8 includes a K47 deletion, T214N, and T352A; the amino acid sequence of the wild-type VSV-G extracellular domain is shown in SEQ ID NO:1; the amino acid sequence of the wild-type VSV-G full-length protein (including the VSV-G signal peptide) is shown in SEQ ID NO:24;
  • the targeted lentiviral vector m-TCP-G comprises a polynucleotide encoding the CD19-TCP-G molecule
  • the targeted lentiviral vector m-TCP-E comprises a polynucleotide encoding the CD19-TCP-E molecule
  • the mutant VSV-G has a lysine deletion at position 47 of its extracellular domain, which weakens or even eliminates its ability to specifically bind to LDL-R, thereby improving the targeting of the lentiviral vector m-TCP-G or m-TCP-E to activate and transduce non-activated T cells.
  • the mutant VSV-G also contains T214N and T352A mutations that enhance its ability to antagonize complement inactivation or prevent complement inactivation, making it more suitable for in vivo targeted transduction of non-activated T cells.
  • HEK-293T cell culture system filter 56 mL of FBS into 500 mL of DMEM/high glucose (10% FBS) and add 4 mL of P/S (double antibody, penicillin ⁇ streptomycin), shake well, and place in a carbon dioxide incubator to preheat for transfection and neutralization.
  • P/S double antibody, penicillin ⁇ streptomycin
  • HEK-293T cells On Day 0, 4.5 ⁇ 10 6 HEK-293T cells were seeded in a 10 cm culture dish. About 48 hours after seeding, when the cell confluence reached 80-90%, the four plasmids were transfected into the packaging HEK-293T cells using PEI reagent, including:
  • the culture medium was renewed after 6 hours, and the culture supernatant was collected 48 hours after transfection, filtered using a 0.45 ⁇ m filter membrane, centrifuged at 50,000 g for 2.5 hours, and the supernatant was discarded; the lentiviral vector m-TCP-E or m-TCP-G was resuspended in 200 ⁇ L F12 medium and frozen at -80°C; at the same time, HEK-293T cells were collected, and the expression of CD19-TCP-E/G molecules in each group of HEK-293T cells was detected by flow cytometry. The results are shown in Figure 1.
  • Flow cytometry antibody for detecting FMC-63 Trade name: PE-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) (Site-specific conjugation) (0.03% Proclin) DMF Filed, Brand: Acro, Product Number: #FM3-PY54A2-200 tests.
  • Opti-MEM alpha Reduced Serum Medium Brand: GIBCO, Catalog Number: #SP0272;
  • HEK-293T cell culture medium DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog Number: #C12430500BT; FBS: Brand: EXCELL, Catalog Number: #FSP500;
  • F12 culture medium Brand: GIBCO, catalog number: #C11330500BT;
  • Syringe filter Brand: SORFA, item number: #622120.
  • the control group lentiviral vector m-CAR was packaged.
  • the envelope plasmid 1 the envelope plasmid 1
  • the pMDLg/pRRE packaging plasmid the pRSV-REV packaging plasmid
  • a main plasmid carrying a polynucleotide encoding a CD19-CAR molecule
  • the structure of the CD19-CAR molecule from N-terminus to C-terminus is: an extracellular antigen binding region, the human CD8 ⁇ hinge region, the human CD8 ⁇ transmembrane region, a human 4-1BB co-stimulatory signaling domain, and a human CD3 ⁇ intracellular signaling domain; the extracellular antigen binding region is the FMC63-scFv;
  • the polynucleotide encoding the CD19-CAR molecule is operably linked to the polynucleotide encoding the human CD8 ⁇ signal peptide, and the human CD8 ⁇ signal peptide is located at the N-terminus of the CD19-CAR molecule;
  • the control group lentiviral vector m-CAR was packaged and frozen at -80°C; at the same time, the packaging cell HEK-293 cells were collected, and the expression of the CD19-CAR molecule in the HEK-293T cells was detected by flow cytometry. The results are shown in Figure 1.
  • the CD19-CAR molecule was expressed in large quantities outside the membrane in the packaging cell HEK-293T cells (positive rate of about 74.76%), while the CD19-TCP-E molecule (positive rate of about 4.21%) and CD19-TCP-G molecule (positive rate of about 1.12%) were rarely expressed outside the membrane in the packaging cell HEK-293T cells; therefore, based on the mechanism of budding of the lentiviral vector in the packaging cells, it can be reasonably inferred that the content of the CD19-TCP-E/G molecule in the viral envelope of the lentiviral vector will also be significantly reduced relative to the CD19-CAR molecule, thereby effectively reducing false transduction when the targeted lentiviral vector m-TCP-E/G is used to transduce T cells; and the ability of the targeted lentiviral vector m
  • the targeted lentiviral vectors m-TCP-E and m-TCP-G and the control group lentiviral vector m-CAR were added to the Nalm-6 cell culture system (1640 medium + 10% FBS) at an MOI of 5, and 2 ⁇ 10 5 CD19 + Nalm-6 cells (human B lymphoid leukemia cells) were transduced respectively; on Day 2, the expression of CD19 in Nalm-6 cells in each group was detected by flow cytometry, and the results are shown in Figure 2.
  • the expression level of CD19 in the Nalm-6 cell culture system to which the control group lentiviral vector m-CAR was added was significantly reduced (positive rate 34.69%), which proves that the control group lentiviral vector m-CAR can effectively bind to the surface antigen CD19 of Nalm-6 cells through the FMC63-scFv contained in its viral envelope, thereby reducing the expression level of CD19 on the surface of the Nalm-6 cells; and in the Nalm-6 cell culture system to which the targeting lentiviral vector m-TCP-E or m-TCP-G was added, the positive rate of CD19 was maintained at 97.07% or 97.61%, respectively, which proves that the viral envelope of the targeting lentiviral vector m-TCP-E/G does not contain or almost does not contain the FMC63-scFv, making it difficult to specifically bind to the Nalm-6 cell surface antigen CD19, and
  • CD19-FITC antibody brand: BD, catalog number: #5554112.
  • Example 2 Detection of TCP molecule expression efficiency after transduction of non-activated T cells with the targeted lentiviral vector m-TCP-E/G and the control lentiviral vector m-7-E
  • envelope plasmid 2 carrying a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding a membrane-expressed anti-CD7 antibody, a pMDLg/pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the CD19-TCP-E main plasmid;
  • the envelope plasmid 2 is synthesized by conventional molecular cloning methods;
  • the control group lentiviral vector m-7-E (a) comprises a polynucleotide encoding the CD19-TCP-E molecule; and (b) the viral envelope comprises a membrane-expressed anti-CD7 antibody;
  • the structure of the polynucleotide encoding the membrane-expressed anti-CD7 antibody from the 5' end to the 3' end is: a polynucleotide encoding the human CD8 ⁇ signal peptide, a polynucleotide encoding a scFv that specifically binds to human CD7 (scFv derived from TH-69, TH69-scFv), a polynucleotide encoding the human CD8 ⁇ hinge region, and a polynucleotide encoding the human CD8 ⁇ transmembrane region;
  • the heavy chain variable region (VH region) of the TH69-scFv is connected to the light chain variable region (VL region) of the TH69-scFv via connecting peptide 2;
  • the amino acid sequence of the VH region of the TH69-scFv is shown in SEQ ID NO: 25; the amino acid sequence of the VL region of the TH69-scFv is shown in SEQ ID NO: 26; the amino acid sequence of the TH69-scFv is shown in SEQ ID NO: 71, the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 72-74, respectively, and the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 75-77, respectively;
  • the packaging method of the targeted lentiviral vector m-TCP-E/G in Example 1 the control lentiviral vector m-7-E and the targeted lentiviral vectors m-TCP-E and m-TCP-G were packaged in the same batch.
  • PBMCs On Day 0, 1 ⁇ 10 6 healthy human non-activated PBMCs were collected from three groups and resuspended in 200 ⁇ L of PBMC culture medium, which included XVT medium, IL-7 at a final concentration of 20 ng/mL, and IL-15 at a final concentration of 20 ng/mL.
  • the expression efficiency of the CD19-TCP-E/G gene delivered by the control lentiviral vector m-7-E, the targeted lentiviral vectors m-TCP-E and m-TCP-G to transduce CD3 + T cells in human non-activated PBMCs was approximately 5.25%, 12.65% and 34.81%, respectively;
  • the targeted lentiviral vectors m-TCP-G and m-TCP-E stably integrated the polynucleotide encoding the CD19-TCP-E/G molecule into the T cell genome, and the efficiency of the CD19-TCP-E/G molecule in T cell membrane expression was significantly improved; this may be because, compared with the anti-CD7 antibody that cannot effectively activate and stimulate non-activated T cells, the T cell activation primary signal molecule anti-CD3 antibody and the secondary signal molecule anti-CD28 antibody can effectively activate and stimulate non-activated T cells in human non-activated PBMCs, and the efficiency of activated T cells in assembling and expressing TCR/CD3 complexes and the CD19-TCP-E/G molecules (which may be incorporated into endogenous TCR/CD3 complexes and/or functionally interact with endogenous TCR/CD3 complexes) is better than that of significantly non-activated
  • the membrane-expressed anti-CD3 antibody used in this example and the UCHT1-scFv is an anti-CD3 ⁇ antibody; during the packaging process of the targeted lentiviral vector m-TCP-E, UCHT1-scFv can specifically bind to CD3 ⁇ contained in the CD19-TCP-E molecule in the packaging cell HEK-293T cells, thereby reducing the content of UCHT1-scFv that can be expressed on the cell membrane of the HEK-293T cells and transferred to the envelope of the targeted lentiviral vector m-TCP-E with budding, thereby reducing the ability of the targeted lentiviral vector m-TCP-E to target and activate and stimulate non-activated T cells, ultimately leading to a decrease in transduction efficiency and/or a decrease in the efficiency of membrane expression of the CD19-TCP-E molecule in T cells with relatively low activation levels;
  • the anti-CD3 ⁇ antibody UCHT1-scFv cannot specifically bind to CD3 ⁇ , and therefore will not lead to a reduction in the content of UCHT1-scFv available for the specific T cell surface antigen CD3 ⁇ in the envelope of the targeted lentiviral vector m-TCP-G, and thus will not affect the ability of the targeted lentiviral vector m-TCP-G to target and activate and stimulate non-activated T cells, and ultimately will not lead to a decrease in transduction efficiency and/or a decrease in the efficiency of the CD19-TCP-G molecule to express outside the membrane in T cells with a relatively high degree of activation.
  • the anti-CD3 antibody or an antigen-binding fragment thereof when using an anti-CD3 antibody or an antigen-binding fragment thereof as a primary signal molecule for T cell activation and simultaneously using the TCP molecule provided by the present invention, in order to maximize the transduction efficiency of the constructed targeted lentiviral vector, the anti-CD3 antibody or antigen-binding fragment thereof used should not specifically bind to the TSP polypeptide contained in the TCP molecule.
  • XVT medium Trade name: PRIME-XV T cell CDM, brand: IRVINE (FUJIFILM), catalog number: #91154;
  • IL-7 Trade Name: IL-7 Protein, Human, Recombinant, Brand: Sino Biological, Catalog Number: #11821-HNAE;
  • IL-15 Trade Name: IL-15 Protein, Human, Recombinant (His Tag), Brand: Sino Biological, Catalog Number: #10360-H07E;
  • Flow cytometry antibody for detecting CD3 Trade name: FITC Mouse Anti-Human CD3; Brand: BIOLEGEND, Item number: #555339.
  • Example 3 Targeted lentiviral vector m-TCP-G can effectively activate and stimulate non-activated T cells
  • the targeted lentiviral vector m-TCP-G was added to the culture medium of one of the non-activated human PBMCs groups of Donor 1 and Donor 2 at an MOI of 5, mixed, and the proliferation of CD3 + T cells in the two groups of cells was continuously counted and recorded.
  • the results are shown in Figure 4.
  • the targeted lentiviral vector m-TCP-G can effectively activate and stimulate the non-activated T cells in the non-activated PBMCs of Donor 1 and Donor 2.
  • the CD19-TCP-T cells prepared by transducing T cells with the targeted lentiviral vector m-TCP-G can efficiently kill the target Nalm-6 cells.
  • Example 5 Targeted lentiviral vector m-TCP-G kills cancer cells in vivo
  • mice On Day 0, 12 NKG immunodeficient female mice aged 4 to 8 weeks (purchased from Saiye Bio) were divided into three groups, with 4 mice in each group, namely control group 1, control group 2, and experimental group;
  • mice Four hours later, 1 ⁇ 10 7 human non-activated PBMCs were injected into the tail vein of mice in control group 2 and experimental group, respectively; and 1 ⁇ 10 6 TU of the viral supernatant of the targeted lentiviral vector m-TCP-G was injected into mice in the experimental group alone;
  • the targeted lentiviral vector m-TCP-G can transduce human PBMCs cells to prepare CD19-TCP-T cells in the experimental group mice, and effectively kill Nalm-6 cells.
  • Example 6 Targeted lentiviral vectors m-TCP-G and m-3-G and control lentiviral vector m-7-G were used to transduce human non-activated PBMCs
  • the control lentiviral vector m-7-G, targeted lentiviral vectors m-3-G and m-TCP-G were packaged in the same batch;
  • the targeted lentiviral vector m-3-G contains a T cell targeting molecule that is a primary signal molecule for T cell activation, the membrane expresses UCHT1-scFv, and does not contain secondary signal molecules for T cell activation and other T cell targeting molecules; (b) contains a polynucleotide encoding the CD19-TCP-G; specifically, the envelope plasmid 1 is replaced with an envelope plasmid (envelope plasmid 3) containing a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding the membrane-expressed UCHT1-scFv.
  • the targeted lentiviral vector m-TCP-G transduced human non-activated PBMCs, in which CD3 + T cells expressed the CD19-TCP-G molecule at a significantly higher efficiency;
  • the envelope of the targeted lentiviral vector m-3-G only contains the antigen-specific first signal that simulates the T cell activation signal, that is, the membrane-expressed anti-CD3 antibody of the T cell activation primary signal molecule, but lacks the T cell activation secondary signal molecules such as anti-CD28 antibodies that simulate the second signal, T cell activation co-stimulatory/secondary signal; therefore, compared with the targeted lentiviral vector m-TCP-G whose envelope contains both T cell activation primary and secondary signal molecules, the ability of the targeted lentiviral vector m-3-G to activate and stimulate non-activated T cells is relatively low, resulting in a low efficiency of membrane expression of the TCP molecule in relatively underactivated T cells.
  • Example 7 Packaging of a targeted lentiviral vector m-86-G containing an anti-CD3 antibody and CD86
  • the polynucleotides encoding membrane-expressed anti-CD3 antibody ⁇ CD86 are, from 5' to 3' end, the following: a polynucleotide encoding the human CD8 ⁇ signal peptide, a polynucleotide encoding the UCHT1-scFv, a polynucleotide encoding the human CD8 ⁇ hinge region, a polynucleotide encoding the human CD8 ⁇ transmembrane region, a polynucleotide encoding the FT2A peptide, a polynucleotide encoding the human CD86 signal peptide, a polynucleotide encoding the human CD86 extracellular domain, and a polynucleotide encoding the human CD86 transmembrane region;
  • the targeted lentiviral vectors m-TCP-G and m-86-G were packaged in the same batch:
  • the packaging envelope comprises the targeted lentiviral vector m-86-G expressing the membrane-expressing anti-CD3 antibody ⁇ CD86; specifically, the envelope plasmid 1 is replaced with an envelope plasmid (envelope plasmid 4) carrying a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding the membrane-expressing anti-CD3 antibody ⁇ CD86.
  • envelope plasmid 4 carrying a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding the membrane-expressing anti-CD3 antibody ⁇ CD86.
  • Day 0 Referring to the method for transducing non-activated human PBMCs with the targeted lentiviral vector m-TCP-E/G in Example 1, 1 ⁇ 10 6 non-activated human PBMCs from healthy donor 1 were transduced using the targeted lentiviral vectors m-86-G and m-TCP-G, respectively, at an MOI of 5.
  • the targeted lentiviral vectors m-TCP-G and m-86-G respectively transduced human non-activated PBMCs, wherein the efficiency of CD3 + T cells expressing the CD19-TCP-G was approximately 12.66% and 16.15%, respectively; the targeted lentiviral vectors m-TCP-G and m-86-G can both effectively transduce CD3 + T cells in human non-activated PBMCs to express the CD19-TCP-G.
  • Example 8 Construction of a targeted lentiviral vector containing a polynucleotide encoding a TCP molecule targeting CD33
  • CD33-TCP-G a TCP molecule targeting human CD33 (Uniprot ID: P20138) (CD33-TCP-G) was constructed; specifically, the FMC63-scFv was replaced with the antigen binding region targeting human CD33;
  • the polynucleotides encoding the CD33-TCP-G molecule are, from the 5' end to the 3' end, the following: a polynucleotide encoding the human CD8 ⁇ signal peptide, a polynucleotide encoding the extracellular antigen binding region targeting human CD33, a polynucleotide encoding the connecting peptide 1, and a polynucleotide encoding the human CD3 ⁇ (CD33-TCP-G);
  • the antigen binding region targeting human CD33 is a scFv (Gemtuzumab-scFv) derived from the anti-human CD33 monoclonal antibody Gemtuzumab; the amino acid sequence of the Gemtuzumab-scFv is shown in SEQ ID NO: 78, the amino acid sequence of the VL region of the Gemtuzumab-scFv is shown in SEQ ID NO: 59; the amino acid sequence of the VH region of the Gemtuzumab-scFv is shown in SEQ ID NO: 60; the amino acid sequences of the HCDR1-3 regions of the Gemtuzumab-scFv are shown in SEQ ID NO: 79-81, respectively; the amino acid sequences of the LCDR1-3 regions of the Gemtuzumab-scFv are shown in SEQ ID NO: 82-84, respectively.
  • m-a33-G was packaged.
  • the targeting vector m-a33-G can effectively transduce CD3 + T cells in non-activated human PBMCs, and the cell membrane expression efficiency of the CD33-TCP-G molecule is approximately 27.84%.
  • CD33-TCP-T cells kill target cells in vitro
  • the CD33-TCP-T cells were used to kill CD33 + target MOLM-13 cells (human acute myeloid leukemia cells) in vitro. On Day 7, the killing efficiency was detected by flow cytometry. The results are shown in FIG10 .
  • the CD33 ⁇ TCP-T cells can effectively kill CD33 + MOLM-13 cells in vitro.
  • CD33 FITC-CD33; Brand: BD, Catalog No.: #561818;

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Abstract

Provided in the present invention are a TCP polypeptide and a viral vector containing a gene encoding the TCP polypeptide. The viral vector further contains a T cell-activation signaling molecule, and can effectively target, activate and transduce T cells.

Description

一种多肽及包含所述多肽基因的病毒载体A polypeptide and a viral vector containing the polypeptide gene 技术领域Technical Field

本发明涉及细胞疗法领域，具体涉及一种多肽及包含所述多肽基因的病毒载体。The present invention relates to the field of cell therapy, and in particular to a polypeptide and a viral vector comprising the polypeptide gene.

背景技术Background Art

在基因工程和细胞疗法领域中，脂质纳米颗粒(Lipid-Nanoparticles，“LNPs”)、类病毒颗粒(Virus-Like Particles，“VLPs”)、腺病毒、腺相关病毒、逆转录病毒载体(Retroviral Vector,RVV)和慢病毒载体(Lentiviral Vector,LVV)等是常用的基因载体之一。In the fields of genetic engineering and cell therapy, lipid nanoparticles (LNPs), virus-like particles (VLPs), adenovirus, adeno-associated virus, retroviral vector (RVV) and lentiviral vector (LVV) are commonly used gene vectors.

LVV和RVV因其能将如CAR基因等目的基因整合至宿主细胞的基因组中，使目的基因在宿主细胞中稳定表达，而被广泛应用于体内外转导T细胞制备CAR-T细胞中。LVV and RVV are widely used in in vitro and in vivo transduction of T cells to prepare CAR-T cells because they can integrate target genes such as CAR genes into the genome of host cells, allowing the target genes to be stably expressed in host cells.

然而，在包装LVV或RVV的过程中，CAR分子在信号肽的作用下可出膜表达在包装细胞的细胞膜上，并随着包装所得的LVV或RVV从包装细胞中出芽(Budding)，成为LVV或RVV的包膜的一部分。However, during the packaging of LVV or RVV, the CAR molecule can be expressed on the cell membrane of the packaging cell under the action of the signal peptide, and buds out from the packaging cell as the packaged LVV or RVV buds out, becoming part of the envelope of the LVV or RVV.

在转导T细胞等宿主细胞时，所述LVV或RVV的包膜与宿主细胞细胞膜发生膜融合，其包膜包含的CAR分子也随之成为宿主细胞细胞膜的一部分，这种CAR分子在病毒载体包膜和宿主细胞细胞膜之间的转移被称为“假转导”(Pseudotransduction)。When transducing host cells such as T cells, the envelope of the LVV or RVV fuses with the host cell membrane, and the CAR molecules contained in its envelope also become part of the host cell membrane. This transfer of CAR molecules between the viral vector envelope and the host cell membrane is called "pseudotransduction".

在假转导中，从病毒包膜转移至宿主细胞细胞膜上的CAR分子只能在短期内被检测到，随后不久便会被宿主细胞降解。In pseudotransduction, the CAR molecule transferred from the viral envelope to the host cell membrane is only detectable for a short period of time and is then degraded by the host cell shortly thereafter.

因此，假转导严重影响检测LVV或RVV转导如T细胞等宿主细胞制备CAR-T细胞等工程化免疫细胞的阳性率的准确性，不利于准确计算细胞疗法，尤其是体外CAR-T细胞疗法所需的CAR-T细胞等工程化免疫细胞的数量，影响疗效。Therefore, false transduction seriously affects the accuracy of detecting the positive rate of LVV or RVV transduction of host cells such as T cells to prepare engineered immune cells such as CAR-T cells, which is not conducive to accurately calculating the number of engineered immune cells such as CAR-T cells required for cell therapy, especially in vitro CAR-T cell therapy, and affects the efficacy.

并且，LVV或RVV还可通过其包膜包含的CAR分子的抗原结合区转导癌细胞等靶细胞，严重影响CAR-T细胞疗法疗效。In addition, LVV or RVV can also transduce target cells such as cancer cells through the antigen binding region of the CAR molecule contained in its envelope, seriously affecting the efficacy of CAR-T cell therapy.

因此，一种包含抗原结合区、但与CAR分子不同，不会分布或极少量分布在LVV或RVV的包膜上的多肽，以及包含所述多肽基因且可有效靶向活化刺激非活化T细胞的LVV或RVV，是减少传统体外CAR-T细胞疗法中的假转导、降低LVV或RVV在患者体内外转导癌细胞等靶细胞的能力,提高转导T细胞的靶向性和转导效率，从而提高细胞疗法疗效的关键。Therefore, a polypeptide comprising an antigen-binding region, but unlike a CAR molecule, not distributed or distributed in very small amounts on the envelope of LVV or RVV, and an LVV or RVV comprising the polypeptide gene and capable of effectively targeting and stimulating non-activated T cells for activation, are the key to reducing false transduction in traditional in vitro CAR-T cell therapy, reducing the ability of LVV or RVV to transduce target cells such as cancer cells in and outside the patient's body, improving the targeting and transduction efficiency of transduced T cells, and thus improving the efficacy of cell therapy.

发明内容Summary of the Invention

有鉴于此，为至少解决上述技术问题之一，本发明一个方面提供了一种病毒载体，In view of this, in order to solve at least one of the above technical problems, the present invention provides a viral vector in one aspect,

(a)所述病毒载体包含编码T细胞受体嵌合蛋白(T Cell Receptor Chimeric Protein，TCP)的多核苷酸；和(a) the viral vector comprises a polynucleotide encoding a T cell receptor chimeric protein (TCP); and

(b)所述病毒载体表面包含T细胞活化信号分子；(b) the surface of the viral vector contains T cell activation signal molecules;

其中，所述TCP包含：Wherein, the TCP includes:

(i)TCR/CD3复合体亚基相关多肽(TCR/CD3 Complex Subunit Related Peptide，TSP),所述TSP包含(1)TCR/CD3复合体亚基、(2)TCR/CD3复合体亚基功能性片段、(3)TCR/CD3复合体亚基变体和(4)TCR/CD3复合体亚基功能性片段的变体中的至少一种；和(i) a TCR/CD3 complex subunit related peptide (TSP), wherein the TSP comprises at least one of (1) a TCR/CD3 complex subunit, (2) a functional fragment of a TCR/CD3 complex subunit, (3) a variant of a TCR/CD3 complex subunit, and (4) a variant of a functional fragment of a TCR/CD3 complex subunit; and

(ii)抗原结合区。(ii) Antigen binding region.

本发明的一些实施例中，所述病毒载体为慢病毒载体(LVV)或逆转录病毒载体(RVV)。In some embodiments of the present invention, the viral vector is a lentiviral vector (LVV) or a retroviral vector (RVV).

本发明的一些实施例中，所述TCR/CD3复合体亚基选自TCRα、TCRβ、TCRγ、TCRδ、CD3γ、CD3δ、CD3ζ和CD3ε中的至少一种。In some embodiments of the present invention, the TCR/CD3 complex subunit is selected from at least one of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ζ and CD3ε.

本发明的一些实施例中，所述TCR/CD3复合体亚基功能性片段包含所述TCR/CD3复合体亚基的胞外区、跨膜区、胞内区、可变区和恒定区中的至少一种。In some embodiments of the present invention, the TCR/CD3 complex subunit functional fragment comprises at least one of the extracellular region, transmembrane region, intracellular region, variable region and constant region of the TCR/CD3 complex subunit.

本发明的一些实施例中，所述TSP包含CD3γ或其功能性片段；In some embodiments of the present invention, the TSP comprises CD3γ or a functional fragment thereof;

优选地，所述TSP包含CD3γ的跨膜区和胞内区；Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3γ;

更优选地，所述TSP包含CD3γ的跨膜区和胞内区以及至少一种以下蛋白的胞外区：TCRα、TCRβ、TCRγ、TCRδ、CD3ε、CD3ζ和CD3δ；More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3γ and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3ζ and CD3δ;

更进一步优选地，所述TSP包含CD3γ的跨膜区和胞内区以及(a)CD3δ的胞外区、(b)CD3ζ的胞外区或(c)CD3ε的胞外区；Still more preferably, the TSP comprises the transmembrane region and intracellular region of CD3γ and the extracellular region of (a) CD3δ, (b) CD3ζ or (c) CD3ε;

最优选地，所述CD3δ的胞外区、CD3ζ的胞外区或CD3ε的胞外区的C-末端位于所述CD3γ的跨膜区的N-末端方向，所述CD3γ的跨膜区的C-末端位于所述CD3γ的胞内区的N-末端方向。Most preferably, the C-terminus of the extracellular region of CD3δ, the extracellular region of CD3ζ or the extracellular region of CD3ε is located towards the N-terminus of the transmembrane region of CD3γ, and the C-terminus of the transmembrane region of CD3γ is located towards the N-terminus of the intracellular region of CD3γ.

本发明的一些实施例中,所述TSP包含CD3ε或其功能性片段；In some embodiments of the present invention, the TSP comprises CD3ε or a functional fragment thereof;

优选地，所述TSP包含CD3ε的跨膜区和胞内区；Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ε;

更优选地，所述TSP包含CD3ε的跨膜区和胞内区以及至少一种以下蛋白的胞外区：TCRα、TCRβ、TCRγ、TCRδ、CD3γ、CD3ζ和CD3δ；More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ε and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3ζ and CD3δ;

更进一步优选地，所述TSP包含CD3ε的跨膜区和胞内区；以及(a)CD3γ的胞外区、(b)CD3ζ的胞外区或(c)CD3δ的胞外区；More preferably, the TSP comprises the transmembrane region and intracellular region of CD3ε; and (a) the extracellular region of CD3γ, (b) the extracellular region of CD3ζ, or (c) the extracellular region of CD3δ;

最优选地，所述CD3γ的胞外区、CD3ζ的胞外区或CD3δ的胞外区的C-末端位于所述CD3ε的跨膜区的N-末端方向，所述CD3ε的跨膜区的C-末端位于所述CD3ε的胞内区的N-末端方向。Most preferably, the C-terminus of the extracellular region of CD3γ, the extracellular region of CD3ζ or the extracellular region of CD3δ is located in the N-terminal direction of the transmembrane region of CD3ε, and the C-terminus of the transmembrane region of CD3ε is located in the N-terminal direction of the intracellular region of CD3ε.

本发明的一些实施例中,所述TSP包含CD3δ或其功能性片段；In some embodiments of the present invention, the TSP comprises CD3δ or a functional fragment thereof;

优选地，所述TSP包含CD3δ的跨膜区和胞内区；Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3δ;

更优选地，所述TSP包含CD3δ的跨膜区和胞内区以及至少一种以下蛋白的胞外区：TCRα、TCRβ、TCRγ、TCRδ、CD3ε、CD3ζ和CD3γ；More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3δ and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3ζ and CD3γ;

更进一步优选地，所述TSP包含CD3δ的跨膜区和胞内区；以及(a)CD3γ的胞外区、(b)CD3ζ的胞外区或(c)CD3ε的胞外区；More preferably, the TSP comprises the transmembrane region and intracellular region of CD3δ; and (a) the extracellular region of CD3γ, (b) the extracellular region of CD3ζ, or (c) the extracellular region of CD3ε;

最优选地，所述CD3γ的胞外区、CD3ζ的胞外区或CD3ε的胞外区的C-末端位于所述CD3δ的跨膜区的N-末端方向，所述CD3δ的跨膜区的C-末端位于所述CD3δ的胞内区的N-末端方向。Most preferably, the C-terminus of the extracellular region of CD3γ, CD3ζ or CD3ε is located towards the N-terminus of the transmembrane region of CD3δ, and the C-terminus of the transmembrane region of CD3δ is located towards the N-terminus of the intracellular region of CD3δ.

本发明的一些实施例中,所述TSP包含CD3ζ或其功能性片段；In some embodiments of the present invention, the TSP comprises CD3ζ or a functional fragment thereof;

优选地，所述TSP包含CD3ζ的跨膜区和胞内区；Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ζ;

更优选地，所述TSP包含CD3ζ的跨膜区和胞内区以及至少一种以下蛋白的胞外区：TCRα、TCRβ、TCRγ、TCRδ、CD3ε、CD3δ和CD3γ；More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ζ and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3δ and CD3γ;

更进一步优选地，所述TSP包含CD3ζ的跨膜区和胞内区；以及(a)CD3γ的胞外区、(b)CD3δ的胞外区或(c)CD3ε的胞外区；More preferably, the TSP comprises the transmembrane region and intracellular region of CD3ζ; and (a) the extracellular region of CD3γ, (b) the extracellular region of CD3δ, or (c) the extracellular region of CD3ε;

最优选地，所述CD3γ的胞外区、CD3δ的胞外区或CD3ε的胞外区的C-末端位于所述CD3ζ的跨膜区的N-末端方向，所述CD3ζ的跨膜区的C-末端位于所述CD3ζ的胞内区的N-末端方向。Most preferably, the C-terminus of the extracellular region of CD3γ, CD3δ or CD3ε is located in the direction of the N-terminus of the transmembrane region of CD3ζ, and the C-terminus of the transmembrane region of CD3ζ is located in the direction of the N-terminus of the intracellular region of CD3ζ.

本发明的一些实施例中，所述TSP包含TCRα或其功能性片段和TCRβ或其功能性片段；In some embodiments of the present invention, the TSP comprises TCRα or a functional fragment thereof and TCRβ or a functional fragment thereof;

优选地，所述TSP包含TCRα的恒定区和TCRβ的恒定区。Preferably, the TSP comprises the constant region of TCRα and the constant region of TCRβ.

本发明的一些实施例中，所述TCRα的恒定区发生突变，所述突变包括所述TCRα恒定区包含半胱氨酸替换；所述突变可增强基于二硫键的链间相互作用；In some embodiments of the present invention, the constant region of the TCRα is mutated, and the mutation includes a cysteine substitution in the TCRα constant region; the mutation can enhance disulfide bond-based interchain interactions;

优选地，所述TCRα恒定区源自人或小鼠的TCRα恒定区，所述人的TCRα恒定区包含如SEQ ID NO:39所示的氨基酸序列，所述小鼠的TCRα恒定区包含如SEQ ID NO:40所示的氨基酸序列；Preferably, the TCRα constant region is derived from a human or mouse TCRα constant region, the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 39, and the mouse TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 40;

所述突变包括所述人的TCRα恒定区的第47位氨基酸苏氨酸T替换为半胱氨酸C(人的TCRα恒定区变体1)或所述小鼠的TCRα恒定区的第47位氨基酸苏氨酸T替换为半胱氨酸C(小鼠的TCRα恒定区变体1)；The mutation includes replacing the 47th amino acid Threonine T of the human TCRα constant region with Cysteine C (human TCRα constant region variant 1) or replacing the 47th amino acid Threonine T of the mouse TCRα constant region with Cysteine C (mouse TCRα constant region variant 1);

所述人的TCRα恒定区变体1包含如SEQ ID NO:41所示的氨基酸序列，所述小鼠的TCRα恒定区变体1包含如SEQ ID NO:42所示的氨基酸序列。The human TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:41, and the mouse TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:42.

本发明的一些实施例中，所述TCRβ恒定区发生突变，所述突变包括所述TCRβ恒定区包含半胱氨酸替换；所述突变可增强基于二硫键的链间相互作用；In some embodiments of the present invention, the TCRβ constant region is mutated, and the mutation includes a cysteine substitution in the TCRβ constant region; the mutation can enhance disulfide bond-based interchain interactions;

优选地，所述TCRβ恒定区源自人的TCRβ恒定区，所述人的TCRβ恒定区包含如SEQ ID NO:43(hTRBC1)或SEQ ID NO:44(hTRBC2)所示的氨基酸序列，所述突变包括所述人的TCRβ恒定区的第56位氨基酸丝氨酸S替换为半胱氨酸C(人的TCRβ恒定区变体1)，所述人的TCRβ的恒定区变体1包含如SEQ ID NO:45或SEQ ID NO:46所示的氨基酸序列。Preferably, the TCRβ constant region is derived from a human TCRβ constant region, and the human TCRβ constant region comprises the amino acid sequence shown in SEQ ID NO: 43 (hTRBC1) or SEQ ID NO: 44 (hTRBC2), and the mutation includes replacing the 56th amino acid serine S of the human TCRβ constant region with cysteine C (human TCRβ constant region variant 1), and the human TCRβ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.

本发明的一些实施例中，所述TCRα恒定区发生突变，所述突变包括所述TCRα恒定区的至少一个不带电氨基酸被替换为疏水氨基酸；所述突变增加了TCRα跨膜区的疏水性，抵消了TCRα跨膜区携带正电荷所导致的不稳定性，使所述TCRα及其与TCRβ形成的二聚体能更稳定地表达在T细胞膜上，进而获得更好的功能；In some embodiments of the present invention, the TCRα constant region undergoes a mutation, wherein the mutation comprises replacing at least one uncharged amino acid in the TCRα constant region with a hydrophobic amino acid; the mutation increases the hydrophobicity of the TCRα transmembrane region, offsetting the instability caused by the positive charge carried by the TCRα transmembrane region, thereby enabling the TCRα and its dimer formed with TCRβ to be more stably expressed on the T cell membrane, thereby achieving better function;

优选地，所述TCRα恒定区源自人的TCRα恒定区，所述人的TCRα恒定区包含如SEQ ID NO:39所示的氨基酸序列，所述突变包括所述人的TCRα恒定区的第115位氨基酸、118位氨基酸、第119位氨基酸中的至少一个氨基酸被疏水氨基酸替换；Preferably, the TCRα constant region is derived from a human TCRα constant region, the human TCRα constant region comprising the amino acid sequence as shown in SEQ ID NO: 39, and the mutation comprises replacement of at least one of amino acids 115, 118, and 119 of the human TCRα constant region with a hydrophobic amino acid;

更优选地，所述突变包括所述人的TCRα恒定区包含至少一种以下突变：第115位氨基酸丝氨酸S替换为亮氨酸L、第118位氨基酸甘氨酸G替换为缬氨酸V、第119位氨基酸苯丙氨酸F替换为被亮氨酸L；More preferably, the mutations include at least one of the following mutations in the human TCRα constant region: substitution of amino acid serine S at position 115 with leucine L, substitution of amino acid glycine G at position 118 with valine V, substitution of amino acid phenylalanine F at position 119 with leucine L;

更进一步优选地，所述突变包括所述人的TCRα恒定区的第115位氨基酸丝氨酸S替换为亮氨酸L、第118位氨基酸甘氨酸G替换为缬氨酸V和第119位氨基酸苯丙氨酸F替换为亮氨酸L(人的TCRα恒定区变体2)，所述人的TCRα恒定区变体2包含如SEQ ID NO:47所示的氨基酸序列。Further preferably, the mutation includes replacing the 115th amino acid serine S of the human TCRα constant region with leucine L, replacing the 118th amino acid glycine G with valine V, and replacing the 119th amino acid phenylalanine F with leucine L (human TCRα constant region variant 2), and the human TCRα constant region variant 2 comprises the amino acid sequence shown in SEQ ID NO:47.

本发明的一些实施例中，所述TCRα恒定区源自人的TCRα恒定区，所述人的TCRα恒定区包含如SEQ ID NO:39所示的氨基酸序列；所述人的TCRα恒定区发生突变，所述突变包括所述人的TCRα恒定区的第47位氨基酸苏氨酸T替换为半胱氨酸C、第115位氨基酸丝氨酸S替换为亮氨酸L、第118位氨基酸甘氨酸G替换为缬氨酸V和第119位氨基酸苯丙氨酸F替换为亮氨酸L(人的TCRα恒定区变体3)，所述人的TCRα恒定区变体3包含如SEQ ID NO:48所示的氨基酸序列；In some embodiments of the present invention, the TCRα constant region is derived from a human TCRα constant region, the human TCRα constant region comprising the amino acid sequence set forth in SEQ ID NO: 39; the human TCRα constant region undergoes a mutation, the mutation comprising substitution of amino acid threonine T at position 47 of the human TCRα constant region with cysteine C, amino acid serine S at position 115 with leucine L, amino acid glycine G at position 118 with valine V, and amino acid phenylalanine F at position 119 with leucine L (human TCRα constant region variant 3), the human TCRα constant region variant 3 comprising the amino acid sequence set forth in SEQ ID NO: 48;

优选地，所述TCRα恒定区和所述TCRβ恒定区源自人的TCRα恒定区和人的TCRβ恒定区；所述人的TCRα恒定区包含如SEQ ID NO:39所示的氨基酸序列，所述人的TCRβ恒定区包含如SEQ ID NO:43或SEQ ID NO:44所示的氨基酸序列；所述人的TCRα恒定区和所述人的TCRβ恒定区发生突变，所述突变包括所述人的TCRα恒定区的第47位氨基酸替换为半胱氨酸、第115位氨基酸丝氨酸S替换为亮氨酸L、第118位氨基酸甘氨酸G替换为缬氨酸V和第119位氨基酸苯丙氨酸F替换为亮氨酸L(人的TCRα恒定区变体3)，以及所述人的TCRβ恒定区的第56位氨基酸丝氨酸S替换为半胱氨酸C(人的TCRβ恒定区变体1)；所述人的TCRα恒定区变体3包含如SEQ ID NO:48所示的氨基酸序列，所述人的TCRβ的恒定区变体1包含如SEQ ID NO:45或SEQ ID NO:46所示的氨基酸序列。Preferably, the TCRα constant region and the TCRβ constant region are derived from human TCRα constant region and human TCRβ constant region; the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO:39, and the human TCRβ constant region comprises the amino acid sequence shown in SEQ ID NO:43 or SEQ ID NO:44; the human TCRα constant region and the human TCRβ constant region are mutated, and the mutation includes the replacement of the 47th amino acid of the human TCRα constant region with cysteine and the 115th amino acid serine S The human TCRα constant region variant 3 comprises the amino acid sequence shown in SEQ ID NO: 48, the human TCRβ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.

本发明的一些实施例中，前述任一种TSP还可包含所述TCR/CD3复合体亚基的铰链区。In some embodiments of the present invention, any of the aforementioned TSPs may further comprise the hinge region of the TCR/CD3 complex subunit.

本发明的一些实施例中，所述抗原结合区通过连接肽可操作地连接至前述任一种TSP的N-末端。In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of any of the aforementioned TSPs via a linker peptide.

本发明的一些实施例中，所述连接肽包括柔性连接肽。In some embodiments of the present invention, the connecting peptide includes a flexible connecting peptide.

本发明的一些实施例中，所述柔性连接肽选自(G4S)n连接肽和包含如SEQ ID NO:23所示的氨基酸序列的连接肽1；其中，n＝1至4；In some embodiments of the present invention, the flexible connecting peptide is selected from a (G 4 S) n connecting peptide and a connecting peptide 1 comprising an amino acid sequence as shown in SEQ ID NO: 23; wherein n=1 to 4;

优选地，所述柔性连接肽为所述连接肽1、(G4S)3连接肽(连接肽2)或连接肽3：GSSGGSGGGGSGGGGSGGGGSSG(SEQ ID NO:63)。Preferably, the flexible connecting peptide is the connecting peptide 1, the (G 4 S) 3 connecting peptide (connecting peptide 2) or the connecting peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 63).

本发明的一些实施例中，所述柔性连接肽是所述连接肽1。In some embodiments of the present invention, the flexible connecting peptide is the connecting peptide 1.

本发明的一些实施例中，所述抗原结合区结合疾病相关抗原。In some embodiments of the present invention, the antigen binding region binds to a disease-associated antigen.

本发明的一些实施例中，所述疾病选自癌症和自身免疫性疾病；所述癌症包括血液癌和实体癌。In some embodiments of the present invention, the disease is selected from cancer and autoimmune diseases; and the cancer includes blood cancer and solid cancer.

本发明的一些实施例中，所述疾病相关抗原选自：In some embodiments of the present invention, the disease-associated antigen is selected from:

TSHR、CD2、CD3、CD4、CD5、CD7、CD8、CD14、CD15、CD19、CD20、CD21、CD23、CD24、CD25、CD28、CD37、CD38、CD40、CD40L、CD44、CD46、CD47、CD52、CD54、CD56、CD70、CD73、CD80、CD97、CD123、CD22、CD126、CD138、DR4、DR5、TAC、TEM1/CD248、VEGF、GUCY2C、EGP40、EGP-2、EGP-4、CDL33、IFNAR1、DLL3、kappa轻链、TIM3、tEGFR、IL-22Ra、IL-2、ErbB3、ErbB4、MUC16、MAGE-A3、MAGE-A6、NKG2DL、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvⅢ、GD2、GD3、BCMA、GPRC5D、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、uPAR、GCC(鸟苷酸环化酶C)、EPCAM、Nectin4、B7H3、KIT、IL-13Ra2、间皮素、IL-1Ra、PSCA、PRSS21、VEGFR2、Lewis-Y、CD24、PDGFR-β、SSEA-4、CD20、AFP、Folate受体α、Her2/neu/ERBB2、MUC1、EGFR、CS1、CD138、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-Ⅰ受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GM1、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD179a、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-1a、MAGE-A1、豆英蛋白、HPV E6/E7、MAGE-A4、MART-1、WT-1、ETV6-AML、精子蛋白17、XAGE1、Tie2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白、存活蛋白和端粒酶、PCTA-1/Galectin 8、MelanA/MARTI、Ras突变体，hTERT、肉瘤易位断点、ML-IAP、TMPRSS2-ETS融合基因/ERG、NA17、PAX3、雄激素受体、CyclinB1、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79A、CD79B、ASGPR、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLLI、PD1、PDL1、PDL2、TGFβ、APRIL、MSLN和NKG2D中的至少一种。TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38 , CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138 , DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3 , tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, uPAR, GCC (guanylate cyclase C), EPCAM, Nectin4, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Le wis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2/neu/ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-Ⅰ receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6 K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6/E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1/ At least one of Galectin 8, MelanA/MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2-ETS fusion gene/ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79A, CD79B, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, and NKG2D.

本发明的一些实施例中，所述疾病相关抗原选自CD19、CD20、CD33、MSLN、CD79B、CD8、ASGPR、BCMA、CEA、uPAR、DLL3、GCC、Nectin4、HER2、Claudin18.2和GUCY2C中的至少一种。In some embodiments of the present invention, the disease-associated antigen is selected from at least one of CD19, CD20, CD33, MSLN, CD79B, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2 and GUCY2C.

本发明的一些实施例中，所述疾病相关抗原是人的疾病相关抗原。In some embodiments of the present invention, the disease-associated antigen is a human disease-associated antigen.

本发明的一些实施例中，所述抗原结合区包含抗体或其抗原结合片段和/或配体或其受体结合片段，所述抗体或其抗原结合片段选自免疫球蛋白(全长抗体)、半抗体、Fab、Fab'、F(ab')2、Fv片段、单链可变区片段(scFv)、二硫键稳定性抗体(dsFv)、抗体的重链可变区(VH)或轻链可变区(VL)、由VH和CH1结构域组成的Fd片段、线性抗体和单域抗体(纳米抗体)中的至少一种。In some embodiments of the present invention, the antigen-binding region comprises an antibody or an antigen-binding fragment thereof and/or a ligand or a receptor-binding fragment thereof, and the antibody or antigen-binding fragment thereof is selected from at least one of an immunoglobulin (full-length antibody), a half antibody, Fab, Fab', F(ab') 2 , an Fv fragment, a single-chain variable region fragment (scFv), a disulfide-stabilized antibody (dsFv), an antibody heavy chain variable region (VH) or a light chain variable region (VL), an Fd fragment consisting of a VH and a CH1 domain, a linear antibody, and a single-domain antibody (nanoantibody).

本发明的一些实施例中，前述任一种TCRα恒定区或其变体可操作地连接至VH区或VL区；前述任一种TCRβ恒定区或其变体可操作地连接至VH区或VL区。In some embodiments of the present invention, any of the aforementioned TCRα constant regions or variants thereof can be operably linked to the VH region or the VL region; any of the aforementioned TCRβ constant regions or variants thereof can be operably linked to the VH region or the VL region.

本发明的一些实施例中，当前述任一种TCRα恒定区或其变体可操作地连接至VH区时，前述任一种TCRβ恒定区或其变体可操作地连接至VL区；当前述任一种TCRα恒定区或其变体可操作地连接至VL区时，前述任一种TCRβ恒定区或其变体可操作地连接至VH区。In some embodiments of the present invention, when any of the aforementioned TCRα constant regions or variants thereof are operably linked to the VH region, any of the aforementioned TCRβ constant regions or variants thereof are operably linked to the VL region; when any of the aforementioned TCRα constant regions or variants thereof are operably linked to the VL region, any of the aforementioned TCRβ constant regions or variants thereof are operably linked to the VH region.

本发明的一些实施例中，所述VH区和VL区源自同种抗体或其抗原结合片段，或配体或其受体结合片段。In some embodiments of the present invention, the VH region and VL region are derived from the same antibody or antigen-binding fragment thereof, or ligand or receptor-binding fragment thereof.

本发明的一些实施例中，所述抗原结合区选自以下抗原结合区中的至少一种：In some embodiments of the present invention, the antigen binding region is selected from at least one of the following antigen binding regions:

(a)结合人CD19的抗原结合区，所述抗原结合区是源自FMC-63的scFv(FMC63-scFv)，所述FMC63-scFv的VH区的氨基酸序列如SEQ ID NO:18所示，所述FMC63-scFv的VL区的氨基酸序列如SEQ ID NO:19所示；(a) an antigen-binding region that binds to human CD19, wherein the antigen-binding region is a scFv derived from FMC-63 (FMC63-scFv), the amino acid sequence of the VH region of the FMC63-scFv being as shown in SEQ ID NO: 18, and the amino acid sequence of the VL region of the FMC63-scFv being as shown in SEQ ID NO: 19;

(b)结合人去唾液酸糖蛋白受体(ASGPR)的抗原结合区，所述抗原结合区是抗人ASGPR的scFv(anti-ASGPR-scFv)；所述anti-ASGPR-scFv的VL区的氨基酸序列如SEQ ID NO:51所示；所述anti-ASGPR-scFv的氨基酸序列如SEQ ID NO:52所示；(b) an antigen-binding region that binds to human asialoglycoprotein receptor (ASGPR), wherein the antigen-binding region is an anti-human ASGPR scFv (anti-ASGPR-scFv); the amino acid sequence of the VL region of the anti-ASGPR-scFv is shown in SEQ ID NO: 51; the amino acid sequence of the anti-ASGPR-scFv is shown in SEQ ID NO: 52;

(c)结合人HER2的抗原结合区，所述抗原结合区是源自抗人HER2的抗体Pertuzumab的scFv(Pertuzumab-scFv)；所述Pertuzumab-scFv的VL区的氨基酸序列如SEQ ID NO:53所示；所述Pertuzumab-scFv的VH区的氨基酸序列如SEQ ID NO:54所示；(c) an antigen-binding region that binds to human HER2, wherein the antigen-binding region is derived from the anti-human HER2 antibody Pertuzumab scFv (Pertuzumab-scFv); the amino acid sequence of the VL region of the Pertuzumab-scFv is shown in SEQ ID NO: 53; the amino acid sequence of the VH region of the Pertuzumab-scFv is shown in SEQ ID NO: 54;

(d)结合人MESOTHELIN(MSLN)的抗原结合区，所述抗原结合区是源自抗人MSLN的抗体PE38的scFv(PE38-scFv)；所述PE38-scFv的VH区的氨基酸序列SEQ ID NO:55所示；所述PE38-scFv的VL区的氨基酸序列如SEQ ID NO:56所示；(d) an antigen-binding region that binds to human mesothelin (MSLN), the antigen-binding region being a scFv derived from the anti-human MSLN antibody PE38 (PE38-scFv); the amino acid sequence of the VH region of the PE38-scFv being shown in SEQ ID NO: 55; and the amino acid sequence of the VL region of the PE38-scFv being shown in SEQ ID NO: 56;

(e)结合人CD8的抗原结合区，所述抗原结合区是抗人CD8的scFv(anti-CD8-scFv)；所述anti-CD8-scFv的VH区的氨基酸序列如SEQ ID NO:57所示；所述anti-CD8-scFv的VL区的氨基酸序列如SEQ ID NO:58所示；(e) an antigen-binding region that binds to human CD8, wherein the antigen-binding region is an anti-human CD8 scFv (anti-CD8-scFv); the amino acid sequence of the VH region of the anti-CD8-scFv is shown in SEQ ID NO: 57; and the amino acid sequence of the VL region of the anti-CD8-scFv is shown in SEQ ID NO: 58;

(f)结合人CD33的抗原结合区，所述抗原结合区是源自抗人CD33的抗体Gemtuzumab的scFv(Gemtuzumab-scFv)；所述Gemtuzumab-scFv的VL区的氨基酸序列如SEQ ID NO:59所示；所述Gemtuzumab-scFv的VH区的氨基酸序列如SEQ ID NO:60所示；和(f) an antigen-binding region that binds to human CD33, wherein the antigen-binding region is derived from the anti-human CD33 antibody Gemtuzumab scFv (Gemtuzumab-scFv); the amino acid sequence of the VL region of the Gemtuzumab-scFv is shown in SEQ ID NO: 59; the amino acid sequence of the VH region of the Gemtuzumab-scFv is shown in SEQ ID NO: 60; and

(g)结合人CD79B的抗原结合区，所述抗原结合区是源自抗人CD79B的抗体SN-8的scFv(SN8-scFv)；所述抗SN8-scFv的VH区的氨基酸序列如SEQ ID NO:61所示；所述SN8-scFv的VL区的氨基酸序列如SEQ ID NO:62所示。(g) an antigen-binding region that binds to human CD79B, wherein the antigen-binding region is derived from the scFv (SN8-scFv) of the anti-human CD79B antibody SN-8; the amino acid sequence of the VH region of the anti-SN8-scFv is shown in SEQ ID NO:61; the amino acid sequence of the VL region of the SN8-scFv is shown in SEQ ID NO:62.

本发明的一些实施例中，前述任一种T细胞受体嵌合蛋白不包含信号肽。In some embodiments of the present invention, any of the aforementioned T cell receptor chimeric proteins does not comprise a signal peptide.

本发明的一些实施例中，前述任一种T细胞受体嵌合蛋白包含信号肽。In some embodiments of the present invention, any of the aforementioned T cell receptor chimeric proteins comprises a signal peptide.

本发明的一些实施例中，所述信号肽只要可介导所述TCP出膜表达便无特别地限定。In some embodiments of the present invention, the signal peptide is not particularly limited as long as it can mediate the membrane expression of TCP.

本发明的一些实施例中，所述信号肽选自以下信号肽：CD8α信号肽、CD28信号肽、IgG信号肽、HLA-A信号肽、CD3γ信号肽、CD3δ信号肽、CD3ζ信号肽和CD3ε信号肽。In some embodiments of the present invention, the signal peptide is selected from the following signal peptides: CD8α signal peptide, CD28 signal peptide, IgG signal peptide, HLA-A signal peptide, CD3γ signal peptide, CD3δ signal peptide, CD3ζ signal peptide and CD3ε signal peptide.

本发明的一些实施例中，所述信号肽为人CD8α信号肽。In some embodiments of the present invention, the signal peptide is a human CD8α signal peptide.

本发明的一些实施例中，所述人CD8α信号肽的氨基酸序列与SEQ ID NO:12具有至少约85％、86％、87％、88％、89％、90％、91％、92％、93、94％、95％、96％、97％、98％、99％或100％同一性。In some embodiments of the present invention, the amino acid sequence of the human CD8α signal peptide is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:12.

本发明的一些实施例中，所述T细胞受体嵌合蛋白还包含共刺激信号传导结构域。In some embodiments of the present invention, the T cell receptor chimeric protein further comprises a co-stimulatory signaling domain.

本发明的一些实施例中，所述共刺激信号传导结构域源自至少一种以下蛋白的共刺激信号传导结构域：In some embodiments of the present invention, the costimulatory signaling domain is derived from the costimulatory signaling domain of at least one of the following proteins:

CD28、4-1BB、CD27、CD2、CD3、CD7、CD8、CD8α、CD8β、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcαRlγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-l、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD40和MyD88。CD28, 4-1BB, CD27, CD2, CD3, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRl γ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88.

本发明的一些实施例中，所述共刺激信号传导结构域源自人4-1BB和/或CD28的共刺激信号传导结构域。In some embodiments of the present invention, the costimulatory signaling domain is derived from the costimulatory signaling domain of human 4-1BB and/or CD28.

本发明的一些实施例中，所述人4-1BB的共刺激信号传导结构域的氨基酸序列与SEQ ID NO:49具有至少约85％、86％、87％、88％、89％、90％、91％、92％、93、94％、95％、96％、97％、98％、99％或100％同一性。In some embodiments of the present invention, the amino acid sequence of the costimulatory signaling domain of human 4-1BB is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:49.

本发明的一些实施例中，前述任一种T细胞受体嵌合蛋白在T细胞中表达时(a)并入内源性TCR/CD3复合体或TCR/CD3复合体亚基或其功能性片段中；或(b)与内源性TCR/CD3复合体或内源性TCR/CD3复合体亚基或其功能性片段发生功能性相互作用。In some embodiments of the present invention, any of the aforementioned T cell receptor chimeric proteins, when expressed in T cells, (a) is incorporated into an endogenous TCR/CD3 complex or a TCR/CD3 complex subunit or a functional fragment thereof; or (b) functionally interacts with an endogenous TCR/CD3 complex or an endogenous TCR/CD3 complex subunit or a functional fragment thereof.

本发明的一些实施例中，前述任一种TCP在T细胞以外的细胞中不出膜表达或所述TCP在T细胞以外的细胞中出膜表达的效率相对于所述TCP在T细胞中出膜表达的效率降低。In some embodiments of the present invention, any of the aforementioned TCPs is not expressed on the membrane in cells other than T cells, or the efficiency of membrane expression of the TCP on cells other than T cells is lower than the efficiency of membrane expression of the TCP on T cells.

本发明的一些实施例中，所述T细胞活化信号分子包括T细胞活化初级信号分子。In some embodiments of the present invention, the T cell activation signaling molecule includes a T cell activation primary signaling molecule.

非活化T细胞(Non-Activated)，是指不扩增、不分化、静息态、未识别抗原、未经T细胞活化信号分子如T细胞活化初级和次级信号分子活化刺激的T细胞，例如处于细胞周期G0阶段的T细胞、静息(Resting/Quiescent)的T细胞或幼稚T细胞。静息T细胞也称为静止T细胞或天然T细胞，是指不具有有丝分裂活性或未暴露于抗原呈递细胞(诸如巨噬细胞或树突细胞)上呈递的同源抗原的T细胞。Non-activated T cells are T cells that are not proliferated, differentiated, in a resting state, do not recognize antigens, and have not been activated by T cell activation signaling molecules such as primary and secondary T cell activation signaling molecules, such as T cells in the G0 phase of the cell cycle, resting/quiescent T cells, or immature T cells. T cells. Resting T cells, also known as quiescent T cells or naive T cells, are T cells that are not mitotically active or have not been exposed to cognate antigens presented on antigen-presenting cells, such as macrophages or dendritic cells.

T细胞活化初级信号分子结合T细胞表面蛋白，参与T细胞受体(T Cell Receptor，“TCR”)介导的T细胞活化(TCR-mediated T Cell Activation)。T cell activation primary signal molecules bind to T cell surface proteins and participate in T cell receptor (T Cell Receptor, "TCR")-mediated T cell activation (TCR-mediated T Cell Activation).

本发明的一些实施例中，所述T细胞活化初级信号分子参与将TCR转化为活性PTK(protein tyrosine kinase)，所述活性PTK可磷酸化一系列底物，从而产生大量下游信号，当这些信号适当整合时(与其他共受体的信号一起)，导致T细胞活化(Smith-Garvin JE,Koretzky GA,Jordan MS.T cell activation.Annu Rev Immunol.2009；27:591-619)。In some embodiments of the present invention, the T cell activation primary signal molecule is involved in converting TCR into active PTK (protein tyrosine kinase), which can phosphorylate a series of substrates to generate a large number of downstream signals. When these signals are properly integrated (together with signals from other co-receptors), they lead to T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

本发明的一些实施例中，所述T细胞活化初级信号分子结合TCR/CD3复合体亚基和TCR/CD3复合体亚基功能性片段中的至少一种；所述TCR/CD3复合体亚基选自CD3ε、CD3γ、CD3δ、CD3ζ、TCRγ、TCRδ、TCRα和TCRβ中的至少一种。In some embodiments of the present invention, the T cell activation primary signal molecule binds to at least one of a TCR/CD3 complex subunit and a functional fragment of a TCR/CD3 complex subunit; the TCR/CD3 complex subunit is selected from at least one of CD3ε, CD3γ, CD3δ, CD3ζ, TCRγ, TCRδ, TCRα and TCRβ.

本发明的一些实施例中，所述TCR/CD3复合体亚基是人的TCR/CD3复合体亚基。In some embodiments of the present invention, the TCR/CD3 complex subunit is a human TCR/CD3 complex subunit.

CD3与TCR在T细胞中形成TCR/CD3复合体，参与辅助T细胞(CD4+T细胞)和细胞毒性T细胞(CD8+T细胞)的活化。CD3 and TCR form a TCR/CD3 complex in T cells, which participates in the activation of helper T cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cells).

本发明的一些实施例中，所述T细胞活化初级信号分子包括抗CD3抗体或其抗原结合片段。In some embodiments of the present invention, the T cell activation primary signal molecule comprises an anti-CD3 antibody or an antigen-binding fragment thereof.

本发明的一些实施例中，所述抗CD3抗体或其抗原结合片段特异性结合人CD3。In some embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof specifically binds to human CD3.

本发明的一些实施例中，所述抗CD3抗体选自OKT3、UCHT1、YTH12.5和TR66中的至少一种。In some embodiments of the present invention, the anti-CD3 antibody is selected from at least one of OKT3, UCHT1, YTH12.5 and TR66.

本发明的一些实施例中，所述抗CD3抗体是SP34。In some embodiments of the present invention, the anti-CD3 antibody is SP34.

本发明的一些实施例中，所述抗CD3抗体或其抗原结合片段不可结合所述TSP。In some embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof cannot bind to the TSP.

本发明的一些实施例中，所述抗CD3抗体是抗CD3ε抗体，所述TSP不是CD3ε、或其功能性片段、或其变体、或其变体的功能性片段。In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3ε antibody, and the TSP is not CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

本发明的一些实施例中，所述抗CD3ε抗体或其抗原结合片段特异性结合人CD3ε(Uniprot ID:P07766)。In some embodiments of the present invention, the anti-CD3ε antibody or its antigen-binding fragment specifically binds to human CD3ε (Uniprot ID: P07766).

本发明的一些实施例中，所述抗CD3ε抗体或其抗原结合片段是UCHT1或其抗原结合片段；In some embodiments of the present invention, the anti-CD3ε antibody or antigen-binding fragment thereof is UCHT1 or an antigen-binding fragment thereof;

优选地，所述UCHT1的抗原结合片段是scFv(UCHT1-scFv)；Preferably, the antigen-binding fragment of UCHT1 is a scFv (UCHT1-scFv);

更优选地，所述UCHT1-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:87-89所示，所述UCHT1-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:90-92所示。More preferably, the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NO: 87-89, respectively, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NO: 90-92, respectively.

本发明的一些实施例中，所述抗CD3ε抗体或其抗原结合片段是OKT3或其抗原结合片段；In some embodiments of the present invention, the anti-CD3ε antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof;

优选地，所述OKT3的抗原结合片段是scFv(OKT3-scFv)；Preferably, the antigen-binding fragment of OKT3 is scFv (OKT3-scFv);

更优选地，所述OKT3-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:104-106所示，所述OKT3-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:107-109所示；More preferably, the amino acid sequences of the HCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NOs: 104-106, respectively, and the amino acid sequences of the LCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NOs: 107-109, respectively;

更进一步优选地，所述OKT3-scFv的VH区的氨基酸序列如SEQ ID NO:119所示，所述OKT3-scFv的VL区的氨基酸序列如SEQ ID NO:120所示。More preferably, the amino acid sequence of the VH region of the OKT3-scFv is shown in SEQ ID NO: 119, and the amino acid sequence of the VL region of the OKT3-scFv is shown in SEQ ID NO: 120.

本发明的一些实施例中，所述抗CD3ε抗体或其抗原结合片段是SP34或其抗原结合片段；In some embodiments of the present invention, the anti-CD3ε antibody or antigen-binding fragment thereof is SP34 or an antigen-binding fragment thereof;

优选地，所述SP34的抗原结合片段是scFv(SP34-scFv)；Preferably, the antigen-binding fragment of SP34 is scFv (SP34-scFv);

更优选地，所述SP34-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:113-115所示，所述SP34-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:116-118所示；More preferably, the amino acid sequences of the HCDR1-3 regions of the SP34-scFv are shown as SEQ ID NOs: 113-115, respectively, and the amino acid sequences of the LCDR1-3 regions of the SP34-scFv are shown as SEQ ID NOs: 116-118, respectively;

更进一步优选地，所述SP34-scFv的VH区的氨基酸序列如SEQ ID NO:111所示，所述SP34-scFv的VL区的氨基酸序列如SEQ ID NO:112所示。More preferably, the amino acid sequence of the VH region of the SP34-scFv is shown in SEQ ID NO: 111, and the amino acid sequence of the VL region of the SP34-scFv is shown in SEQ ID NO: 112.

本发明的一些实施例中，当所述抗CD3抗体或其抗原结合片段是所述抗CD3ε抗体或其抗原结合片段，所述TSP是：In some embodiments of the present invention, when the anti-CD3 antibody or antigen-binding fragment thereof is the anti-CD3ε antibody or antigen-binding fragment thereof, the TSP is:

(a)CD3γ、或其功能性片段、或其变体、或其变体的功能性片段；(a) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;

(b)CD3δ、或其功能性片段、或其变体、或其变体的功能性片段；或(b) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or

(c)CD3ζ，或其功能性片段、或其变体、或其变体的功能性片段。(c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

本发明的一些实施例中，所述抗CD3抗体是抗CD3γ抗体，所述TSP不是CD3γ、或其功能性片段、或其变体、或其变体的功能性片段。In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3γ antibody, and the TSP is not CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

本发明的一些实施例中，当所述抗CD3抗体或其抗原结合片段是所述抗CD3γ抗体或其抗原结合片段时，所述TSP是：In some embodiments of the present invention, when the anti-CD3 antibody or antigen-binding fragment thereof is the anti-CD3γ antibody or antigen-binding fragment thereof, the TSP is:

(a)CD3ε、或其功能性片段、或其变体、或其变体的功能性片段；(a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;

(b)CD3δ、或其功能性片段、或其变体、或其变体的功能性片段；或(b) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or

(c)CD3ζ，或其功能性片段、或其变体、或其变体的功能性片段。(c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

本发明的一些实施例中，所述抗CD3抗体是抗CD3δ抗体，所述TSP不是CD3δ、或其功能性片段、或其变体、或其变体的功能性片段。In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3δ antibody, and the TSP is not CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

本发明的一些实施例中，所述抗CD3δ抗体或其抗原结合片段是TR66或其抗原结合片段；In some embodiments of the present invention, the anti-CD3δ antibody or antigen-binding fragment thereof is TR66 or an antigen-binding fragment thereof;

优选地，所述TR66的抗原结合片段是scFv(TR66-scFv)。Preferably, the antigen-binding fragment of TR66 is scFv (TR66-scFv).

本发明的一些实施例中，当所述抗CD3抗体或其抗原结合片段是所述抗CD3δ抗体或其抗原结合片段时，所述TSP是：In some embodiments of the present invention, when the anti-CD3 antibody or antigen-binding fragment thereof is the anti-CD3δ antibody or antigen-binding fragment thereof, the TSP is:

(a)CD3ε、或其功能性片段、或其变体、或其变体的功能性片段；(a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;

(b)CD3γ、或其功能性片段、或其变体、或其变体的功能性片段；或(b) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or

(c)CD3ζ，或其功能性片段、或其变体、或其变体的功能性片段。(c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

本发明的一些实施例中，所述抗CD3抗体是抗CD3ζ抗体，所述TSP不是CD3ζ、或其功能性片段、或其变体、或其变体的功能性片段。In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3ζ antibody, and the TSP is not CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

本发明的一些实施例中，所述抗CD3ζ抗体或其抗原结合片段是YTH12.5或其抗原结合片段；In some embodiments of the present invention, the anti-CD3ζ antibody or antigen-binding fragment thereof is YTH12.5 or an antigen-binding fragment thereof;

优选地，所述YTH12.5的抗原结合片段是scFv(YTH12.5-scFv)。Preferably, the antigen-binding fragment of YTH12.5 is scFv (YTH12.5-scFv).

本发明的一些实施例中，当所述抗CD3抗体或其抗原结合片段是所述抗CD3ζ抗体或其抗原结合片段，所述TSP是：In some embodiments of the present invention, when the anti-CD3 antibody or antigen-binding fragment thereof is the anti-CD3ζ antibody or antigen-binding fragment thereof, the TSP is:

(a)CD3ε、或其功能性片段、或其变体、或其变体的功能性片段；(a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;

(b)CD3γ、或其功能性片段、或其变体、或其变体的功能性片段；或(b) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or

(c)CD3δ，或其功能性片段、或其变体、或其变体的功能性片段。(c) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

本发明的一些实施例中，所述T细胞活化初级信号分子包括抗TCRα抗体或其抗原结合片段、抗TCRβ抗体或其抗原结合片段、抗TCRγ抗体或其抗原结合片段和抗TCRδ抗体或其抗原结合片段中的至少一种。In some embodiments of the present invention, the T cell activation primary signal molecule includes at least one of an anti-TCRα antibody or an antigen-binding fragment thereof, an anti-TCRβ antibody or an antigen-binding fragment thereof, an anti-TCRγ antibody or an antigen-binding fragment thereof, and an anti-TCRδ antibody or an antigen-binding fragment thereof.

本发明的一些实施例中，当所述T细胞活化初级信号分子包括抗TCRα抗体或其抗原结合片段时，所述TSP不是TCRα、或其功能性片段、或其变体、或其变体的功能性片段。In some embodiments of the present invention, when the T cell activation primary signal molecule comprises an anti-TCRα antibody or an antigen-binding fragment thereof, the TSP is not TCRα, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

本发明的一些实施例中，当所述T细胞活化初级信号分子包括抗TCRβ抗体或其抗原结合片段时，所述TSP不是TCRβ、或其功能性片段、或其变体、或其变体的功能性片段。In some embodiments of the present invention, when the T cell activation primary signal molecule comprises an anti-TCRβ antibody or an antigen-binding fragment thereof, the TSP is not TCRβ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

本发明的一些实施例中，当所述T细胞活化初级信号分子包括抗TCRγ抗体或其抗原结合片段时，所述TSP不是TCRγ、或其功能性片段、或其变体、或其变体的功能性片段。In some embodiments of the present invention, when the T cell activation primary signal molecule comprises an anti-TCRγ antibody or an antigen-binding fragment thereof, the TSP is not TCRγ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

本发明的一些实施例中，当所述T细胞活化初级信号分子包括抗TCRδ抗体或其抗原结合片段时，所述TSP不是TCRδ、或其功能性片段、或其变体、或其变体的功能性片段。In some embodiments of the present invention, when the T cell activation primary signal molecule comprises an anti-TCRδ antibody or an antigen-binding fragment thereof, the TSP is not TCRδ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

本发明的一些实施例中，所述T细胞活化信号分子还包括T细胞活化次级信号分子。In some embodiments of the present invention, the T cell activation signal molecule further includes a T cell activation secondary signal molecule.

T细胞活化次级信号分子(Secondary Signal)，又称共刺激信号分子(Co-Stimulatory Signal)，与其他T细胞表面受体结合，提供避免无反应(anergy)和有效的T细胞活化所必需的额外信号(Smith-Garvin JE,Koretzky GA,Jordan MS.T cell activation.Annu Rev Immunol.2009；27:591-619)。T cell activation secondary signal molecules (Secondary Signal), also known as co-stimulatory signal molecules (Co-Stimulatory Signal), bind to other T cell surface receptors to provide additional signals necessary to avoid anergy and effective T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

本发明的一些实施例中，所述T细胞活化次级信号分子结合CD28。In some embodiments of the present invention, the T cell activation secondary signaling molecule binds to CD28.

本发明的一些实施例中，所述CD28是人的CD28(Uniprot ID:P10747)。In some embodiments of the present invention, the CD28 is human CD28 (Uniprot ID: P10747).

尽管其他细胞表面受体(共刺激受体)也可通过TCR增强活化信号，比起其他共刺激受体，CD28介导的共刺激作用更强(Smith-Garvin JE,Koretzky GA,Jordan MS.T cell activation.Annu Rev Immunol.2009；27:591-619)。Although other cell surface receptors (co-stimulatory receptors) can also enhance activation signals through TCR, CD28-mediated co-stimulation is stronger than other co-stimulatory receptors (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

本发明的一些实施例中，所述T细胞活化次级信号分子选自抗CD28抗体或其抗原结合片段和CD28配体或其受体结合片段中的至少一种。In some embodiments of the present invention, the T cell activation secondary signal molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof.

本发明的一些实施例中，所述CD28配体或其受体结合片段包括CD80或其受体结合片段和CD86或其受体结合片段。In some embodiments of the present invention, the CD28 ligand or its receptor binding fragment includes CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.

本发明的一些实施例中，所述CD80和CD86是人CD80和CD86。In some embodiments of the present invention, the CD80 and CD86 are human CD80 and CD86.

本发明的一些实施例中，所述人CD80的氨基酸序列与SEQ ID NO:85具有至少约85％、86％、87％、88％、89％、90％、91％、92％、93、94％、95％、96％、97％、98％、99％或100％同一性。In some embodiments of the present invention, the amino acid sequence of human CD80 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:85.

本发明的一些实施例中，In some embodiments of the present invention,

(1)所述人CD80信号肽的氨基酸序列如SEQ ID NO:27所示；(1) The amino acid sequence of the human CD80 signal peptide is shown in SEQ ID NO: 27;

(2)所述人CD80胞外域的氨基酸序列如SEQ ID NO:28所示；(2) The amino acid sequence of the human CD80 extracellular domain is shown in SEQ ID NO: 28;

(3)所述人CD80跨膜区的氨基酸序列如SEQ ID NO:29所示。(3) The amino acid sequence of the human CD80 transmembrane region is shown in SEQ ID NO: 29.

本发明的一些实施例中，所述人CD86的氨基酸序列与SEQ ID NO:86具有至少约85％、86％、87％、88％、89％、90％、91％、92％、93、94％、95％、96％、97％、98％、99％或100％同一性。In some embodiments of the present invention, the amino acid sequence of human CD86 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:86.

本发明的一些实施例中，所述T细胞活化次级信号分子包括抗CD28抗体或其抗原结合片段；In some embodiments of the present invention, the T cell activation secondary signaling molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof;

优选地，所述抗CD28抗体或其抗原结合片段是源自15E8的scFv(15E8-scFv)，所述15E8-scFv的氨基酸序列如SEQ ID NO:17所示；所述15E8-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:93-95所示，所述15E8-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:96-98所示。Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is a scFv derived from 15E8 (15E8-scFv), and the amino acid sequence of the 15E8-scFv is shown in SEQ ID NO: 17; the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO: 93-95, respectively, and the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO: 96-98, respectively.

本发明的一些实施例中，所述抗CD28抗体或其抗原结合片段选自CD28.2、10F3和TGN1412中的至少一种。In some embodiments of the present invention, the anti-CD28 antibody or antigen-binding fragment thereof is selected from at least one of CD28.2, 10F3 and TGN1412.

本发明的一些实施例中，所述T细胞活化次级信号分子在包含可结合CD28的抗CD28抗体或其抗原结合片段和CD28配体或其受体结合片段中的至少一种的同时，还包括选自ICOS(inducible costimulator，“ICOS”)配体(ICOSL)或其受体结合片段、4-1BB配体(4-1BBL)或其受体结合片段和OX40配体(OX40L)或其受体结合片段中的至少一种配体或其受体结合片段。In some embodiments of the present invention, the T cell activation secondary signal molecule, while comprising at least one of an anti-CD28 antibody or an antigen-binding fragment thereof that can bind to CD28 and a CD28 ligand or a receptor-binding fragment thereof, also includes at least one ligand or receptor-binding fragment selected from ICOS (inducible costimulator, "ICOS") ligand (ICOSL) or a receptor-binding fragment thereof, 4-1BB ligand (4-1BBL) or a receptor-binding fragment thereof, and OX40 ligand (OX40L) or a receptor-binding fragment thereof.

与在非活化和活化的T细胞上均稳定表达的CD28不同，ICOS诱导性地表达在活化的T细胞上(Hutloff A,Dittrich AM,Beier KC,Eljaschewitsch B,Kraft R,et al.ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28.Nature 1999；397:263-6.[PubMed:9930702])(Smith-Garvin JE,Koretzky GA,Jordan MS.T cell activation.Annu Rev Immunol.2009；27:591-619)。缺乏ICOS将导致与CD28敲除模型相似、但不及CD28敲除模型严重的免疫反应受损，这意味着这两种分子可能在相似的通路发挥作用(Coyle AJ,Lehar S,Lloyd C,Tian J,Delaney T,et al.The CD28-related molecule ICOS is required for effective T cell-dependent immune responses.Immunity 2000；13:95-105.[PubMed:10933398])(Smith-Garvin JE,Koretzky GA,Jordan MS.T cell activation.Annu Rev Immunol.2009；27:591-619)。Unlike CD28, which is stably expressed on both non-activated and activated T cells, ICOS is inducibly expressed on activated T cells (Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, et al. ICOS is an inducible T-cell costimulator structurally and functionally related to CD28. Nature 1999; 397: 263-6. [PubMed: 9930702])(Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619). Lack of ICOS results in impaired immune responses similar to but less severe than those in the CD28 knockout model, suggesting that the two molecules may act in similar pathways (Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity 2000; 13:95-105. [PubMed:10933398])(Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27:591-619).

在CD28家族之外的共刺激受体，TNFR家族成员OX40(CD134)和4-1BB(CD137)通过与其配体OX40L和4-1BBL的结合提供共刺激信号(Smith-Garvin JE,Koretzky GA,Jordan MS.T cell activation.Annu Rev Immunol.2009；27:591-619)。In addition to the CD28 family of co-stimulatory receptors, TNFR family members OX40 (CD134) and 4-1BB (CD137) provide co-stimulatory signals by binding to their ligands OX40L and 4-1BBL (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

本发明的一些实施例中，所述T细胞活化次级信号分子还可结合CD27、HVEM、LIGHT、CD40、DR3、GITR、CD30、TIM1、SLAM、CD2和CD226中的至少一种。In some embodiments of the present invention, the T cell activation secondary signaling molecule can also bind to at least one of CD27, HVEM, LIGHT, CD40, DR3, GITR, CD30, TIM1, SLAM, CD2 and CD226.

本发明的一些实施例中，所述T细胞活化次级信号分子还可选自B7-H2、CD70、LIGHT、HVEM、CD40L、TL1A、GITRL、CD30L、TIM4、SLAM、CD48、CD58、CD155和CD112中的至少一种。In some embodiments of the present invention, the T cell activation secondary signaling molecule may also be selected from at least one of B7-H2, CD70, LIGHT, HVEM, CD40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155 and CD112.

本发明的一些实施例中，所述T细胞活化信号分子直接地或间接地与跨膜多肽(Transmembrane Polypeptide)相连，展示在所述病毒载体的表面。In some embodiments of the present invention, the T cell activation signal molecule is directly or indirectly connected to a transmembrane polypeptide and displayed on the surface of the viral vector.

本发明的一些实施例中，所述跨膜多肽选自以下蛋白的跨膜区：In some embodiments of the present invention, the transmembrane polypeptide is selected from the transmembrane regions of the following proteins:

CD2、CD3、CD4、CD5、CD7、CD8、CD8α、CD8β、CD9、CD16、CD22、CD27、CD28、CD28H、CD30、CD33、CD37、CD40、CD45、CD64、CD80、CD86、CD84、CD154、CD166、CD226、CD244、4-1BB、OX40、ICOS、ICAM-1、CTLA-4、PD-1、LAG-3、GITR、HVEM、DAP10、DAP12、TIM-1、LIGHT、ICOS、OX40、2B4、BTLA、DNAM-1、DR3、FcERIγ、IL7、IL12、IL15、SLAM、KIR2DL4、KIR2DS1、KIR2DS2、NKG2C、NKG2D和CS1；CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD 37. CD40, CD45, CD64, CD80, CD86, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1;

优选地，所述跨膜多肽是CD8α跨膜区。Preferably, the transmembrane polypeptide is the CD8α transmembrane region.

本发明的一些实施例中，所述跨膜多肽是人CD8α跨膜区。In some embodiments of the present invention, the transmembrane polypeptide is the human CD8α transmembrane region.

本发明的一些实施例中，所述人CD8α跨膜区与SEQ ID NO:15具有至少约85％、86％、87％、88％、89％、90％、91％、92％、93、94％、95％、96％、97％、98％、99％或100％同一性。In some embodiments of the present invention, the human CD8α transmembrane region has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:15.

本发明的一些实施例中，所述T细胞活化信号分子通过连接结构域，间接地与所述跨膜多肽相连，展示在所述病毒载体的表面；In some embodiments of the present invention, the T cell activation signaling molecule is indirectly linked to the transmembrane polypeptide via a linker domain and is displayed on the surface of the viral vector;

优选地，所述连接结构域选自：Preferably, the linker domain is selected from:

(a)免疫球蛋白铰链区，所述免疫球蛋白铰链区选自野生型或经修饰的IgG1、IgG2、IgG3、IgG4、IgA和IgD铰链区；(a) an immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge region;

(b)铰链区，所述铰链区选自以下蛋白的野生型或经修饰的铰链区：CD28、CD7、CD8、CD8α、CD8β、CD3、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154；(b) a hinge region selected from the wild-type or modified hinge region of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

(c)Fc结构域的全部或一部分，所述Fc结构域选自CH1结构域、CH2结构域和CH3结构域中的一种或多种；(c) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain;

(d)Ⅱ型C-凝集素的茎区，所述Ⅱ型C-凝集素选自CD23、CD69、CD72、CD94、NKG2A和NKG2D的茎区；和(d) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and

(e)柔性连接肽；(e) flexible linker peptide;

更优选地，所述连接结构域是CD8α铰链区。More preferably, the connecting domain is the CD8α hinge region.

本发明的一些实施例中，所述连接结构域是人CD8α铰链区。In some embodiments of the present invention, the connecting domain is the human CD8α hinge region.

本发明的一些实施例中，所述人CD8α铰链区与SEQ ID NO:14具有至少约85％、86％、87％、88％、89％、90％、91％、92％、93、94％、95％、96％、97％、98％、99％或100％同一性。In some embodiments of the present invention, the human CD8α hinge region is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:14.

本发明的一些实施例中，所述病毒载体表面包含糖蛋白，所述糖蛋白选自水疱性口炎病毒属毒株的包膜糖蛋白及其变体、狒狒内源性逆转录病毒BaEV的包膜糖蛋白及其变体、猫内源性逆转录病毒的包膜糖蛋白RD114及其变体和长臂猿白血病病毒的包膜糖蛋白GALV及其变体。In some embodiments of the present invention, the surface of the viral vector comprises a glycoprotein, and the glycoprotein is selected from the envelope glycoprotein of the vesicular stomatitis virus strain and its variants, the envelope glycoprotein of the baboon endogenous retrovirus BaEV and its variants, the envelope glycoprotein RD114 of the feline endogenous retrovirus and its variants, and the envelope glycoprotein GALV of the gibbon ape leukemia virus and its variants.

本发明的一些实施例中，所述跨膜多肽是所述糖蛋白，所述糖蛋白直接地或间接地连接至所述T细胞活化信号分子；In some embodiments of the present invention, the transmembrane polypeptide is the glycoprotein, and the glycoprotein is directly or indirectly linked to the T cell activation signaling molecule;

优选地，所述糖蛋白通过多肽连接子(Polypeptide Linker)，间接地连接至所述T细胞活化信号分子。Preferably, the glycoprotein is indirectly connected to the T cell activation signal molecule through a polypeptide linker.

本发明的一些实施例中，所述T细胞活化初级信号分子直接地或间接地连接至所述T细胞活化次级信号分子；In some embodiments of the present invention, the T cell activation primary signaling molecule is directly or indirectly linked to the T cell activation secondary signaling molecule;

优选地，所述T细胞活化初级信号分子通过多肽连接子，间接地连接至所述T细胞活化次级信号分子。Preferably, the T cell activation primary signal molecule is indirectly linked to the T cell activation secondary signal molecule via a polypeptide linker.

本发明的一些实施例中，所述多肽连接子是柔性连接肽；In some embodiments of the present invention, the polypeptide linker is a flexible connecting peptide;

优选地，所述柔性连接肽选自(G4S)n连接肽、连接肽1：GSTSGSGKPGSGEGSTKG(SEQ ID NO:23)和连接肽3：GSSGGSGGGGSGGGGSGGGGSSG(SEQ ID NO:63)；其中，n＝1至4。Preferably, the flexible connecting peptide is selected from (G 4 S) n connecting peptide, connecting peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23) and connecting peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 63); wherein n=1 to 4.

本发明的一些实施例中，In some embodiments of the present invention,

(a)所述糖蛋白通过(G4S)n连接肽，间接地连接至所述抗CD3抗体或其抗原结合片段，所述抗CD3抗体或其抗原结合片段通过(G4S)n连接肽，间接地连接至所述抗CD28抗体或其抗原结合片段、(ii)CD80胞外域(iii)或CD86胞外域；和/或(a) the glycoprotein is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a ( G4S ) n linker peptide, and the anti-CD3 antibody or antigen-binding fragment thereof is indirectly linked to the anti - CD28 antibody or antigen-binding fragment thereof, (ii) the CD80 extracellular domain, (iii) or the CD86 extracellular domain via a (G4S)n linker peptide; and/or

(b)所述糖蛋白通过(G4S)n连接肽，间接地连接至(i)所述抗CD28抗体或其抗原结合片段、(ii)CD80胞外域(iii)或CD86胞外域；所述抗CD28抗体或其抗原结合片段、CD80胞外域或CD86胞外域通过(G4S)n连接肽，间接地连接至所述抗CD3抗体或其抗原结合片段；(b) the glycoprotein is indirectly linked to (i) the anti-CD28 antibody or antigen-binding fragment thereof, (ii) the CD80 extracellular domain, (iii) the CD86 extracellular domain via a ( G4S ) n connecting peptide; the anti-CD28 antibody or antigen-binding fragment thereof, the CD80 extracellular domain, or the CD86 extracellular domain is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a ( G4S ) n connecting peptide;

优选地，n＝3；Preferably, n=3;

优选地，所述抗CD3抗体或其抗原结合片段是所述UCHT1-scFv；Preferably, the anti-CD3 antibody or antigen-binding fragment thereof is the UCHT1-scFv;

优选地，所述抗CD28抗体或其抗原结合片段是所述15E8-scFv。Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is the 15E8-scFv.

本发明的一些实施例中，所述糖蛋白选自水疱性口炎病毒属毒株的包膜糖蛋白及其变体中的至少一种。In some embodiments of the present invention, the glycoprotein is selected from at least one of the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof.

本发明的一些实施例中，所述水疱性口炎病毒属毒株的包膜糖蛋白及其变体包括以下包膜糖蛋白及其变体：水疱性口炎病毒属Indiana毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Cocal毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Maraba毒株的包膜糖蛋及其变体、水疱性口炎病毒属Morreton毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Alagoas毒株的包膜糖蛋白及其变体、水疱性口炎病毒属New Jersey毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Carajas毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Chandipura毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Eptesicus毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Isfahan毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Jurona毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Malpais毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Perinet毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Piry毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Radi毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Rhinolopus毒株的包膜糖蛋白及其变体和水疱性口炎病毒属Yug Bogdanovac毒株的包膜糖蛋白及其变体。In some embodiments of the present invention, the envelope glycoprotein and variants thereof of the vesicular stomatitis virus strain include the following envelope glycoproteins and variants thereof: envelope glycoprotein and variants of the Indiana strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Cocal strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Maraba strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Morreton strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Alagoas strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the New Jersey strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Carajas strain of the vesicular stomatitis virus, envelope glycoprotein and variants thereof of the Chandipura strain of the vesicular stomatitis virus Membrane glycoprotein and variants thereof, envelope glycoprotein of Eptesicus strain and variants thereof, envelope glycoprotein of Isfahan strain and variants thereof, envelope glycoprotein of Jurona strain and variants thereof, envelope glycoprotein of Malpais strain and variants thereof, envelope glycoprotein of Perinet strain and variants thereof, envelope glycoprotein of Piry strain and variants thereof, envelope glycoprotein of Radi strain and variants thereof, envelope glycoprotein of Rhinolopus strain and variants thereof, and envelope glycoprotein of Yug Bogdanovac strain and variants thereof.

水疱性口炎病毒属毒株(Vesicular Stomatitis Virus)的包膜糖蛋白(VSV-G)，如Indiana毒株和Cocal毒株的包膜糖蛋白可结合广泛存在于多种细胞表面的低密度脂蛋白受体(Low Density Lipoprotein Receptor,“LDL-R”)而具有广泛的感染性。The envelope glycoprotein (VSV-G) of Vesicular Stomatitis Virus strains (VSV-G), such as the Indiana strain and the Cocal strain, can bind to the low-density lipoprotein receptor (LDL-R) that is widely present on the surface of various cells and has a wide range of infectivity.

所述水疱性口炎病毒属Indiana毒株的包膜糖蛋白的胞外域包含如SEQ ID NO:1所示的氨基酸序列；所述水疱性口炎病毒属Cocal毒株的包膜糖蛋白的胞外域包含如SEQ ID NO:2所示的氨基酸序列。The extracellular domain of the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus contains the amino acid sequence shown in SEQ ID NO:1; the extracellular domain of the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus contains the amino acid sequence shown in SEQ ID NO:2.

本发明的一些实施例中，所述野生型VSV-G的全长蛋白(包括VSV-G信号肽)的氨基酸序列如SEQ ID NO:24所示；In some embodiments of the present invention, the amino acid sequence of the full-length protein of wild-type VSV-G (including the VSV-G signal peptide) is shown in SEQ ID NO: 24;

其中，如SEQ ID NO:24的第1位-第16位所示的氨基酸序列：Wherein, the amino acid sequence shown at positions 1 to 16 of SEQ ID NO: 24:

MKCLLYLAFLFIGVNC为所述野生型VSV-G的信号肽的氨基酸序列。MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of the wild-type VSV-G.

本发明的一些实施例中，所述野生型Cocal-G的全长蛋白(包含Cocal-G信号肽)的氨基酸序列如SEQ ID NO:99所示；In some embodiments of the present invention, the amino acid sequence of the full-length wild-type Cocal-G protein (including the Cocal-G signal peptide) is shown in SEQ ID NO: 99;

其中，如SEQ ID NO:99的第1位-第17位所示的序列：Among them, the sequence shown in positions 1 to 17 of SEQ ID NO: 99:

MNFLLLTFIVLPLCSHA为所述野生型Cocal-G的信号肽的氨基酸序列。MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of the wild-type Cocal-G.

野生型VSV-G的胞外域：

Extracellular domain of wild-type VSV-G:

野生型Cocal-G的胞外域：
Extracellular domain of wild-type Cocal-G:

野生型VSV-G的全长蛋白(包含其信号肽)：
Full-length protein of wild-type VSV-G (including its signal peptide):

野生型Cocal-G的全长蛋白(包含其信号肽)：
Full-length wild-type Cocal-G protein (including its signal peptide):

活化的T细胞表达LDL-R，因此，人工合成的、具有生物安全性的LVV或RVV通常使用野生型VSV-G构建其包膜糖蛋白(VSV-G型LVV或RVV)，转导活化的T细胞。Activated T cells express LDL-R; therefore, artificially synthesized, biosafe LVV or RVV usually uses wild-type VSV-G to construct its envelope glycoprotein (VSV-G type LVV or RVV) to transduce activated T cells.

本发明的一些实施例中，所述糖蛋白为水疱性口炎病毒属Indiana毒株或Cocal毒株的包膜糖蛋白或其变体；In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus or a variant thereof;

所述糖蛋白的胞外域包含如SEQ ID NO:1或SEQ ID NO:2所示、或与如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列。The extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

本发明的一些实施例中，前述任一种糖蛋白发生第一突变，使所述糖蛋白结合糖蛋白受体的能力相对于发生所述第一突变前降低或丧失。In some embodiments of the present invention, any of the aforementioned glycoproteins undergoes a first mutation, which reduces or loses the ability of the glycoprotein to bind to a glycoprotein receptor relative to before the first mutation occurs.

LDL-R广泛表达于多种细胞表面，如活化的T细胞、肝细胞、心肌细胞和内皮细胞等，因此，VSV-G型LVV或RVV也可通过结合LDL-R转导其他细胞，转导T细胞的靶向性较低。LDL-R is widely expressed on the surface of multiple cells, such as activated T cells, hepatocytes, cardiomyocytes, and endothelial cells. Therefore, VSV-G type LVV or RVV can also transduce other cells by binding to LDL-R, but the targeting of transduced T cells is relatively low.

通过减弱VSV-G结合LDL-R的能力，同时使其包膜包含抗CD3抗体和抗CD28抗体等T细胞活化初级和次级信号分子，可有效提高VSV-G型LVV或RVV靶向活化并转导T细胞的能力。By weakening the ability of VSV-G to bind to LDL-R and at the same time making its envelope contain primary and secondary signal molecules for T cell activation such as anti-CD3 antibodies and anti-CD28 antibodies, the ability of VSV-G type LVV or RVV to target, activate and transduce T cells can be effectively improved.

本发明的一些实施例中，所述糖蛋白为水疱性口炎病毒属Indiana毒株或Cocal毒株的包膜糖蛋白或其变体；所述糖蛋白受体是低密度脂蛋白受体LDL-R；所述糖蛋白发生第一突变，使所述糖蛋白结合LDL-R的能力相对于发生所述第一突变前降低或丧失；In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus, or a variant thereof; the glycoprotein receptor is the low-density lipoprotein receptor (LDL-R); the glycoprotein undergoes a first mutation, such that the ability of the glycoprotein to bind to the LDL-R is reduced or lost relative to before the first mutation occurs;

所述糖蛋白的胞外域包含如SEQ ID NO:1或SEQ ID NO:2所示、或与如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列。The extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

本发明的一些实施例中，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a)位于SEQ ID NO:1或SEQ ID NO:2的第8位氨基酸的替换或缺失、第9位氨基酸的替换或缺失、第10位氨基酸的替换或缺失、第47位氨基酸的替换或缺失、第50位氨基酸的替换或缺失、第51位氨基酸的替换或缺失、第183位氨基酸的替换或缺失、第179位氨基酸的替换或缺失、第180位氨基酸的替换或缺失、第182位氨基酸的替换或缺失、第184位氨基酸的替换或缺失、第209位氨基酸的替换或缺失、第347位氨基酸的替换或缺失、第350位氨基酸的替换或缺失、第352位氨基酸的替换或缺失、第353位氨基酸的替换或缺失、第354位氨基酸的替换、第1-18位氨基酸缺失、第19-36位氨基酸缺失、第37-51位氨基酸缺失、第314-384位氨基酸缺失、第321-374位氨基酸缺失、第331-364位氨基酸缺失、第344-354位氨基酸缺失、第345-353位氨基酸缺失；和(a) substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 209 of SEQ ID NO: 1 or SEQ ID NO: 2. 347, substitution or deletion of amino acid 350, substitution or deletion of amino acid 352, substitution or deletion of amino acid 353, substitution of amino acid 354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; and

(b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第8位氨基酸的替换或缺失、第9位氨基酸的替换或缺失、第10位氨基酸的替换或缺失、第47位氨基酸的替换或缺失、第50位氨基酸的替换或缺失、第51位氨基酸的替换或缺失、第183位氨基酸的替换或缺失、第179位氨基酸的替换或缺失、第180位氨基酸的替换或缺失、第182位氨基酸的替换或缺失、第184位氨基酸的替换或缺失、第209位氨基酸的替换或缺失、第347位氨基酸的替换或缺失、第350位氨基酸的替换或缺失、第352位氨基酸的替换或缺失、第353位氨基酸的替换或缺失、第354位氨基酸的替换、第1-18位氨基酸缺失、第19-36位氨基酸缺失、第37-51位氨基酸缺失、第314-384位氨基酸缺失、第321-374位氨基酸缺失、第331-364位氨基酸缺失、第344-354位氨基酸缺失、第345-353位氨基酸缺失。(b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 185, substitution or deletion of amino acid at position 186, substitution or deletion of amino acid at position 187, substitution or deletion of amino acid at position 188, substitution or deletion of amino acid at position 189, substitution or deletion of amino acid at position 190, substitution or deletion of amino acid at position 191, substitution or deletion of amino acid at position 192, substitution or deletion of amino acid at position 193, substitution or deletion of amino acid at position 194, substitution or deletion of amino acid at position 195, substitution or deletion of amino acid at position 196, substitution or deletion of amino acid at position 197, substitution or deletion of amino acid at position 198, substitution or deletion of amino acid at position 199, substitution or deletion of amino acid at position 200, substitution or deletion of amino acid at position 201, substitution or deletion of amino acid at position 202, substitution or deletion of amino acid at position 203, substitution or deletion of amino acid at position 204 The present invention also includes substitution or deletion of the amino acid at position 350, substitution or deletion of the amino acid at position 352, substitution or deletion of the amino acid at position 353, substitution of the amino acid at position 354, deletion of amino acids at positions 1-18, deletion of amino acids at positions 19-36, deletion of amino acids at positions 37-51, deletion of amino acids at positions 314-384, deletion of amino acids at positions 321-374, deletion of amino acids at positions 331-364, deletion of amino acids at positions 344-354, and deletion of amino acids at positions 345-353.

本发明的一些实施例中，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a)位于SEQ ID NO:1的H8的替换或缺失、N9的替换或缺失、Q10的替换或缺失、K47的替换或缺失，K50的替换或缺失、A51的替换或缺失、S183的替换或缺失、S179的替换或缺失、N180的替换或缺失、I182的替换或缺失、M184的替换或缺失、Y209的替换或缺失、I347的替换或缺失、T350的替换或缺失、T352的替换或缺失、E353的替换或缺失、R354的替换、第1-18位氨基酸缺失、第19-36位氨基酸缺失、第37-51位氨基酸缺失、第314-384位氨基酸缺失、第321-374位氨基酸缺失、第331-364位氨基酸缺失、第344-354位氨基酸缺失、第345-353位氨基酸缺失；和(a) substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 1; and

(b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的H8的替换或缺失、N9的替换或缺失、Q10的替换或缺失、K47的替换或缺失，K50的替换或缺失、A51的替换或缺失、S183的替换或缺失、S179的替换或缺失、N180的替换或缺失、I182的替换或缺失、M184的替换或缺失、Y209的替换或缺失、I347的替换或缺失、T350的替换或缺失、T352的替换或缺失、E353的替换或缺失、R354的替换、第1-18位氨基酸缺失、第19-36位氨基酸缺失、第37-51位氨基酸缺失、第314-384位氨基酸缺失、第321-374位氨基酸缺失、第331-364位氨基酸缺失、第344-354位氨基酸缺失、第345-353位氨基酸缺失。(b) After optimal global alignment with SEQ ID NO: 1, the substitution or deletion at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, Substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids at positions 1-18, deletion of amino acids at positions 19-36, deletion of amino acids at positions 37-51, deletion of amino acids at positions 314-384, deletion of amino acids at positions 321-374, deletion of amino acids at positions 331-364, deletion of amino acids at positions 344-354, deletion of amino acids at positions 345-353.

本发明的一些实施例中，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a)位于SEQ ID NO:1的H8的替换、N9的替换、Q10的替换、K47的替换、K47的缺失、K50的替换、A51的替换、S183的替换、S179的替换、N180的替换、I182的替换、M184的替换、Y209的替换、I347的替换、T350的替换、T352的替换、E353的替换、R354的替换、第1-18位氨基酸缺失、第19-36位氨基酸缺失、第37-51位氨基酸缺失、第314-384位氨基酸缺失、第321-374位氨基酸缺失、第331-364位氨基酸缺失、第344-354位氨基酸缺失、第345-353位氨基酸缺失；和(a) substitution of H8, substitution of N9, substitution of Q10, substitution of K47, deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 1; and

(b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的H8的替换、N9的替换、Q10的替换、K47的替换、K47的缺失、K50的替换、A51的替换、S183的替换、S179的替换、N180的替换、I182的替换、M184的替换、Y209的替换、I347的替换、T350的替换、T352的替换、E353的替换、R354的替换、第1-18位氨基酸缺失、第19-36位氨基酸缺失、第37-51位氨基酸缺失、第314-384位氨基酸缺失、第321-374位氨基酸缺失、第331-364位氨基酸缺失、第344-354位氨基酸缺失、第345-353位氨基酸缺失。(b) After optimal global alignment with SEQ ID NO:1, the following residues are located: replacement of H8, replacement of N9, replacement of Q10, replacement of K47, deletion of K47, replacement of K50, replacement of A51, replacement of S183, replacement of S179, replacement of N180, replacement of I182, replacement of M184, replacement of Y209, replacement of I347, replacement of T350, replacement of T352, replacement of E353, replacement of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO:1.

本发明的一些实施例中，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a)位于SEQ ID NO:1或SEQ ID NO:2的第331-364位氨基酸缺失、第344-354位氨基酸缺失、K47的替换、K47的缺失、R354的替换；和(a) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, or a substitution of R354 in SEQ ID NO: 1 or SEQ ID NO: 2; and

(b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第331-364位氨基酸缺失、第344-354位氨基酸缺失、K47的替换、K47的缺失、R354的替换；(b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, and substitution of R354 are located at positions equivalent to SEQ ID NO: 1 or SEQ ID NO: 2;

优选地，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a)位于SEQ ID NO:1或SEQ ID NO:2的第331-第364位氨基酸缺失、第344-354位氨基酸缺失、K47Q、R354Q、K47缺失；和(a) a deletion of amino acids 331 to 364, a deletion of amino acids 344 to 354, K47Q, R354Q, or K47 in SEQ ID NO: 1 or SEQ ID NO: 2; and

(b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第331-第364位氨基酸缺失、第344-354位氨基酸缺失、K47Q、R354Q、K47缺失。(b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, amino acid deletions at positions 331 to 364, amino acid deletions at positions 344 to 354, K47Q, R354Q, and K47 deletions are located at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

本发明的一些实施例中，所述第一突变包括所述氨基酸序列包含以下氨基酸的突变：In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises the following amino acids:

(a)位于SEQ ID NO:1或SEQ ID NO:2的第47位氨基酸赖氨酸缺失；或(a) the amino acid lysine at position 47 of SEQ ID NO: 1 or SEQ ID NO: 2 is missing; or

(b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第47位氨基酸赖氨酸缺失。(b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, the lysine residue at position 47 corresponding to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is missing.

本发明的一些实施例中，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a)位于SEQ ID NO:1的K47的替换、K47缺失、I182的替换、R354的替换、Y209的替换；(a) Substitution of K47, deletion of K47, substitution of I182, substitution of R354, and substitution of Y209 in SEQ ID NO: 1;

(b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的K47的替换、K47缺失、I182的替换、R354的替换、Y209的替换；(b) After optimal global alignment with SEQ ID NO: 1, the substitutions at K47, K47 deletion, I182 substitution, R354 substitution, and Y209 substitution are equivalent to those in SEQ ID NO: 1;

(c)位于SEQ ID NO:2的K47的替换、K47缺失、V182的替换、R354的替换、Y209的替换；和(c) substitution of K47, deletion of K47, substitution of V182, substitution of R354, substitution of Y209 at SEQ ID NO: 2; and

(d)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的K47的替换、K47缺失、V182的替换、R354的替换、Y209的替换；(d) After optimal global alignment with SEQ ID NO: 2, the substitutions at K47, K47 deletion, V182 substitution, R354 substitution, and Y209 substitution are located at positions equivalent to those in SEQ ID NO: 2;

优选地，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a)位于SEQ ID NO:1的K47Q或K47A、K47缺失、I182E或I182D、R354Q或R354A、Y209Q；(a) K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q in SEQ ID NO: 1;

(b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的K47Q或K47A、K47缺失、I182E或I182D、R354Q或R354A、Y209Q；(b) After optimal global alignment with SEQ ID NO: 1, the residues located at K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, and Y209Q are equivalent to those in SEQ ID NO: 1;

(c)位于SEQ ID NO:2的K47Q或K47A、K47缺失、V182E或V182D、R354Q或R354A、Y209Q；和(c) K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q at SEQ ID NO: 2; and

(d)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的K47Q或K47A、K47缺失、V182E或V182D、R354Q或R354A、Y209Q。(d) After optimal global alignment with SEQ ID NO: 2, it is located at K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q equivalent to SEQ ID NO: 2.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:33或SEQ ID NO:34所示的氨基酸序列。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 33 or SEQ ID NO: 34.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:3所示或与如SEQ ID NO:3所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:1，SEQ ID NO:3包含K47缺失。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:3; relative to SEQ ID NO:1, SEQ ID NO:3 comprises a K47 deletion.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:4所示或与如SEQ ID NO:4所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:1，SEQ ID NO:4包含R354Q。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:4; relative to SEQ ID NO:1, SEQ ID NO:4 comprises R354Q.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:33所示或与如SEQ ID NO:33所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:2，SEQ ID NO:33包含K47缺失。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:33 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:33; relative to SEQ ID NO:2, SEQ ID NO:33 comprises a K47 deletion.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:34所示或与如SEQ ID NO:34所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:2，SEQ ID NO:34包含R354Q。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:34 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:34; relative to SEQ ID NO:2, SEQ ID NO:34 comprises R354Q.

本发明的一些实施例中，发生前述任一种第一突变的糖蛋白仍保留膜融合和内体/溶酶体逃逸的能力。In some embodiments of the present invention, the glycoprotein having any of the aforementioned first mutations still retains the ability to fuse membranes and escape from endosomal/lysosomal regions.

本发明的一些实施例中，所述糖蛋白还发生第二突变，使所述糖蛋白拮抗被补体失活的能力相对于发生所述第二突变前增强或不被补体失活。In some embodiments of the present invention, the glycoprotein further undergoes a second mutation, which enhances the ability of the glycoprotein to antagonize inactivation by complement, or prevents inactivation by complement, compared to before the second mutation.

本发明的一些实施例中，发生所述第二突变的糖蛋白为前述任一种糖蛋白。In some embodiments of the present invention, the glycoprotein having the second mutation is any of the aforementioned glycoproteins.

补体系统由一系列的蛋白质组成，属于先天免疫系统的一部分。补体(complemnt，C)存在于正常人和动物的血清、组织液和细胞膜表面，经活化后具有酶的活性，可发生复杂的级联反应。补体系统通过一连串的酵素(酶)相互切割启动，最终在目标微生物上形成类似孔洞的膜攻击复合物，使微生物破裂而死亡。补体成分能被抗原抗体复合物或者抗体激活，通过溶胞、调理、吞噬以及介导炎症反应来清除免疫复合物，表现出相应的生物学功能。补体广泛参与机体抗微生物感染的防御反应以及免疫调节，同时也介导免疫病理性损伤反应，是体内具有重要生物学作用的效应系统和效应方法系统。The complement system is composed of a series of proteins and is part of the innate immune system. Complement (C) is present in the serum, tissue fluid, and cell membrane surfaces of normal humans and animals. Once activated, it possesses enzymatic activity and can undergo a complex cascade reaction. The complement system is initiated through a series of enzymes that cut each other, ultimately forming a membrane attack complex that resembles a hole on the target microorganism, causing the microorganism to rupture and die. Complement components can be activated by antigen-antibody complexes or antibodies, and clear immune complexes through lysis, opsonization, phagocytosis, and mediation of inflammatory responses, demonstrating corresponding biological functions. Complement is widely involved in the body's defense response against microbial infection and immune regulation, and also mediates immunopathological damage responses. It is an effector system and effector method system with important biological functions in the body.

起调节作用的补体成分以可溶性或膜结合形式存在，主要包括备解素(properdin,P因子)、C1抑制物(C1 inhibitor,C1INH)、I因子、H因子、C4结合蛋白(C4 binding protein,C4BP)、S蛋白(S protein)、SP40/40、膜辅助蛋白因子(membrane cofactor protein,MCP)、衰变加速因子(decay accelerating factor,DAF)、同源限制因子(homologous restriction factor,HRF)和膜反应性溶解抑制物(membrane inhibitor of reactive lysis,MIRL)等。The regulatory complement components exist in soluble or membrane-bound forms, including properdin (P factor), C1 inhibitor (C1INH), factor I, factor H, C4 binding protein (C4BP), S protein, SP40/40, membrane cofactor protein (MCP), decay accelerating factor (DAF), homologous restriction factor (HRF) and membrane inhibitor of reactive lysis (MIRL).

假型LVV或RVV进入血清后可能会被补体识别、失活，难以高效达到靶细胞发挥作用；因此，在被应用于体内制备CAR-T或TCP-T细胞等工程化T细胞时，假型LVV或RVV转导非活化T细胞的效率较低。After entering the serum, pseudotyped LVV or RVV may be recognized and inactivated by complement, making it difficult to efficiently reach target cells and exert their effects; therefore, when used in vivo to prepare engineered T cells such as CAR-T or TCP-T cells, the efficiency of pseudotyped LVV or RVV in transducing non-activated T cells is low.

通过使VSV-G等病毒糖蛋白发生所述第二突变，提高VSV-G等病毒糖蛋白拮抗被补体失活的能力，进而使假型LVV或RVV更适合被应用于体内制备CAR-T细胞或TCP-T细胞等工程化T细胞。By causing the second mutation in viral glycoproteins such as VSV-G, the ability of viral glycoproteins such as VSV-G to antagonize complement inactivation is improved, thereby making pseudotyped LVV or RVV more suitable for use in the in vivo preparation of engineered T cells such as CAR-T cells or TCP-T cells.

本发明的一些实施例中，发生所述第二突变的糖蛋白为水疱性口炎病毒属Indiana毒株或Cocal毒株的包膜糖蛋白或其变体，所述糖蛋白的胞外域包含如SEQ ID NO:1或SEQ ID NO:2所示、或与如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％的同一性的氨基酸序列。In some embodiments of the present invention, the glycoprotein that undergoes the second mutation is the envelope glycoprotein of the Indiana strain or Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

本发明的一些实施例中，所述第二突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：In some embodiments of the present invention, the second mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

(a)位于SEQ ID NO:1或SEQ ID NO:2的第214位氨基酸；(a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

(b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第214位氨基酸；(b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at the amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

(c)位于SEQ ID NO:1或SEQ ID NO:2的第352位氨基酸；(c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

(d)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第352位氨基酸；(d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

(e)位于SEQ ID NO:1或SEQ ID NO:2的第50位氨基酸；(e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

(f)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第50位氨基酸；(f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at the 50th amino acid position equivalent to SEQ ID NO: 1 or SEQ ID NO: 2;

(g)位于SEQ ID NO:1或SEQ ID NO:2的第146位氨基酸；和(g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

(h)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第146位氨基酸；(h) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at amino acid position 146 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

优选地，所述氨基酸的突变包括氨基酸的缺失、插入和替换中的至少一种。Preferably, the amino acid mutation includes at least one of amino acid deletion, insertion and substitution.

更优选地，所述第二突变包括所述氨基酸序列包含至少一个以下氨基酸的替换：More preferably, the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids:

(a)位于SEQ ID NO:1或SEQ ID NO:2的第214位氨基酸；(a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

(b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第214位氨基酸；(b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at the amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

(c)位于SEQ ID NO:1或SEQ ID NO:2的第352位氨基酸；(c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

(d)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第352位氨基酸；(d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

(e)位于SEQ ID NO:1或SEQ ID NO:2的第50位氨基酸；(e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

(f)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第50位氨基酸；(f) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 50th amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

(g)位于SEQ ID NO:1或SEQ ID NO:2的第146位氨基酸；和(g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

(h)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第146位氨基酸。(h) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 146th amino acid position equivalent to SEQ ID NO: 1 or SEQ ID NO: 2.

本发明的一些实施例中，所述第二突变包括如SEQ ID NO:1所示或与如SEQ ID NO:1所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％的同一性的氨基酸序列包含至少一种以下位点突变：In some embodiments of the present invention, the second mutation comprises an amino acid sequence as set forth in SEQ ID NO: 1 or at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO: 1, comprising at least one of the following position mutations:

(a)位于SEQ ID NO:1的T214的替换、T352的替换、K50的替换、S146的替换；和(a) substitution of T214, substitution of T352, substitution of K50, and substitution of S146 in SEQ ID NO: 1; and

(b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的T214的替换、T352的替换、K50的替换、S146的替换；(b) After optimal global alignment with SEQ ID NO: 1, the substitutions at T214, T352, K50, and S146 are equivalent to those in SEQ ID NO: 1;

优选地，所述第二突变包括所述氨基酸序列包含至少一种以下位点突变：Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence:

(a)位于SEQ ID NO:1的T214N、T352A、K50T、S146T；和(a) T214N, T352A, K50T, S146T located in SEQ ID NO: 1; and

(b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的T214N、T352A、K50T、S146T。(b) After optimal global alignment with SEQ ID NO:1, T214N, T352A, K50T, and S146T are located equivalent to SEQ ID NO:1.

本发明的一些实施例中，所述第二突变包括所述氨基酸序列包含任一种以下位点突变的组合：In some embodiments of the present invention, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

(a)位于SEQ ID NO:1的(1)T214和T352的替换；或(2)T214、T352、K50和S146的替换；和(a) substitution of (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1; and

(b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的(1)T214和T352的替换；或(2)T214、T352、K50和S146的替换；(b) substitutions at positions equivalent to (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1;

优选地，所述第二突变包括所述氨基酸序列包含任一种以下位点突变的组合：Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

(a)位于SEQ ID NO:1的(1)T214N和T352A；或(2)T214N、T352A、K50T和S146T；和(a) (1) T214N and T352A; or (2) T214N, T352A, K50T, and S146T located in SEQ ID NO: 1; and

(b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的(1)T214N和T352A；或(2)T214N、T352A、K50T和S146T。(b) After optimal global alignment with SEQ ID NO:1, located at (1) T214N and T352A; or (2) T214N, T352A, K50T and S146T equivalent to SEQ ID NO:1.

本发明的一些实施例中，所述第二突变包括如SEQ ID NO:2所示或与如SEQ ID NO:2所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％的同一性的氨基酸序列包含至少一种以下位点突变：In some embodiments of the present invention, the second mutation comprises an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to the amino acid sequence as set forth in SEQ ID NO: 2, comprising at least one of the following position mutations:

(a)位于SEQ ID NO:2的K214的替换、T352的替换、K50的替换、S146的替换；和(a) substitution of K214, substitution of T352, substitution of K50, and substitution of S146 in SEQ ID NO: 2; and

(b)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的K214的替换、T352的替换、K50的替换、S146的替换；(b) After optimal global alignment with SEQ ID NO: 2, the substitutions at K214, T352, K50, and S146 are equivalent to those in SEQ ID NO: 2;

优选地，所述第二突变包括所述氨基酸序列包含至少一种以下位点突变：Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence:

(a)位于SEQ ID NO:2的K214N、T352A、K50T、S146T；和(a) K214N, T352A, K50T, S146T located in SEQ ID NO: 2; and

(b)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的K214N、T352A、K50T、S146T。(b) After optimal global alignment with SEQ ID NO: 2, K214N, T352A, K50T, and S146T are located equivalent to SEQ ID NO: 2.

本发明的一些实施例中，所述第二突变包括所述氨基酸序列包含任一种以下位点突变的组合：In some embodiments of the present invention, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

(a)位于SEQ ID NO:2的(1)K214和T352的替换；或(2)K214、T352、K50和S146的替换；和(a) substitution of (1) K214 and T352; or (2) K214, T352, K50, and S146 at SEQ ID NO:2; and

(b)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的(1)K214和T352的替换；或(2)K214、T352、K50和S146的替换；(b) substitutions at positions corresponding to (1) K214 and T352 of SEQ ID NO: 2 after optimal global alignment with SEQ ID NO: 2; or (2) substitutions at positions corresponding to K214, T352, K50, and S146 of SEQ ID NO: 2;

优选地，所述第二突变包括所述氨基酸序列包含任一种以下位点突变的组合：Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

(a)位于SEQ ID NO:2的(1)K214N和T352A；或(2)K214N、T352A、K50T和S146T；和(a) (1) K214N and T352A; or (2) K214N, T352A, K50T, and S146T at SEQ ID NO:2; and

(b)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的(1)K214N和T352A；或(2)K214N、T352A、K50T和S146T。(b) After optimal global alignment with SEQ ID NO:2, located at (1) K214N and T352A; or (2) K214N, T352A, K50T and S146T equivalent to SEQ ID NO:2.

本发明的一些实施例中，所述糖蛋白为水疱性口炎病毒属Indiana毒株的包膜糖蛋白或其变体；In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus or a variant thereof;

所述糖蛋白的胞外域包含如SEQ ID NO:1所示或与如SEQ ID NO:1所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；The extracellular domain of the glycoprotein comprises an amino acid sequence as set forth in SEQ ID NO:1 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO:1;

所述糖蛋白发生前述任一种第一突变，使所述糖蛋白结合LDL-R的能力相对于所述第一突变前降低或丧失；所述糖蛋白还可发生前述任一种第二突变，使所述糖蛋白拮抗被补体失活的能力相对于所述第二突变前增强或不被补体失活。The glycoprotein undergoes any of the aforementioned first mutations, so that the ability of the glycoprotein to bind to LDL-R is reduced or lost relative to before the first mutation; the glycoprotein may also undergo any of the aforementioned second mutations, so that the ability of the glycoprotein to antagonize inactivation by complement is enhanced relative to before the second mutation, or is not inactivated by complement.

本发明的一些实施例中，所述糖蛋白为水疱性口炎病毒属Cocal毒株的包膜糖蛋白或其变体；In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus or a variant thereof;

所述糖蛋白的胞外域包含如SEQ ID NO:2所示或与如SEQ ID NO:2所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；The extracellular domain of the glycoprotein comprises an amino acid sequence as set forth in SEQ ID NO:2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO:2;

所述糖蛋白发生前述任一种第一突变，使所述糖蛋白结合LDL-R的能力相对于所述第一突变前降低或丧失；所述糖蛋白还可发生前述任一种第二突变，使所述糖蛋白拮抗被补体失活的能力相对于所述第二突变前增强或不被补体失活。The glycoprotein undergoes any of the aforementioned first mutations, so that the ability of the glycoprotein to bind to LDL-R is reduced or lost relative to before the first mutation; the glycoprotein may also undergo any of the aforementioned second mutations, so that the ability of the glycoprotein to antagonize inactivation by complement is enhanced relative to before the second mutation, or is not inactivated by complement.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37或SEQ ID NO:38所示的氨基酸序列。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 38.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:8所示或与如SEQ ID NO:8所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:1,SEQ ID NO:8包含K47缺失、T214N和T352A。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:8; relative to SEQ ID NO:1, SEQ ID NO:8 comprises K47 deletion, T214N and T352A.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:9所示或与如SEQ ID NO:9所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:1,SEQ ID NO:9包含K47缺失、T214N、T352A、K50T和S146T。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:9; relative to SEQ ID NO:1, SEQ ID NO:9 comprises K47 deletion, T214N, T352A, K50T and S146T.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:10所示或与如SEQ ID NO:10所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:1,SEQ ID NO:10包含R354Q、T214N和T352A。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 10; relative to SEQ ID NO: 1, SEQ ID NO: 10 comprises R354Q, T214N and T352A.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:11所示或与如SEQ ID NO:11所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:1,SEQ ID NO:11包含R354Q、T214N、T352A、K50T和S146T。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 11; relative to SEQ ID NO: 1, SEQ ID NO: 11 comprises R354Q, T214N, T352A, K50T and S146T.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:35所示或与如SEQ ID NO:35所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:2，SEQ ID NO:35包含K47缺失、K214N、T352A、K50T和S146T。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:35 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:35; relative to SEQ ID NO:2, SEQ ID NO:35 comprises K47 deletion, K214N, T352A, K50T and S146T.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:36所示或与如SEQ ID NO:36所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:2，SEQ ID NO:36包含K47缺失、K214N和T352A。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:36 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:36; relative to SEQ ID NO:2, SEQ ID NO:36 comprises K47 deletion, K214N and T352A.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:37所示或与如SEQ ID NO:37所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:2，SEQ ID NO:37包含R354Q、K214N、T352A、K50T和S146T。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:37 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:37; relative to SEQ ID NO:2, SEQ ID NO:37 comprises R354Q, K214N, T352A, K50T and S146T.

本发明的一些实施例中，所述糖蛋白的胞外域包含如SEQ ID NO:38所示或与如SEQ ID NO:38所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列；相对于SEQ ID NO:2，SEQ ID NO:38包含R354Q、K214N和T352A。In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:38 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:38; relative to SEQ ID NO:2, SEQ ID NO:38 comprises R354Q, K214N and T352A.

本发明的一些实施例中，发生前述任一种第二突变的糖蛋白仍保留膜融合和内体/溶酶体逃逸的能力。In some embodiments of the present invention, the glycoprotein having any of the aforementioned second mutations still retains the ability to fuse membranes and escape from endosomal/lysosomal regions.

本发明的一些实施例中，发生前述任一种第一突变和第二突变的糖蛋白仍保留膜融合和内体/溶酶体逃逸的能力。In some embodiments of the present invention, the glycoprotein having any of the aforementioned first and second mutations still retains the ability to fuse with membranes and escape from endosomal/lysosomes.

本发明的一些实施例中，相对于表面不包含所述T细胞活化信号分子的对照载体1，前述任一种病毒载体与非活化T细胞接触后，所述T细胞受体嵌合蛋白在T细胞中出膜表达的效率更高。In some embodiments of the present invention, compared with the control vector 1 whose surface does not contain the T cell activation signal molecule, after any of the aforementioned viral vectors contacts with non-activated T cells, the efficiency of the T cell receptor chimeric protein in T cell extracellular expression is higher.

本发明的一些实施例中，相对于表面不包含所述T细胞活化信号分子的对照载体1，前述任一种病毒载体与非活化T细胞接触后，制备所得的TCP-T细胞的杀伤效率更高。In some embodiments of the present invention, compared with the control vector 1 whose surface does not contain the T cell activation signal molecule, the TCP-T cells prepared after any of the aforementioned viral vectors contact with non-activated T cells have a higher killing efficiency.

本发明的一些实施例中，所述对照载体1表面包含至少一种T细胞靶向分子；In some embodiments of the present invention, the control carrier 1 comprises at least one T cell targeting molecule on its surface;

优选地，所述T细胞靶向分子结合CD5或CD7；Preferably, the T cell targeting molecule binds to CD5 or CD7;

更优选地，所述T细胞靶向分子结合CD7；More preferably, the T cell targeting molecule binds to CD7;

更进一步优选地，所述T细胞靶向分子选自抗CD7抗体或其抗原结合片段和CD7配体或其受体结合片段中的至少一种；任选地，所述抗CD7抗体或其抗原结合片段是源自单克隆抗体TH-69的scFv(TH69-scFv)；所述TH69-scFv的氨基酸序列如SEQ ID NO:71所示；所述TH69-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:72-74所示，所述TH69-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:75-77所示。Further preferably, the T cell targeting molecule is selected from at least one of an anti-CD7 antibody or an antigen-binding fragment thereof and a CD7 ligand or a receptor-binding fragment thereof; optionally, the anti-CD7 antibody or an antigen-binding fragment thereof is a scFv (TH69-scFv) derived from the monoclonal antibody TH-69; the amino acid sequence of the TH69-scFv is as shown in SEQ ID NO: 71; the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO: 72-74, respectively, and the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are as shown in SEQ ID NO: 75-77, respectively.

本发明的一些实施例中，前述任一种对照载体1为除不包含编码前述任一种T细胞活化信号分子外，其余结构和/或特征与前述任一种病毒载体相同的载体。In some embodiments of the present invention, any of the aforementioned control vectors 1 is a vector having the same structure and/or characteristics as any of the aforementioned viral vectors except that it does not contain the protein encoding any of the aforementioned T cell activation signaling molecules.

本发明的一些实施例中，前述任一种病毒载体表面不包含或仅包含少量所述抗原结合区；所述“少量”是相对于包含编码其他多肽的多核苷酸的对照载体2而言；所述其他多肽包含前述任一种抗原结合区但不包含前述任一种TSP。In some embodiments of the present invention, the surface of any of the aforementioned viral vectors does not contain or contains only a small amount of the antigen binding region; the "small amount" is relative to the control vector 2 containing a polynucleotide encoding other polypeptides; the other polypeptides contain any of the aforementioned antigen binding regions but do not contain any of the aforementioned TSPs.

本发明的一些实施例中，前述任一种病毒载体与T细胞接触后，相对于包含编码其他多肽的多核苷酸的对照载体2，所述病毒载体不发生假转导或假转导减少；所述其他多肽包含前述任一种抗原结合区但不包含前述任一种TSP。In some embodiments of the present invention, after any of the aforementioned viral vectors contacts T cells, the viral vector does not undergo pseudo-transduction or pseudo-transduction is reduced compared to the control vector 2 comprising a polynucleotide encoding other polypeptides; the other polypeptide comprises any of the aforementioned antigen binding regions but does not comprise any of the aforementioned TSPs.

本发明的一些实施例中，相对于不包含编码其他多肽的多核苷酸的对照载体2，前述任一种病毒载体(a)结合靶细胞表面抗原的能力降低或不结合靶细胞表面抗原；和/或(b)转导靶细胞的能力降低或不转导靶细胞；所述靶细胞表面抗原可结合所述抗原结合区，所述其他多肽包含前述任一种抗原结合区但不包含前述任一种TSP。In some embodiments of the present invention, relative to a control vector 2 that does not contain a polynucleotide encoding other polypeptides, any of the aforementioned viral vectors (a) has a reduced ability to bind to target cell surface antigens or does not bind to target cell surface antigens; and/or (b) has a reduced ability to transduce target cells or does not transduce target cells; the target cell surface antigen can bind to the antigen binding region, and the other polypeptide comprises any of the aforementioned antigen binding regions but does not comprise any of the aforementioned TSPs.

本发明的一些实施例中，所述其他多肽连接至信号肽。In some embodiments of the present invention, the other polypeptide is linked to a signal peptide.

本发明的一些实施例中，所述其他多肽包括嵌合抗原受体，所述嵌合抗原受体的N-末端可操作地连接至信号肽的C-末端。In some embodiments of the present invention, the other polypeptide comprises a chimeric antigen receptor, wherein the N-terminus of the chimeric antigen receptor is operably linked to the C-terminus of the signal peptide.

本发明的一些实施例中，所述信号肽选自前述任一种信号肽。In some embodiments of the present invention, the signal peptide is selected from any one of the aforementioned signal peptides.

本发明的一些实施例中，所述嵌合抗原受体选自一代、二代、三代和四代嵌合抗原受体中的至少一种。In some embodiments of the present invention, the chimeric antigen receptor is selected from at least one of first-generation, second-generation, third-generation and fourth-generation chimeric antigen receptors.

本发明的一些实施例中，所述嵌合抗原受体从N-末端至C-末端的结构依次包括胞外抗原结合区、铰链区、跨膜区、共刺激信号传导结构域、胞内信号传导结构域。In some embodiments of the present invention, the structure of the chimeric antigen receptor from N-terminus to C-terminus includes, in sequence, an extracellular antigen binding region, a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signaling domain.

本发明的一些实施例中，所述靶细胞选自癌细胞和自身免疫性疾病相关细胞中的至少一种。In some embodiments of the present invention, the target cell is selected from at least one of cancer cells and autoimmune disease-related cells.

本发明的一些实施例中，所述自身免疫性疾病包括：系统性红斑狼疮、类风湿性关节炎、多发性硬化症、炎症性肠病、干燥综合征、重症肌无力、乳糜泻、1型糖尿病、弥漫性毒性甲状腺肿、阿狄森病、自身免疫性血管炎、恶性贫血、皮肌炎、多肌炎和硬皮病。In some embodiments of the present invention, the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Sjögren's syndrome, myasthenia gravis, celiac disease, type 1 diabetes, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis and scleroderma.

本发明的一些实施例中，所述靶细胞为癌细胞，所述癌细胞包括实体癌细胞和血液癌细胞。In some embodiments of the present invention, the target cells are cancer cells, including solid cancer cells and blood cancer cells.

本发明的一些实施例中，所述血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤、高级别B细胞淋巴瘤和多发性骨髓瘤。In some embodiments of the present invention, the blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, high-grade B-cell lymphoma and multiple myeloma.

本发明的一些实施例中，所述癌细胞是血液癌细胞；In some embodiments of the present invention, the cancer cells are blood cancer cells;

优选地，所述血液癌是CD19+血液癌；Preferably, the blood cancer is a CD19 + blood cancer;

更优选地，所述CD19+血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤和高级别B细胞淋巴瘤。More preferably, the CD19 + blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin's lymphoma and high-grade B-cell lymphoma.

本发明的一些实施例中，所述癌细胞是血液癌细胞，所述血液癌选自CD19+血液癌和CD33+血液癌；In some embodiments of the present invention, the cancer cell is a blood cancer cell, and the blood cancer is selected from CD19 + blood cancer and CD33 + blood cancer;

优选地，Preferably,

所述CD19+血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤和高级别B细胞淋巴瘤；The CD19 + blood cancer is selected from the group consisting of marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, and high-grade B-cell lymphoma;

所述CD33+血液癌选自多发性骨髓瘤(MM)、急性髓性白血病(AML)、慢性髓性白血病(CML)和急性单核细胞白血病(AMoL)。The CD33 + blood cancer is selected from the group consisting of multiple myeloma (MM), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and acute monocytic leukemia (AMoL).

另一个方面，本发明还提供了一种TCP，所述TCP为本发明提供的前述任一种TCP。In another aspect, the present invention further provides a TCP, which is any of the aforementioned TCPs provided by the present invention.

另一个方面，本发明还提供了一种多核苷酸，所述多核苷酸编码本发明提供的前述任一种TCP。In another aspect, the present invention further provides a polynucleotide encoding any of the aforementioned TCPs provided by the present invention.

本发明的一些实施例中，所述多核苷酸是分离的(Isolated)。In some embodiments of the present invention, the polynucleotide is isolated.

另一个方面，本发明还提供一种转导T细胞的方法，包括使用本发明提供的前述任一种病毒载体与T细胞接触。In another aspect, the present invention also provides a method for transducing T cells, comprising contacting any one of the aforementioned viral vectors provided by the present invention with T cells.

本发明的一些实施例中，所述T细胞选自活化的T细胞和非活化T细胞中的至少一种。In some embodiments of the present invention, the T cells are selected from at least one of activated T cells and non-activated T cells.

本发明的一些实施例中，所述接触发生在受试者体内和/或体外；所述受试者是被给予经所述转导T细胞的方法转导的T细胞和/或本发明提供的前述任一种病毒载体的个体。In some embodiments of the present invention, the contact occurs in vivo and/or in vitro in a subject; the subject is an individual who is administered T cells transduced by the method for transducing T cells and/or any of the aforementioned viral vectors provided by the present invention.

另一个方面，本发明还提供一种工程化T细胞，所述工程化T细胞包含本发明提供的编码前述任一种TCP分子的所述多核苷酸和/或表达前述任一种TCP分子。In another aspect, the present invention also provides an engineered T cell, which comprises the polynucleotide encoding any one of the aforementioned TCP molecules provided by the present invention and/or expresses any one of the aforementioned TCP molecules.

本发明的一些实施例中，所述工程化T细胞由本发明提供的前述任一种病毒载体接触T细胞制备所得。In some embodiments of the present invention, the engineered T cells are prepared by contacting T cells with any of the aforementioned viral vectors provided by the present invention.

本发明的一些实施例中，所述T细胞选自活化的T细胞和非活化T细胞中的至少一种。In some embodiments of the present invention, the T cells are selected from at least one of activated T cells and non-activated T cells.

本发明的一些实施例中，所述接触发生在受试者体内和/或体外，所述受试者是被给予所述工程化T细胞和/或本发明提供的前述任一种病毒载体的个体。In some embodiments of the present invention, the contacting occurs in vivo and/or in vitro in a subject, and the subject is an individual who is administered the engineered T cells and/or any of the aforementioned viral vectors provided by the present invention.

本发明的一些实施例中，所述给予选自经口、鼻、静脉内、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉、病灶内、持续释放系统和植入装置进行给予中的至少一种。In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implantation device administration.

另一个方面，本发明还提供了一种组合物，所述组合物包含药学上可接受的赋形剂或载体以及(a)本发明提供的前述任一种病毒载体或(b)前述任一种工程化T细胞。In another aspect, the present invention also provides a composition comprising a pharmaceutically acceptable excipient or carrier and (a) any one of the aforementioned viral vectors provided by the present invention or (b) any one of the aforementioned engineered T cells.

另一个方面，本发明还提供了本发明提供的前述任一种TCP、多核苷酸、病毒载体、工程化T细胞或组合物在制备预防和/或治疗癌症的药物中的应用。In another aspect, the present invention also provides the use of any of the aforementioned TCPs, polynucleotides, viral vectors, engineered T cells or compositions provided by the present invention in the preparation of a drug for preventing and/or treating cancer.

另一个方面，本发明还提供一种治疗受试者患有的癌症或杀伤受试者癌细胞的方法，包括向所述受试者给予本发明提供的前述任一种病毒载体、工程化T细胞或组合物。In another aspect, the present invention also provides a method for treating cancer in a subject or killing cancer cells in a subject, comprising administering to the subject any one of the aforementioned viral vectors, engineered T cells or compositions provided by the present invention.

本发明的一些实施例中，所述癌症包括实体癌和血液癌。In some embodiments of the present invention, the cancer includes solid cancer and blood cancer.

本发明的一些实施例中，所述癌症为血液癌。In some embodiments of the present invention, the cancer is a blood cancer.

本发明的一些实施例中，所述血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤、高级别B细胞淋巴瘤和多发性骨髓瘤。In some embodiments of the present invention, the blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, high-grade B-cell lymphoma and multiple myeloma.

本发明的一些实施例中，所述血液癌是CD19+血液癌。In some embodiments of the present invention, the blood cancer is a CD19 + blood cancer.

本发明的一些实施例中，所述CD19+血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤和高级别B细胞淋巴瘤。In some embodiments of the present invention, the CD19 + blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma and high-grade B-cell lymphoma.

本发明的一些实施例中，所述血液癌选自CD19+血液癌和CD33+血液癌；In some embodiments of the present invention, the blood cancer is selected from CD19 + blood cancer and CD33 + blood cancer;

优选地，Preferably,

所述CD19+血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤和高级别B细胞淋巴瘤；The CD19 + blood cancer is selected from the group consisting of marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, and high-grade B-cell lymphoma;

所述CD33+血液癌选自多发性骨髓瘤(MM)、急性髓性白血病(AML)、慢性髓性白血病(CML)和急性单核细胞白血病(AMoL)。The CD33 + blood cancer is selected from the group consisting of multiple myeloma (MM), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and acute monocytic leukemia (AMoL).

本发明的一些实施例中，所述癌细胞表达选自CD19、CD20、CD33、MSLN、CD79B、CD8、ASGPR、BCMA、CEA、uPAR、DLL3、GCC、Nectin4、HER2、Claudin18.2和GUCY2C中的至少一种抗原。In some embodiments of the present invention, the cancer cells express at least one antigen selected from CD19, CD20, CD33, MSLN, CD79B, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2 and GUCY2C.

本发明的一些实施例中，所述给予选自经口、鼻、静脉内、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉、病灶内、持续释放系统和植入装置中的至少一种。In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implant device.

本发明的有益效果包括：The beneficial effects of the present invention include:

本发明提供的病毒载体包含(a)编码前述任一种TCP的多核苷酸，可有效减少所述病毒载体转导靶细胞时的假转导；(b)包含T细胞活化初级信号分子，如抗CD3抗体或其抗原结合片段，还可进一步包含T细胞活化次级信号分子，如抗CD28抗体或其抗原结合片段；相比其他T细胞靶向分子，如抗CD7抗体或其抗原结合片段，可更有效地活化刺激T细胞，使所述TCP分子在T细胞中表达效率更高、杀伤效率更好；The viral vector provided by the present invention comprises (a) a polynucleotide encoding any of the aforementioned TCPs, which can effectively reduce false transduction when the viral vector transduces target cells; (b) comprises a primary signaling molecule for T cell activation, such as an anti-CD3 antibody or an antigen-binding fragment thereof, and can further comprise a secondary signaling molecule for T cell activation, such as an anti-CD28 antibody or an antigen-binding fragment thereof; compared with other T cell targeting molecules, such as an anti-CD7 antibody or an antigen-binding fragment thereof, the viral vector can more effectively activate and stimulate T cells, thereby achieving higher expression efficiency and better killing efficiency of the TCP molecule in T cells;

更重要的是，本发明的发明人首次发现，当所述T细胞活化初级信号分子为抗CD3抗体或其抗原结合片段且不能结合所述TCP包含的TSP时，例如，当所述抗CD3抗体或其抗原结合片段源自抗CD3ε抗体UCHT1(如所述UCHT1-scFv)时，TSP为CD3γ而非CD3ε，所述病毒载体的转导效率更高。More importantly, the inventors of the present invention discovered for the first time that when the primary signal molecule for T cell activation is an anti-CD3 antibody or an antigen-binding fragment thereof and cannot bind to the TSP contained in the TCP, for example, when the anti-CD3 antibody or its antigen-binding fragment is derived from the anti-CD3ε antibody UCHT1 (such as the UCHT1-scFv), the TSP is CD3γ rather than CD3ε, and the transduction efficiency of the viral vector is higher.

定义：definition:

“T细胞”：T细胞是人免疫系统中重要的几种白细胞之一，并在后天免疫应答中发挥着重要的作用。T细胞的主要功能之一是免疫介导的细胞死亡，这一功能主要由两种T细胞亚型完成：CD8+T细胞(Cytotoxic T Cell，细胞毒性T细胞)和CD4+T细胞(Helper T Cell，辅助T细胞)。T cells: T cells are one of several important white blood cells in the human immune system and play a crucial role in acquired immune responses. One of the primary functions of T cells is immune-mediated cell death, a function primarily performed by two T cell subtypes: CD8 + T cells (cytotoxic T cells) and CD4 + T cells (helper T cells).

本发明的一些实施例中，T细胞是CD4+/CD8-、CD4-/CD8+、CD4+/CD8+、CD4-/CD8-T细胞或其组合。本发明的一些实施例中，CD4+T细胞在表达TCP分子并结合靶细胞如CD19+癌细胞后产生IL-2、IFN、TNF或其组合。本发明的一些实施例中，CD8+T细胞在表达TCP分子并与靶细胞结合后裂解抗原特异性靶细胞。In some embodiments of the present invention, the T cells are CD4 + / CD8- , CD4- /CD8 + , CD4 + /CD8 + , CD4- / CD8- T cells, or combinations thereof. In some embodiments of the present invention, CD4 + T cells produce IL-2, IFN, TNF, or a combination thereof after expressing TCP molecules and binding to target cells, such as CD19 + cancer cells. In some embodiments of the present invention, CD8 + T cells lyse antigen-specific target cells after expressing TCP molecules and binding to target cells.

“T细胞受体嵌合蛋白”：即T Cell Receptor Chimeric Protein，“TCP”，是一种重组(recombinant)蛋白，所述TCP分子包括来源于组成TCR/CD3复合体的各种多肽及其变体，如TCR/CD3复合体亚基或其功能性片段及其变体，以及可特异性结合至少一种抗原的抗原结合区；所述TCP分子一般能够通过其包含的抗原结合区，结合至靶细胞表面抗原。"T Cell Receptor Chimeric Protein": T Cell Receptor Chimeric Protein, "TCP", is a recombinant protein. The TCP molecule includes various polypeptides and variants thereof that constitute the TCR/CD3 complex, such as TCR/CD3 complex subunits or their functional fragments and variants, and an antigen binding region that can specifically bind to at least one antigen; the TCP molecule is generally capable of binding to target cell surface antigens through the antigen binding region it contains.

“TCR/CD3复合体亚基或其功能性片段”：TCR/CD3复合体亚基选自TCRα、TCRβ、TCRγ、TCRδ、CD3γ、CD3ζ、CD3δ和CD3ε中的至少一种；而其功能性片段则是所述TCR/CD3复合体亚基胞外、跨膜、胞内、可变区和恒定区等各个结构域足以完成各自功能的最小单位。"TCR/CD3 complex subunit or its functional fragment": TCR/CD3 complex subunit is selected from at least one of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3ζ, CD3δ and CD3ε; and its functional fragment is the minimum unit of the TCR/CD3 complex subunit, including the extracellular, transmembrane, intracellular, variable and constant domains, which are sufficient to perform their respective functions.

本发明的一些实施例中，本发明提供的TCP分子在T细胞中可通过(a)并入内源性TCR/CD3复合体、内源性TCR/CD3复合体亚基或其功能性片段；和/或(b)与内源性TCR/CD3复合体、内源性TCR/CD3复合体亚基或其功能性片段发生功能性相互作用,例如,与内源性CD3亚基或其功能性片段形成TCR/CD3复合体或其功能性片段，进而出膜表达并锚定在T细胞细胞膜上。In some embodiments of the present invention, the TCP molecules provided by the present invention can be (a) incorporated into endogenous TCR/CD3 complexes, endogenous TCR/CD3 complex subunits or functional fragments thereof in T cells; and/or (b) functionally interact with endogenous TCR/CD3 complexes, endogenous TCR/CD3 complex subunits or functional fragments thereof, for example, forming a TCR/CD3 complex or a functional fragment thereof with an endogenous CD3 subunit or a functional fragment thereof, thereby being expressed outside the membrane and anchored on the cell membrane of the T cell.

与CAR分子不同的是，由于T细胞以外的细胞不包含TCRα、TCRβ、CD3γ、CD3ε、CD3γ、CD3ζ和CD3δ等TCR/CD3复合体亚基，因此，即使在信号肽的作用下，所述TCP分子也不会或相对地极少在T细胞以外的细胞，如常见的包装细胞HEK-293T细胞中出膜表达，因此，也不会在出芽过程中，随之转移至包装所得的慢病毒载体或逆转录病毒载体的包膜上。Unlike CAR molecules, cells other than T cells do not contain TCR/CD3 complex subunits such as TCRα, TCRβ, CD3γ, CD3ε, CD3γ, CD3ζ and CD3δ. Therefore, even under the action of signal peptides, the TCP molecule will not or relatively rarely be expressed in cells other than T cells, such as the common packaging cells HEK-293T cells. Therefore, it will not be transferred to the envelope of the packaged lentiviral vector or retroviral vector during the budding process.

“CD19”：CD19是CAR-T疗法中最广泛使用的靶标，已被验证在治疗B细胞急性淋巴细胞白血病(B-ALL)、慢性淋巴细胞白血病(CLL)和B细胞淋巴瘤方面有效且安全。CD19在整个B细胞发育阶段中广泛且专一性地表达，直到终末分化为浆细胞。因此，CD19对B细胞恶性肿瘤具有完美的覆盖作用，这使得CAR-T-19治疗取得了非常高的完全缓解率(CRR)(Wei,J.,Han,X.,Bo,J.et al.Target selection for CAR-T therapy.J Hematol Oncol 12,62(2019))。"CD19": CD19 is the most widely used target in CAR-T therapy and has been proven to be effective and safe in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma. CD19 is widely and specifically expressed throughout the developmental stages of B cells until terminal differentiation into plasma cells. Therefore, CD19 has perfect coverage for B-cell malignancies, which has led to a very high complete remission rate (CRR) achieved by CAR-T-19 therapy (Wei, J., Han, X., Bo, J. et al. Target selection for CAR-T therapy. J Hematol Oncol 12, 62 (2019)).

“CD33”：CD33是一种由膜远端免疫球蛋白可变区(IgV)和膜近端免疫球蛋白恒定区(IgC)结构域组成的唾液粘附蛋白，因其在急性髓性白血病(AML)细胞上的几乎普遍表达，已成为一个可行的靶标。值得注意的是，CD33也存在于前体/成熟髓系细胞和造血干细胞(HSCs)上，但对人类髓系细胞的发育和功能并非必需，这为采用策略去除所有CD33阳性恶性和正常造血细胞群体提供了可能，随后可以通过使用工程化的CD33阴性HSCs进行造血恢复(Freeman R,Shahid S,Khan AG,et al.Developing a membrane-proximal CD33-targeting CAR T cell.J Immunother Cancer.2024 May 20；12(5):e009013.)。"CD33": CD33 is a sialoadhesive protein composed of a membrane-proximal immunoglobulin variable region (IgV) and a membrane-proximal immunoglobulin constant region (IgC) domain. It has become a viable target due to its near-universal expression on acute myeloid leukemia (AML) cells. Notably, CD33 is also present on precursor/mature myeloid cells and hematopoietic stem cells (HSCs) but is not essential for the development and function of human myeloid cells. This provides the possibility of adopting strategies to eliminate all CD33-positive malignant and normal hematopoietic cell populations, followed by hematopoietic restoration by using engineered CD33-negative HSCs (Freeman R, Shahid S, Khan AG, et al. Developing a membrane-proximal CD33-targeting CAR T cell. J Immunother Cancer. 2024 May 20;12(5):e009013.).

“柔性接连肽”：即Flexible Linkers，柔性连接肽通常在相连的结构域需要一定程度的移动或相互作用时应用(Chen X,Zaro JL,Shen WC.,Fusion protein linkers:property,design and functionality.Adv Drug Deliv Rev.2013Oct；65(10):1357-69.)。柔性连接肽通常由小的、非极性的(例如Gly)或者极性的(例如Ser或者Thr)氨基酸组成(Argos P.An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion.J Mol Biol.1990；211:943–958.)。这些小尺寸的氨基酸提供了灵活性，同时也允许相连的功能性结构域的移动。常用的柔性连接肽参见Chen X,Zaro JL,Shen WC.,Fusion protein linkers:property,design and functionality.Adv Drug Deliv Rev.2013 Oct；65(10):1357-69，其以引用的方式整体并入本文中。本发明对于连接所述TSP和抗原结合区的柔性连接肽没有特别的限定，只要其可为所述抗原结合区和TSP之间提供一定的灵活性即可，包括但不限于(G4S)n连接肽、连接肽1：GSTSGSGKPGSGEGSTKG(SEQ ID NO:23)或连接肽3：GSSGGSGGGGSGGGGSGGGGSSG(SEQ ID NO:63)；其中，n＝1至4，任选n＝3,即(G4S)3连接肽(连接肽2)。"Flexible linkers": Flexible linkers are usually used when the connected domains need to move or interact to a certain extent (Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69.). Flexible linkers are usually composed of small, non-polar (such as Gly) or polar (such as Ser or Thr) amino acids (Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990; 211: 943–958.). These small amino acids provide flexibility while also allowing the movement of the connected functional domains. Commonly used flexible linker peptides can be found in Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69, which is incorporated herein by reference in its entirety. The present invention has no particular limitation on the flexible linker peptide connecting the TSP and the antigen-binding region, as long as it can provide a certain degree of flexibility between the antigen-binding region and the TSP, including but not limited to (G 4 S) n linker peptide, linker peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23) or linker peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 63); wherein n=1 to 4, optionally n=3, i.e., (G 4 S) 3 linker peptide (linker peptide 2).

“抗体”：是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。"Antibody" refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to specifically bind to an antigen. "Antibody" herein encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

本文“抗体”包括一种典型的“四链抗体”,其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链指的是从其N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成的多肽链；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链指的是从其N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。The "antibody" herein includes a typical "four-chain antibody", which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to a polypeptide chain composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain from its N-terminus to its C-terminus; and, when the full-length antibody is of the IgE isotype, it optionally further includes a heavy chain constant region CH4 domain; the light chain refers to a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) from its N-terminus to its C-terminus; the heavy chains and the light chains are linked by disulfide bonds to form a "Y"-shaped structure.

在抗体的上下文中，术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链(分别为VH区和VL区)的可变区通常具有相似的结构，每个结构域包含四个保守框架区(FR)和三个互补决定区(CDR)。(参见例如Kindt等人，Kuby Immunology，第6版，W.H.Freeman and Co.，第91页(2007))。单个VH区或VL区可以足以赋予抗原结合特异性。此外，结合特定抗原的抗体可使用来自结合该特定抗原的抗体的VH区或VL区分离，以分别筛选互补VL区或VH区的文库。参见例如Portolano等人，J.Immunol.150:880-887(1993)；Clarkson等人，Nature352:624-628(1991)。In the context of antibodies, the term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains (VH and VL regions, respectively) of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL region may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL region from an antibody that binds to that specific antigen to screen for a library of complementary VL or VH regions, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

与“高变区”或“HVR”同义的术语“互补决定区”和“CDR”在本领域中已知是指抗体可变区内的氨基酸的非连续序列，其赋予抗原特异性和/或结合亲和力。一般来讲，每个重链可变区中有三个CDR(HCDR1、HCDR2、HCDR3)，并且每个轻链可变区中有三个CDR(LCDR1、LCDR2、LCDR3)。The terms "complementarity determining region" and "CDR", synonymous with "hypervariable region" or "HVR", are known in the art to refer to non-contiguous sequences of amino acids within an antibody variable region that confer antigen specificity and/or binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

本发明的一些实施例中，CDR区的确定是根据IMGT编号方案、Kabat编号方案、Martin编号方案、AbM编号方案、Chothia编号方案或Contact编号方案。In some embodiments of the present invention, the CDR regions are identified according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, or the Contact numbering scheme.

本发明的一些实施例中，CDR区的确定是根据Kabat编号方案。In some embodiments of the present invention, the CDR regions are identified according to the Kabat numbering scheme.

本文“抗体”还包括单链可变区片段(Single-Chain Fragment Variable，“scFv”)。The term “antibody” in this article also includes single-chain variable region fragments (Single-Chain Fragment Variable, “scFv”).

本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus Dromedarius)、双峰驼(Camelus Bactrianus)、大羊驼(Lama Glama)、原驼(Lama Guanicoe)和羊驼(Vicugna Pacos)等产生的重链抗体(Heavy-Chain Antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig New Antigen Receptor,IgNAR)。The term "antibody" in this article also includes antibodies that do not contain light chains, such as heavy-chain antibodies (HCAbs) produced by dromedary camels (Camelus Dromedarius), Bactrian camels (Camelus Bactrianus), llamas (Lama Glama), guanacos (Lama Guanicoe) and alpacas (Vicugna Pacos), as well as immunoglobulin new antigen receptors (Ig New Antigen Receptor, IgNAR) found in cartilaginous fish such as sharks.

本文术语“VHH结构域”和“单域抗体”(Single Domain Antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。In this article, the terms "VHH domain" and "single domain antibody" (sdAb) have the same meaning and are used interchangeably. They refer to the construction of a single domain antibody (sdAb) consisting solely of a single heavy chain variable region, obtained by cloning the variable region of a heavy chain antibody. SdAbs are minimal antigen-binding fragments with full functionality. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single heavy chain variable region.

本文“抗体”还包括单克隆抗体或其抗原结合部分。单克隆抗体或其抗原结合部分可以是非人的、嵌合的、人源化的或人的，优选地人源化的或人的。免疫球蛋白结构和功能例如在Harlow等人编辑，Antibodies:A Laboratory Manua，第14章(Cold Spring Harbor Laboratory,Cold Spring Harbor,1988)中进行了综述。The term "antibody" herein also includes monoclonal antibodies or antigen-binding portions thereof. Monoclonal antibodies or antigen-binding portions thereof may be non-human, chimeric, humanized or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、猴科(例如食蟹猴和恒河猴)、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。The "antibodies" herein can be derived from any animal, including but not limited to humans and non-human animals, which can be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, monkeys (such as cynomolgus monkeys and rhesus monkeys), alpacas, sheep, rabbits, mice, rats or cartilaginous fish (such as sharks).

本文“抗体”还包括抗体的重链可变区(VH)或轻链可变区(VL)。"Antibody" herein also includes the heavy chain variable region (VH) or light chain variable region (VL) of the antibody.

本文中，“抗原结合片段”：是指不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。Herein, "antigen-binding fragment" refers to a fragment that does not have the entire structure of an intact antibody and only contains a portion or a partial variant of the intact antibody, wherein the portion or partial variant has the ability to bind to an antigen.

示例性的，本文中，“抗体或其抗原结合片段”包括但不限于免疫球蛋白(全长抗体)、半抗体、Fab、Fab'、F(ab')2、Fv片段、单链可变区片段(scFv)、二硫键稳定性抗体(dsFv)、抗体的重链可变区(VH)或轻链可变区(VL)、由VH和CH1结构域组成的Fd片段、线性抗体和单域抗体(纳米抗体)；所述scFv的重链(VH)通过连接肽与轻链(VL)连接。Exemplary, herein, "antibody or antigen-binding fragment thereof" includes but is not limited to immunoglobulin (full-length antibody), half antibody, Fab, Fab', F(ab') 2 , Fv fragment, single-chain variable region fragment (scFv), disulfide bond-stabilized antibody (dsFv), the heavy chain variable region (VH) or light chain variable region (VL) of an antibody, an Fd fragment consisting of a VH and a CH1 domain, a linear antibody and a single-domain antibody (nanobody); the heavy chain (VH) of the scFv is connected to the light chain (VL) by a connecting peptide.

本发明的一些实施例中，对scFv从N-末端至C-末端包含VH区或VL区的顺序没有特别的限定，如从N-末端至C-末端包含VH-Linker-VL或VL-Linker-VH；所述连接肽可选自柔性连接肽。In some embodiments of the present invention, there is no particular limitation on the order in which the scFv comprises the VH region or the VL region from the N-terminus to the C-terminus, such as VH-Linker-VL or VL-Linker-VH from the N-terminus to the C-terminus; the connecting peptide can be selected from a flexible connecting peptide.

“配体”：在受体-配体结合中，配体通常为与受体上的位点结合产生信号的分子，所述结合通常导致复杂结构的构象变化，从而诱导相关的生理活性。"Ligand": In receptor-ligand binding, a ligand is generally a molecule that binds to a site on a receptor to generate a signal, such binding typically resulting in a conformational change in the complex structure, thereby inducing the relevant physiological activity.

“受体结合片段”：是指不具备完整配体的全部结构，仅包含完整配体的局部或局部的变体，所述局部或局部的变体具备结合受体的能力。示例性的，本文“受体结合片段”包括但不限于所述配体的的胞外域、功能性片段、表位、结合区和可变区。"Receptor binding fragment" refers to a fragment that lacks the full structure of a complete ligand and contains only a portion or partial variant of the complete ligand, which possesses the ability to bind to the receptor. Exemplarily, "receptor binding fragment" herein includes, but is not limited to, the extracellular domain, functional fragment, epitope, binding region, and variable region of the ligand.

“内吞作用”(Endocytosis)指的是物质进入细胞的一种过程，在内吞作用中，待被摄入的物质被质膜的一个区域包围，随后质膜在细胞内出芽(Budding)形成一个包含被摄入物质的囊泡。内吞作用可被分为四类：受体介导的内吞作用(Receptor-Mediated Endocytosis，又名Clathrin-Mediated Endocytosis，网格蛋白介导的内吞作用)、小窝(Caveolae)、胞饮作用(Pinocytosis)和吞噬作用(Phagocytosis)(Marsh M，Endocytosis.Oxford University Press.p.vii.，2001)。Endocytosis refers to the process by which substances enter cells. During endocytosis, the substance to be taken in is surrounded by a region of the plasma membrane. The plasma membrane then buds into the cell to form a vesicle containing the taken in substance. Endocytosis can be divided into four categories: receptor-mediated endocytosis (also known as clathrin-mediated endocytosis), caveolae, pinocytosis, and phagocytosis (Marsh M, Endocytosis. Oxford University Press. p. vii., 2001).

“嵌合抗原受体”：即Chimeric Antigen Receptor(CAR)，是指经改造以在淋巴细胞等免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其至少包含(1)细胞外的抗原结合区，例如scFv或VHH；(2)锚定CAR分子进入免疫效应细胞的跨膜区，和(3)胞内信号传导结构域；CAR的细胞外结构还可进一步包含铰链区，细胞内结构还可进一步包含一个或多个共刺激分子组成共刺激信号传导结构域。CAR分子能利用细胞外的抗原结合区以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。"Chimeric Antigen Receptor": Chimeric Antigen Receptor (CAR) refers to an artificial cell surface receptor that has been modified to be expressed on immune effector cells such as lymphocytes and specifically bind to antigens, which at least contains (1) an extracellular antigen binding region, such as scFv or VHH; (2) a transmembrane region that anchors the CAR molecule into the immune effector cell, and (3) an intracellular signaling domain; the extracellular structure of the CAR may further include a hinge region, and the intracellular structure may further include one or more costimulatory molecules to form a costimulatory signaling domain. CAR molecules can use the extracellular antigen binding region to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner.

“嵌合”：术语“嵌合”是指非内源的并且包含接合或连接在一起的序列的组合的任何核酸分子或蛋白质，所述序列在自然界中并非天然接合或连接在一起的。例如，嵌合核酸分子可包含编码来自多个不同基因的各种结构域的核酸。又如，嵌合核酸分子可包含来源于不同来源的调控序列和编码序列，或者来源于相同来源但以不同于天然存在的方式排列的调控序列和编码序列。"Chimeric": The term "chimeric" refers to any nucleic acid molecule or protein that is non-endogenous and comprises a combination of sequences joined or linked together that are not naturally joined or linked together in nature. For example, a chimeric nucleic acid molecule can comprise nucleic acids encoding various domains from multiple different genes. As another example, a chimeric nucleic acid molecule can comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature.

“抗原”：术语“抗原”和“Ag”是指能够诱导免疫响应的分子。诱导的免疫响应可包括抗体产生和/或特异性免疫感受态细胞的激活。包括蛋白质、糖蛋白和糖脂在内的大分子可用作抗原。抗原可以来源于重组或基因组DNA。如本文所设想的那样，抗原不需要(i)仅由基因的全长核苷酸序列编码或(ii)完全由基因编码。抗原可以生成或合成，或者抗原可来源于生物样品。这样的生物样品可包括但不限于组织样品、肿瘤样品、细胞或生物流体。"Antigen": The terms "antigen" and "Ag" refer to a molecule capable of inducing an immune response. The induced immune response may include the production of antibodies and/or the activation of specific immune competent cells. Macromolecules including proteins, glycoproteins and glycolipids can be used as antigens. Antigens can be derived from recombinant or genomic DNA. As contemplated herein, an antigen need not be (i) encoded solely by the full-length nucleotide sequence of a gene or (ii) fully encoded by a gene. Antigens can be generated or synthesized, or the antigen can be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

“降低”：当提及所述糖蛋白或其变体结合其受体的能力“降低”时，术语“降低”包括完全消除所述糖蛋白或其变体结合其受体的能力，以及显著降低所述结合能力。在具体实施方案中，“显著降低”是指相对于野生型病毒糖蛋白而言；“降低”选自降低至少95％、至少90％、至少85％、至少80％、至少75％、至少70％、至少65％、至少60％、至少55％、至少50％、至少45％、至少40％、至少35％、至少30％、至少25％、至少20％、至少15％、至少10％、至少5％、至少4％、至少3％、至少2％和至少1％。"Reduction": When referring to the ability of the glycoprotein or its variant to bind to its receptor "reduction", the term "reduction" includes completely eliminating the ability of the glycoprotein or its variant to bind to its receptor, as well as significantly reducing the binding ability. In a specific embodiment, "significant reduction" refers to a reduction relative to the wild-type viral glycoprotein; "reduction" is selected from a reduction of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 4%, at least 3%, at least 2% and at least 1%.

“核酸”：指包括包含核苷酸的聚合物的任何化合物和/或物质，如多核苷酸。本文中，“核酸”、“多核苷酸”和“基因”同义使用。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5'至3'。在本文中，术语“核酸”涵盖脱氧核糖核酸(DNA),包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA),特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。“核酸”可以是线性的或环状的。此外，“核酸”包括有正义链(编码链)和反义链(模板链)二者，以及单链和双链形式。而且，本文所述的“核酸”可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖、磷酸骨架键合或化学修饰的残基所修饰的核苷酸碱基。"Nucleic acid" refers to any compound and/or substance including a polymer containing nucleotides, such as a polynucleotide. As used herein, "nucleic acid," "polynucleotide," and "gene" are used synonymously. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. As used herein, the term "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. "Nucleic acid" can be linear or circular. In addition, "nucleic acid" includes both a sense strand (coding strand) and an antisense strand (template strand), as well as single-stranded and double-stranded forms. Furthermore, the "nucleic acids" described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include nucleotide bases modified with derivatized sugars, phosphate backbone linkages, or chemically modified residues.

“核酸载体”：即表示携带、容纳或表达任何核酸的载体。所述核酸载体可具有诸如表达、包装、假型化或转导的特有功能。如果核酸载体适合用作克隆载体或穿梭载体，则其还可具有操纵功能。载体的结构可包括制作可行且适合于特定用途的任何期望的形式。此类形式包括例如环状形式诸如质粒和噬菌粒，以及线性或分支形式。核酸载体可由例如DNA或RNA构成，以及含有部分或全部核苷酸衍生物、类似物和模拟物。此类核酸载体可从自然来源获得、重组产生或以化学方式合成。"Nucleic acid vector" means a vector that carries, contains or expresses any nucleic acid. The nucleic acid vector may have specific functions such as expression, packaging, pseudotyping or transduction. If the nucleic acid vector is suitable for use as a cloning vector or shuttle vector, it may also have a manipulation function. The structure of the vector may include any desired form that is feasible to manufacture and suitable for a particular use. Such forms include, for example, circular forms such as plasmids and phagemids, as well as linear or branched forms. Nucleic acid vectors may be composed of, for example, DNA or RNA, as well as contain some or all nucleotide derivatives, analogs and mimetics. Such nucleic acid vectors may be obtained from natural sources, recombinantly produced or chemically synthesized.

“转基因”：即Transgene，又称负荷基因(Payload gene)，如本文所用，术语“转基因”是指编码感兴趣的蛋白质(例如，前述任一种TCP等)的基因或多核苷酸，所述感兴趣的蛋白质表达在宿主细胞/靶细胞中是期望的并且已通过基因工程技术转移到细胞中。转基因可以编码有治疗意义的蛋白质以及作为报告子、标签、标志物、自杀蛋白等的蛋白质。转基因可以来自天然来源、天然基因的修饰、或者重组或合成分子。在某些实施方案中，转基因是病毒载体的组分。"Transgene": Transgene, also known as payload gene (Payload gene). As used herein, the term "transgene" refers to a gene or polynucleotide encoding a protein of interest (e.g., any of the aforementioned TCPs, etc.), the expression of which is desired in host cells/target cells and has been transferred into cells by genetic engineering technology. Transgenes can encode therapeutic proteins as well as proteins that serve as reporters, tags, markers, suicide proteins, etc. Transgenes can come from natural sources, modifications of natural genes, or recombinant or synthetic molecules. In certain embodiments, the transgene is a component of a viral vector.

“表达盒”：如本文所用，术语“表达盒”是指包含至少一种转基因和控制其在宿主细胞中表达的调控序列(例如，启动子、3'UTR)的载体核酸的独特组分。串联表达盒是指包含至少两种转基因的载体核酸的组分，所述转基因处于用于串联表达所述至少两种转基因的一组相同调控序列的控制下。在某些实施方案中，串联表达盒包含至少两种处于相同启动子控制下的转基因。在某些实施方案中，第一转基因和第二转基因被内部核糖体进入位点(IRES)、弗林蛋白酶切割位点或自切割病毒2A肽分开，以允许由单个mRNA共表达两种蛋白质。"Expression cassette": As used herein, the term "expression cassette" refers to a unique component of a vector nucleic acid that comprises at least one transgene and regulatory sequences (e.g., promoter, 3'UTR) that control its expression in a host cell. A tandem expression cassette refers to a component of a vector nucleic acid that comprises at least two transgenes that are under the control of a set of identical regulatory sequences for tandem expression of the at least two transgenes. In certain embodiments, the tandem expression cassette comprises at least two transgenes under the control of the same promoter. In certain embodiments, the first transgene and the second transgene are separated by an internal ribosome entry site (IRES), a furin cleavage site, or a self-cleaving viral 2A peptide to allow co-expression of two proteins from a single mRNA.

如本文所用，术语“肽”、“多肽”和“蛋白质”可互换使用，并且是指由通过肽键共价连接的氨基酸残基组成的化合物。As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds.

“编码”：是指特定多核苷酸序列(诸如DNA、cDNA和mRNA序列)在生物过程中用作合成其他聚合物和大分子的模板的固有特性，所述模板具有限定的核苷酸序列(即，rRNA、tRNA和mRNA)或限定的氨基酸序列以及由此产生的生物特性。因此，如果对应于多核苷酸的mRNA的转录和翻译在细胞或其他生物系统中产生蛋白质，则该多核苷酸编码该蛋白质。编码链和非编码链都可称为编码蛋白质或多核苷酸的其他产物。除非另外指明，否则“编码氨基酸序列的核苷酸序列”包括彼此为简并型式并且编码相同氨基酸序列的所有核苷酸序列。"Encoding": refers to the inherent property of a specific polynucleotide sequence (such as DNA, cDNA and mRNA sequences) used as a template for synthesizing other polymers and macromolecules in biological processes, wherein the template has a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. Therefore, if the transcription and translation of the mRNA corresponding to the polynucleotide produces a protein in a cell or other biological system, the polynucleotide encodes the protein. Both the coding strand and the non-coding strand can be referred to as encoding proteins or other products of the polynucleotide. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.

“自切割肽”或“自裂解肽”或“2A肽”：是指被配置成从单个开放阅读框生成两种或更多种蛋白质的自裂解肽，包括FT2A肽、F2A肽、E2A肽、T2A肽和P2A肽等。2A肽是在真核细胞中介导翻译期间多肽的“裂解”的18至22个残基长的病毒寡肽。“2A肽”可指代具有不同氨基酸序列的肽。在本发明公开中，应当理解，在病毒载体包含两个或更多个2A肽的情况下，2A肽可彼此相同或不同。用于设计和使用2A肽的详细方法由Szymczak-Workman等人(2012)ColdSpring Harb.Protoc.2012:199-204提供。"Self-cleaving peptide" or "self-cleaving peptide" or "2A peptide": refers to a self-cleaving peptide that is configured to generate two or more proteins from a single open reading frame, including FT2A peptide, F2A peptide, E2A peptide, T2A peptide and P2A peptide, etc. 2A peptides are 18 to 22 residues long viral oligopeptides that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. "2A peptide" can refer to peptides with different amino acid sequences. In the present disclosure, it should be understood that when a viral vector contains two or more 2A peptides, the 2A peptides may be the same or different from each other. Detailed methods for designing and using 2A peptides are provided by Szymczak-Workman et al. (2012) Cold Spring Harb. Protoc. 2012: 199-204.

“外源性”：指来源于生物体外部的任何分子，包括核酸、蛋白质、多肽或小分子化合物等。相比之下，术语“内源性”是指来源于生物体内部的任何分子(即生物体自然产生的)。"Exogenous" refers to any molecule that originates from outside an organism, including nucleic acids, proteins, peptides, or small molecule compounds. In contrast, the term "endogenous" refers to any molecule that originates from within an organism (i.e., produced naturally by the organism).

“启动子”：如本文所用的术语“启动子”被定义为由起始多核苷酸序列特异性转录所需的细胞合成机制或引入的合成机制识别的DNA序列。如本文所用，术语“启动子/调控序列”意指表达与启动子/调控序列可操作地连接的基因产物所需的核酸序列。在一些情况下，该序列可以是核心启动子序列，并且在其他情况下，该序列可包含增强子序列和表达基因产物所需的其他调控元件。启动子/调控序列可以是例如以组织特异性方式表达基因产物的序列。"Promoter": As used herein, the term "promoter" is defined as a DNA sequence that is recognized by the cellular synthetic machinery or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence. As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence required for expression of a gene product operably linked to the promoter/regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter/regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

“组成型”启动子是当与编码或指定基因产物的多核苷酸可操作地连接时在细胞的大多数或所有生理条件下引起细胞中产生基因产物的核苷酸序列。A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

“诱导型”启动子是当与编码或指定基因产物的多核苷酸可操作地连接时，基本上仅在细胞中存在对应于该启动子的诱导物时才引起细胞中产生基因产物的核苷酸序列。An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

“组织特异性”启动子是当与编码或由基因指定的多核苷酸可操作地连接时，基本上仅在细胞是对应于该启动子的组织类型的细胞时才引起细胞中产生基因产物的核苷酸序列。A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is of the tissue type corresponding to the promoter.

“病毒包膜”：指的是多种病毒的最外层(HURLBERT,RONALD E.,Fundamentals of Microbiology,102.Chapter #11:Viruses.Archived from the original on 2008-11-10.)。当病毒在宿主细胞中穿梭时，病毒包膜保护其生命周期内的遗传物质。并不是所有病毒都有病毒包膜。多种人类致病病毒被包裹在脂质双层中，它们通过使病毒包膜与细胞膜融合感染靶细胞。"Viral envelope" refers to the outermost layer of many viruses (Hurlbert, Ronald E., Fundamentals of Microbiology, 102. Chapter #11: Viruses. Archived from the original on 2008-11-10.). The viral envelope protects the viral genetic material during its life cycle as it navigates through host cells. Not all viruses have a viral envelope. Many human pathogenic viruses are enclosed in a lipid bilayer and infect target cells by fusing their viral envelope with the cell membrane.

“慢病毒”：慢病毒是复杂的逆转录病毒，除了常见的逆转录病毒基因gag、pol和env外，其还含有其他具有调控或结构功能的基因。较高的复杂性使得病毒能够调节其生命周期，如其在潜伏感染过程中一样。慢病毒属于可感染分裂(dividing)细胞和非分裂(non-dividing)细胞的逆转录病毒属。慢病毒的实例包括但不限于HIV(人免疫缺陷病毒，包括HIV Ⅰ型和HIV Ⅱ型)、马传染性贫血病毒、猫免疫缺陷病毒(FIV)、牛免疫缺陷病毒(BIV)和猿免疫缺陷病毒(SIV)。"Lentivirus": Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. This greater complexity allows the virus to regulate its life cycle, as it does during latent infection. Lentiviruses belong to a genus of retroviruses that can infect both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type I and HIV type II), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

“慢病毒载体”：即Lentiviral Vector，是来源于慢病毒的载体，并且包含一种或多种慢病毒包装蛋白和/或表达由载体携带的一种或多种基因所必需的慢病毒蛋白。通过基因编辑、基因工程等技术手段对HIV等慢病毒的毒力基因进行多次减毒而产生，例如，删除基因env、vif、vpr、vpu和nef，使慢病毒载体具有生物安全性。"Lentiviral vector" is a vector derived from a lentivirus and contains one or more lentiviral packaging proteins and/or lentiviral proteins necessary for the expression of one or more genes carried by the vector. Lentiviral vectors are produced by multiple attenuation of virulence genes of lentiviruses such as HIV through gene editing and genetic engineering techniques. For example, deletion of the env, vif, vpr, vpu, and nef genes makes lentiviral vectors biosafe.

如本文所用，术语“慢病毒载体”旨在表示包括病毒包膜、具有慢病毒的至少一种特征并且能够侵入靶细胞的、不具备自我复制能力的慢病毒颗粒。As used herein, the term "lentiviral vector" is intended to mean a lentiviral particle that includes a viral envelope, has at least one characteristic of a lentivirus, and is capable of invading target cells without the ability to replicate itself.

常用的假型慢病毒载体包括所谓的第三代慢病毒载体包装系统。第三代慢病毒载体包装系统包括四种质粒，通常来说，一般包括转移质粒和三种包装质粒：包含编码目的基因(Gene of Interest，“GOI”)，如转基因的转移质粒/主质粒、GagPol质粒、Rev质粒和包膜质粒(包含如VSV-G或其变体或Cocal-G或其变体等病毒糖蛋白基因)。Commonly used pseudotyped lentiviral vectors include the so-called third-generation lentiviral packaging systems. Third-generation lentiviral packaging systems typically consist of four plasmids: a transfer plasmid (containing the gene of interest, "GOI"), such as a transgene; a GagPol plasmid; a Rev plasmid; and an envelope plasmid (containing the viral glycoprotein gene, such as VSV-G or its variants or Cocal-G or its variants).

“转移质粒”(Transfer Vector)包含慢病毒载体骨架基因组和转基因。转移质粒通常具有一个或多个侧接有长末端重复(LTRs)序列的转基因，这有助于将转移质粒包含的转基因整合到宿主基因组中。LTRs负责病毒基因组的逆转录和整合过程。通过这些序列，慢病毒可以将转基因整合到宿主细胞的基因组中。出于安全原因，转移质粒通常被设计成使所产生的病毒载体无法自我复制，例如，转移质粒缺乏在宿主细胞中产生具有感染能力的慢病毒载体所必需的基因元件。此外，可将转移质粒设计成缺失3’LTR，从而使病毒“自身失活(Self-Inactivating)。相较于传统的第二代假型慢病毒载体包装系统(通常是包含编码Gag、Pol、Rev和Tat的核酸的单一包装质粒和单独的包膜质粒)，通过在转移质粒上添加与异源启动子(例如，CMV或RSV启动子)融合的嵌合5'LTR以来将TAT基因从第三代假型慢病毒载体包装系统中消除。转移质粒通常包含位于5’LTR下游的Ψ序列(Psi序列，又称Ψ包装信号)，Ψ序列负责将转基因RNA打包(Packaged into)到病毒载体中。Ψ序列确保了只有包含转基因的RNA被包装进入病毒载体。转移质粒还可选可包含内部核糖体进入位点(Internal Ribosome Entry Site,“IRES”)，以允许在一个mRNA上同时翻译两个或多个开放阅读框(ORF)，从而实现多基因表达。一些转移质粒，如本发明的一些实施例中使用的慢病毒主质粒/转移质粒，还可包含选择标记基因，如抗生素抗性基因(如PuroR，编码嘌呤霉素抗性)或荧光蛋白基因(如GFP)，用于筛选或追踪转导细胞。The "transfer plasmid" contains the lentiviral vector backbone genome and the transgene. The transfer plasmid usually has one or more transgenes flanked by long terminal repeat (LTRs) sequences, which facilitate the integration of the transgene contained in the transfer plasmid into the host genome. LTRs are responsible for the reverse transcription and integration processes of the viral genome. Through these sequences, the lentivirus can integrate the transgene into the genome of the host cell. For safety reasons, the transfer plasmid is usually designed so that the resulting viral vector cannot replicate itself, for example, the transfer plasmid lacks the genetic elements necessary to produce an infectious lentiviral vector in the host cell. In addition, the transfer plasmid can be designed to lack the 3'LTR, thereby making the virus "self-inactivating". Compared with the traditional second-generation pseudotype lentiviral vector packaging system (usually a single packaging plasmid containing nucleic acids encoding Gag, Pol, Rev and Tat and a separate envelope plasmid), the TAT gene is eliminated from the third-generation pseudotype lentiviral vector packaging system by adding a chimeric 5'LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) to the transfer plasmid. The transfer plasmid usually contains a Ψ sequence (Psi sequence, also known as Ψ packaging signal) located downstream of the 5'LTR. The Ψ sequence is responsible for packaging the transgenic RNA (Pack aged into) into the viral vector. The Ψ sequence ensures that only RNA containing the transgene is packaged into the viral vector. The transfer plasmid may also optionally contain an internal ribosome entry site (Internal Ribosome Entry Site, "IRES") to allow simultaneous translation of two or more open reading frames (ORFs) on one mRNA, thereby achieving multi-gene expression. Some transfer plasmids, such as the lentiviral master plasmid/transfer plasmid used in some embodiments of the present invention, may also contain a selection marker gene, such as an antibiotic resistance gene (such as PuroR, encoding puromycin resistance) or a fluorescent protein gene (such as GFP), for screening or tracking transduced cells.

关于慢病毒载体包装系统中的转移质粒，具体可参见Dull,et al.,J.Virol.72:8463-71(1998)；Miyoshi,et al.,J.Virol.72:8150-57(1998)。For details about the transfer plasmid in the lentiviral vector packaging system, see Dull, et al., J. Virol. 72: 8463-71 (1998); Miyoshi, et al., J. Virol. 72: 8150-57 (1998).

第三代慢病毒载体系统通常还包括三种包装质粒：GagPol质粒、Rev质粒和包膜质粒。包膜质粒通常携带病毒糖蛋白基因，野生型VSV-G或Cocal-G是常用的病毒糖蛋白之一；病毒糖蛋白基因可操作地连接到启动子，启动子通常为CMV启动子，启动病毒糖蛋白基因的转录。第三代慢病毒载体系统还包括两个包装质粒，一个包含编码Gag蛋白和Pol蛋白的基因(GagPol包装质粒)，而另一个包含编码Rev蛋白的基因(Rev质粒)作为进一步的安全特征，这是对所谓的第二代包装系统的单个包装质粒的改进。Gag基因编码包含慢病毒结构蛋白的Gag多蛋白前体，其包含基质、衣壳和核衣壳；Pol基因编码提供复制所必需的慢病毒酶功能的Pol多蛋白前体，其包含蛋白酶、逆转录酶和整合酶；Rev基因编码Rev蛋白，其结合Rev响应元件(RRE)以允许在病毒复制期间未剪接和单剪接的HIV RNA的核输出。Gag和Pol多蛋白前体在病毒载体制备期间被切割。Rev蛋白结合病毒RNA上的Rev响应元件(RRE)序列，通过与宿主细胞的核输出机制相互作用，促进未完全剪切的病毒RNA从细胞核转运到细胞质。这些未剪切的RNA在细胞质中可以被翻译成病毒结构蛋白和酶类，或者组装成新的病毒载体。Third-generation lentiviral vector systems typically also include three packaging plasmids: a GagPol plasmid, a Rev plasmid, and an envelope plasmid. The envelope plasmid typically carries a viral glycoprotein gene, with wild-type VSV-G or Cocal-G being one of the commonly used viral glycoproteins. The viral glycoprotein gene is operably linked to a promoter, typically a CMV promoter, to initiate transcription of the viral glycoprotein gene. Third-generation lentiviral vector systems also include two packaging plasmids, one containing genes encoding Gag and Pol proteins (GagPol packaging plasmid), and the other containing a gene encoding Rev protein (Rev plasmid) as a further safety feature, which is an improvement over the single packaging plasmid of the so-called second-generation packaging system. The Gag gene encodes the Gag polyprotein precursor, which contains the lentiviral structural proteins and includes the matrix, capsid, and nucleocapsid. The Pol gene encodes the Pol polyprotein precursor, which provides the lentiviral enzyme functions necessary for replication and includes protease, reverse transcriptase, and integrase. The Rev gene encodes the Rev protein, which binds to the Rev response element (RRE) to allow nuclear export of unspliced and singly spliced HIV RNA during viral replication. The Gag and Pol polyprotein precursors are cleaved during viral vector preparation. The Rev protein binds to the Rev response element (RRE) sequence on the viral RNA and, by interacting with the host cell's nuclear export machinery, promotes the transport of incompletely spliced viral RNA from the nucleus to the cytoplasm. These unspliced RNAs can be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral vectors.

示例性的，所述包装质粒包括但不限于pMD2.G、pRSV-rev、pMDLG-pRRE和pRRL-GOI。Exemplarily, the packaging plasmid includes but is not limited to pMD2.G, pRSV-rev, pMDLG-pRRE and pRRL-GOI.

慢病毒载体和慢病毒载体骨架基因组是本领域中已知的，参见Naldini,etal.，(1996)Science272：263-7；Zufferey等人，(1998)J.Virol.72：9873-9880；Dull等人，(1998)J.Virol.72：8463-8471、美国专利号6,013,516和美国专利号5,994,136，其各自以引用的方式整体并入本文。Lentiviral vectors and lentiviral vector backbone genomes are known in the art, see Naldini, et al., (1996) Science 272:263-7; Zufferey et al., (1998) J. Virol. 72:9873-9880; Dull et al., (1998) J. Virol. 72:8463-8471, U.S. Pat. No. 6,013,516 and U.S. Pat. No. 5,994,136, each of which is incorporated herein by reference in its entirety.

相较于假型慢病毒载体包装系统，假型逆转录病毒载体包装系统通常不包含Rev质粒，这是由于源自摩洛尼鼠白血病病毒(Moloney Murine Leukemia Virus，“MMLV”)等逆转录病毒的基因组RNA可以自然地从细胞核中转运到细胞质进行翻译和组装，因此无需依赖于特定的核输出机制，如Rev蛋白等。假型逆转录病毒载体包装系统通常包含一种转移质粒和两种包装质粒：包膜质粒和GagPol包装质粒。转移质粒包含的转基因序列被长末端重复序列(LTRs)夹在两侧，这些LTR序列促进了转移质粒序列整合到宿主基因组中。通常，在病毒转导过程中，在LTRs之间并包括LTRs的序列将被整合至宿主基因组中。MMLV或鼠干细胞病毒(Murine Stem Cell Virus,“MSCV”)的包含其各自的LTRs的骨架基因组通常被应运用于构建假型逆转录病毒载体包装系统中的转移质粒。GagPol包装质粒包含Gag基因和Pol基因；包膜质粒通常包含编码病毒糖蛋白，如VSV-G或Cocal-G的多核苷酸。本发明的一些实施例中，包膜质粒还可包含编码所述T细胞活化初级信号分子和/或T细胞活化次级信号分子的核酸。Compared to pseudotyped lentiviral vector packaging systems, pseudotyped retroviral vector packaging systems usually do not contain Rev plasmids. This is because the genomic RNA derived from retroviruses such as Moloney Murine Leukemia Virus (MMLV) can be naturally transported from the cell nucleus to the cytoplasm for translation and assembly, so there is no need to rely on specific nuclear export mechanisms such as Rev protein. Pseudotyped retroviral vector packaging systems usually contain a transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transgene sequence contained in the transfer plasmid is sandwiched on both sides by long terminal repeat sequences (LTRs). These LTR sequences promote the integration of the transfer plasmid sequence into the host genome. Typically, during viral transduction, the sequences between and including the LTRs will be integrated into the host genome. The backbone genome of MMLV or murine stem cell virus (MSCV), including their respective LTRs, is typically used to construct transfer plasmids in pseudotyped retroviral vector packaging systems. The GagPol packaging plasmid contains the Gag gene and the Pol gene; the envelope plasmid typically contains a polynucleotide encoding a viral glycoprotein, such as VSV-G or Cocal-G. In some embodiments of the present invention, the envelope plasmid may also contain a nucleic acid encoding the T cell activation primary signaling molecule and/or the T cell activation secondary signaling molecule.

在一些实施方案中，用限定比率的转移质粒、GagPol质粒、包膜质粒和Rev质粒转染生产细胞。在一些实施方案中，各质粒的比率通过质量确定，其只要是能包装出具有生物活性的非整合慢病毒载体便无特别的限定。在一些实施方案中，转移质粒和GagPol质粒中每一种的质量高于包膜质粒和Rev质粒中每一种的质量。在一些实施方案中，转移质粒、GagPol质粒、包膜质粒和Rev质粒的限定比率为约1:1:1:1至约9:4:2:2；本发明的一些实施例中，可以是包膜质粒包含编码所述T细胞活化初级信号分子和/或T细胞活化次级信号分子的核酸。In some embodiments, the production cells are transfected with a defined ratio of transfer plasmids, GagPol plasmids, envelope plasmids, and Rev plasmids. In some embodiments, the ratio of each plasmid is determined by mass, and there is no particular limitation as long as it can package a non-integrated lentiviral vector with biological activity. In some embodiments, the mass of each of the transfer plasmid and the GagPol plasmid is higher than the mass of each of the envelope plasmid and the Rev plasmid. In some embodiments, the defined ratio of the transfer plasmid, GagPol plasmid, envelope plasmid, and Rev plasmid is about 1:1:1:1 to about 9:4:2:2; in some embodiments of the present invention, the envelope plasmid may contain a nucleic acid encoding the T cell activation primary signaling molecule and/or the T cell activation secondary signaling molecule.

在一些实施方案中，包膜质粒包含编码前述任一种VSV-G或其变体或Cocal-G或其变体和如本文所公开的所述T细胞活化初级信号分子和/或T细胞活化次级信号分子的串联表达盒。在具体实施方案中，包含在包膜质粒中的串联表达盒包含编码第一信号肽的多核苷酸、编码所述T细胞活化初级信号分子和/或T细胞活化次级信号分子的多核苷酸、编码内部核糖体进入位点(IRES)、弗林蛋白酶切割位点或病毒2A肽中的一者的多核苷酸、编码第二信号肽的多核苷酸以及编码VSV-G或其变体或Cocal-G或其变体的多核苷酸。在某些实施方案中，编码VSV-G或其变体或Cocal-G或其变体的多核苷酸位于编码所述T细胞活化初级信号分子和/或T细胞活化次级信号分子的多核苷酸的5'端。在其他实施方案中，编码VSV-G或其变体或Cocal-G或其变体的多核苷酸位于编码所述T细胞活化初级信号分子和/或T细胞活化次级信号分子的多核苷酸的3'端。编码所述T细胞活化初级信号分子和/或T细胞活化次级信号分子的多核苷酸和编码VSV-G或其变体或Cocal-G或其变体的多核苷酸在串联盒中被编码IRES、弗林蛋白酶切割位点或病毒2A肽的多核苷酸分开，该多核苷酸允许由单个mRNA共表达两种蛋白。在某些实施方案中，病毒2A肽是猪捷申病毒-1(P2A)、明脉扁刺蛾(Thosea asigna)病毒(T2A)、马甲型鼻病毒(E2A)、口蹄疫病毒(F2A)或它们的变体。In some embodiments, the envelope plasmid comprises a tandem expression cassette encoding any one of the aforementioned VSV-G or its variants or Cocal-G or its variants and the T cell activation primary signal molecule and/or T cell activation secondary signal molecule as disclosed herein. In a specific embodiment, the tandem expression cassette contained in the envelope plasmid comprises a polynucleotide encoding a first signal peptide, a polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule, a polynucleotide encoding an internal ribosome entry site (IRES), a furin cleavage site or one of the viral 2A peptides, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding VSV-G or its variants or Cocal-G or its variants. In certain embodiments, the polynucleotide encoding VSV-G or its variants or Cocal-G or its variants is located at the 5' end of the polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule. In other embodiments, the polynucleotide encoding VSV-G or its variants or Cocal-G or its variants is located at the 3' end of the polynucleotide encoding the T cell activation primary signal molecule and/or the T cell activation secondary signal molecule. The polynucleotide encoding the T cell activation primary signaling molecule and/or the T cell activation secondary signaling molecule and the polynucleotide encoding VSV-G or its variant or Cocal-G or its variant are separated in a tandem cassette by a polynucleotide encoding an IRES, a furin cleavage site, or a viral 2A peptide, which allows co-expression of the two proteins from a single mRNA. In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A), or variants thereof.

慢病毒/逆转录病毒载体包装系统的使用依赖于“包装细胞系”。一般来说，包装细胞系是在将转移质粒、一个或多个包装质粒导入细胞时，其细胞能够产生不具有自我复制能力、能感染/转导靶细胞的慢病毒载体或逆转录病毒载体的细胞系。由JM Coffin,SM Hughes等人编写的Cold Spring Harbour Laboratory Press，1997，第447页中提供了可用包装系的概述，以引用的方式整体并入本文。The use of lentiviral/retroviral vector packaging systems relies on a "packaging cell line." Generally speaking, a packaging cell line is a cell line that, when a transfer plasmid or one or more packaging plasmids are introduced into the cell, produces a non-replication-competent lentiviral or retroviral vector capable of infecting/transducing target cells. An overview of available packaging lines is provided in J.M. Coffin, S.M. Hughes, et al., Cold Spring Harbour Laboratory Press, 1997, p. 447, which is incorporated herein by reference in its entirety.

示例性的，可使用包括化学介导的转染方法、物理介导的转染方法或生物介导的转染方法等转染方法将各种质粒导入包装细胞系中，例如，化学介导的转染方法包括使用磷酸钙、DEAE-葡聚糖或PEI(Polyethylenimine，聚乙烯亚胺转染试剂)等化学试剂转染，物理介导的转染方法包括使用电穿孔等转染方法。Exemplarily, various plasmids can be introduced into the packaging cell line using transfection methods including chemical-mediated transfection methods, physical-mediated transfection methods or biological-mediated transfection methods. For example, chemical-mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran or PEI (Polyethylenimine, polyethyleneimine transfection reagent), and physical-mediated transfection methods include transfection methods such as electroporation.

可用于制备本发明公开的病毒载体的生产/宿主/包装细胞包括人胚肾(HEK)293细胞及其衍生物。生产细胞可以是贴壁细胞系诸如HEK293T生产细胞，或悬浮细胞系诸如HEK293T/17SF生产细胞。Production/host/packaging cells that can be used to prepare the viral vectors disclosed herein include human embryonic kidney (HEK) 293 cells and their derivatives. The production cells can be adherent cell lines such as HEK293T production cells, or suspension cell lines such as HEK293T/17SF production cells.

示例性的，所述包装细胞/宿主细胞选自CHO细胞、BHK细胞、MDCK细胞、C3H-10T1/2细胞、FLY细胞、Psi-2细胞、BOSC23细胞、PA317细胞、WEHI细胞、COS细胞、BSC-1细胞、BSC-40细胞、BMT-10细胞、VERO细胞、W138细胞、MRC5细胞、A549细胞、HT1080细胞、HEK-293细胞、B-50细胞、3T3细胞、NIH3T3细胞、HepG2细胞、Saos-2细胞、Huh7细胞、HeLa细胞、W163细胞和211细胞；Exemplarily, the packaging cell/host cell is selected from CHO cells, BHK cells, MDCK cells, C3H-10T1/2 cells, FLY cells, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells and 211 cells;

优选地，所述包装细胞/宿主细胞为HEK-293T细胞。Preferably, the packaging cell/host cell is a HEK-293T cell.

“逆转录病毒”和“逆转录病毒载体”：即Retrovirus和Retroviral Vector。“逆转录病毒”是指具有单链正义RNA分子的RNA病毒。逆转录病毒包含逆转录酶和整合酶。进入靶细胞后，逆转录病毒利用其逆转录酶将其RNA分子转录成DNA分子。随后，利用整合酶将DNA分子整合到宿主细胞基因组中。整合到宿主细胞基因组中后，来自逆转录病毒的序列被称为前病毒(例如，前病毒的序列或前病毒序列)。逆转录病毒载体通常指源自逆转录病毒的假型逆转录病毒载体，示例性地，来源与γ-逆转录病毒。与可转导分裂和非分裂细胞的慢病毒载体不同，逆转录病毒载体仅能转导分裂细胞，且其能够携带的外源性转基因一般相对较小。关于慢病毒载体和逆转录病毒载体的对比与讨论，具体可见：Stripecke,R.,Kasahara,N.(2007).Lentiviral and Retroviral Vector Systems.In:Hunt,K.K.,Vorburger,S.A.,Swisher,S.G.(eds)Gene Therapy for Cancer.Cancer Drug Discovery and Development.Humana Press。"Retrovirus" and "Retroviral Vector": Retrovirus and Retroviral Vector. "Retrovirus" refers to an RNA virus with a single-stranded positive-sense RNA molecule. Retroviruses contain reverse transcriptase and integrase. After entering the target cell, the retrovirus uses its reverse transcriptase to transcribe its RNA molecule into a DNA molecule. Subsequently, the DNA molecule is integrated into the host cell genome using integrase. After integration into the host cell genome, the sequence from the retrovirus is called a provirus (e.g., a proviral sequence or a proviral sequence). Retroviral vectors generally refer to pseudotyped retroviral vectors derived from retroviruses, illustratively from γ-retroviruses. Unlike lentiviral vectors that can transduce dividing and non-dividing cells, retroviral vectors can only transduce dividing cells, and the exogenous transgenes they can carry are generally relatively small. For a comparison and discussion of lentiviral vectors and retroviral vectors, see: Stripecke, R., Kasahara, N. (2007). Lentiviral and Retroviral Vector Systems. In: Hunt, K.K., Vorburger, S.A., Swisher, S.G. (eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

慢病毒载体和逆转录病毒载体可将穿梭基因等外源性负荷基因稳定地整合到靶细胞的染色体中，从而允许靶细胞长期表达递送的穿梭基因，为基因疗法提供了巨大的优势。此外，它们不转移病毒基因，因此避免了产生可被细胞毒性T细胞破坏的转导细胞的问题。并且它们具有相对较大的克隆能力，足以满足大多数预期的临床应用。Lentiviral and retroviral vectors offer significant advantages for gene therapy by stably integrating exogenous cargo genes, such as shuttle genes, into the chromosomes of target cells, allowing for long-term expression of the delivered shuttle genes. Furthermore, they do not transfer viral genes, thus avoiding the problem of generating transduced cells that can be destroyed by cytotoxic T cells. Furthermore, they possess relatively large cloning capacities, sufficient for most anticipated clinical applications.

“包膜糖蛋白”：是指包被在病毒外层的糖蛋白，在对病毒的吸附和穿入宿主细胞、致病性、下调宿主表面蛋白质的表达以及增加病毒包装和出芽过程中起着重要的作用。"Envelope glycoprotein" refers to the glycoprotein coated on the outer layer of the virus, which plays an important role in the adsorption and penetration of the virus into host cells, pathogenicity, downregulation of host surface protein expression, and increase in virus packaging and budding.

“变体”：变体是指与非突变体(野生型)的氨基酸序列具有至少约50％同一性的突变体，“至少约50％同一性”指与非突变体(野生型)的氨基酸序列的约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％相同；或者，变体是指编码变体的核酸序列与编码非突变体(野生型)的核酸序列具有至少50％同一性的突变体，“至少50％同一性”是指编码变体的核酸序列与编码非突变体(野生型)的核酸序列具有至少50％、至少60％、至少70％、至少80％、至少90％、至少91％、至少92％、至少93、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或至少100％同一性。"Variant": A variant refers to a mutant having at least about 50% identity to the amino acid sequence of a non-mutant (wild type), and "at least about 50% identity" refers to about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of the non-mutant (wild type); or, a variant refers to a variant that is identical to the nucleic acid sequence encoding the non-mutant (wild type). "At least 50% identity" means that the nucleic acid sequence encoding the variant is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the nucleic acid sequence encoding the non-mutant (wild type).

本发明的一些实施例中，变体包括相对于非突变体，包含保守取代的突变体。“保守取代”在本领域中被认为是用一个氨基酸取代具有相似特性的另一个氨基酸。示例性地，保守取代是本领域熟知的(参见例如WO97/09433，第10页，1997年3月13日公布；Lehninger,Biochemistry，第二版；Worth Publishers,Inc.NY:NY(1975)，第71-77页；Lewin,Genes IV,Oxford University Press,NY and Cell Press,Cambridge,MA(1990)，第8页)。In some embodiments of the present invention, variants include mutants comprising conservative substitutions relative to non-mutants. "Conservative substitutions" are considered in the art to be substitutions of one amino acid with another amino acid having similar properties. For example, conservative substitutions are well known in the art (see, for example, WO97/09433, page 10, published on March 13, 1997; Lehninger, Biochemistry, 2nd edition; Worth Publishers, Inc. NY: NY (1975), pages 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), page 8).

“药学上可接受的赋形剂或载体”：药学上可接受的赋形剂或载体包括但不限于稀释剂、增溶剂、乳化剂、保存液、防腐剂和/或佐剂。辅料优选地在所采用的剂量和浓度下对接受者无毒或基本上无毒。这类辅料包括但并不限于：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。在某些实施方案中，药物组合物可含有用于改善、维持或保留例如组合物的pH、渗透性、粘度、澄清度、颜色、等渗性、气味、无菌性、稳定性、溶解或释放速率、吸收或渗透的物质。可视预期的给予途径、递送方式和所需的剂量来确定最佳的药物组合物。"Pharmaceutically acceptable excipient or carrier": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, preservatives, and/or adjuvants. Excipients are preferably nontoxic or substantially nontoxic to the recipient at the dosages and concentrations employed. Such excipients include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, pharmaceutical compositions may contain substances for improving, maintaining, or preserving, for example, the pH, osmotic properties, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition. The optimal pharmaceutical composition can be determined based on the intended route of administration, mode of delivery, and desired dosage.

用于体内给予的药物组合物通常以无菌制剂的形式提供。通过经无菌过滤膜过滤来实现灭菌。在组合物冻干时，可在冻干、复溶、稀释之前或之后使用此方法进行灭菌。可选择本发明的药物组合物用于肠胃外递送。用于肠胃外给予的组合物可以冻干形式或在溶液中储存。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。肠胃外组合物通常放在具有无菌进入孔的容器中，例如具有皮下注射针可刺穿的塞子的静脉内溶液袋或小瓶。或者，可选择组合物用于吸入或通过消化道(诸如经口)递送。所述药学上可接受的组合物的制备在本领域的技术范围内。其它药物组合物将为本领域技术人员显而易见，包括在持续或控制释放递送配制物中包含抗体的配制物。用于配制多种其它持续或可控传递方式的技术(诸如脂质体载剂、生物易蚀微粒或多孔珠粒和积存注射)也为本领域技术人员所知。Pharmaceutical compositions for in vivo administration are typically provided as sterile formulations. Sterilization is achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization, reconstitution, or dilution. The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, they can be prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposomal carriers, bioerodible microparticles or porous beads, and depot injection) are also known to those skilled in the art.

药物组合物一经配制，就以溶液、悬浮液、凝胶、乳液、固体、晶体或以冻干粉末的形式储存在无菌小瓶中。所述配制物可储存成即用形式或在给予前复溶的形式(例如，冻干)。本发明还提供用于产生单剂量给予单位的试剂盒。本发明的试剂盒可各自含有具有干燥蛋白的第一容器和具有含水配制物的第二容器。在本发明的某些实施方案中，提供含有单腔和多腔预填充注射器(例如，液体注射器和冻干注射器)的试剂盒。Once the pharmaceutical composition is formulated, it is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form (e.g., lyophilized) that is redissolved before administration. The present invention also provides a test kit for producing a single-dose administration unit. The test kit of the present invention can each contain a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, a test kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilizing syringe) is provided.

“受试者”：如本文所用，“受试者”、“患者”和“个体”同义使用，包括但不限于，哺乳动物、如人或非人哺乳动物，例如家畜、农业动物或野生动物，以及鸟类和水生动物。“患者”是罹患疾病、病症或病况，或有发展疾病、病症或病况的风险，或在其他方面需要本文所提供的任一种病毒载体、TCP-T细胞、组合物或治疗方法的受试者。"Subject": As used herein, "subject," "patient," and "individual" are used synonymously and include, but are not limited to, mammals, such as humans or non-human mammals, such as domestic animals, agricultural animals, or wild animals, as well as birds and aquatic animals. A "patient" is a subject who suffers from a disease, disorder, or condition, or is at risk of developing a disease, disorder, or condition, or who is otherwise in need of any of the viral vectors, TCP-T cells, compositions, or treatment methods provided herein.

“疾病”是受试者的健康状态，其中受试者不能维持稳态，并且其中如果疾病未改善，则受试者的健康继续恶化。相反，受试者的“病症”或“不良状况”是其中受试者能够维持稳态的健康状态，但其中受试者的健康状态不如不存在病症或不良状况时有利。在不治疗的情况下，病症或不良状况不一定导致受试者的健康状态进一步降低。A "disease" is a state of health in a subject in which the subject is unable to maintain homeostasis and in which the subject's health continues to deteriorate if the disease does not improve. In contrast, a "disorder" or "adverse condition" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's health is less favorable than it would be in the absence of the disorder or adverse condition. Without treatment, a disorder or adverse condition does not necessarily result in a further deterioration in the subject's health.

“癌症”：如本文所用的术语“癌症”被定义为以异常细胞的快速且不受控制的生长为特征的疾病。异常细胞可形成实体瘤或者构成血液恶性肿瘤。癌细胞可以局部扩散或通过血流和淋巴系统扩散到身体的其他部位。各种癌症的实例包括但不限于血液癌，如B淋巴细胞恶性肿瘤和多发性骨髓瘤等；实体癌，如乳腺癌、前列腺癌、卵巢癌、宫颈癌、皮肤癌、胰腺癌、结直肠癌、肾癌、肝癌、脑癌、淋肺癌等。"Cancer": As used herein, the term "cancer" is defined as a disease characterized by the rapid, uncontrolled growth of abnormal cells. Abnormal cells may form solid tumors or constitute hematological malignancies. Cancer cells may spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, hematological cancers, such as B-lymphocyte malignancies and multiple myeloma; and solid cancers, such as breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, and lymphoma.

“治疗”：指向受试者，采用本文所述的治疗方法以达到至少一种阳性治疗效果(比如，癌细胞数目减少、肿瘤体积减小、癌细胞浸润至周边器官的速率降低或肿瘤转移或肿瘤生长的速率降低)。有效治疗患者的治疗方法可根据多种因素(比如患者的疾病状态、年龄、体重以及疗法激发受试者的抗癌反应能力)而变。"Treatment" refers to the use of a treatment method described herein to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor size, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth) in a subject. The treatment method that effectively treats a patient may vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the treatment to elicit an anti-cancer response in the subject.

如本文所使用的“治疗”包括与治疗相关的任何有益或期望的效果。“治疗”并不一定指示完全根除或治愈疾病或病状，或其相关症状。As used herein, "treatment" includes any beneficial or desired effect associated with treatment."Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or its associated symptoms.

将采用含有本发明提供的任一种所述工程化T细胞和/或病毒载体的药物组合物的治疗有效量将取决于例如治疗程度和目标。本领域技术人员将了解，用于治疗的适当剂量水平将部分取决于所递送的分子、适应症、给予途径和患者情况(体重、体表或器官大小)和/或状况(年龄和一般健康状况)而变化。在某些实施方式中，临床医生可滴定剂量并改变给予途径来获得最佳的治疗效果。The therapeutically effective amount of the pharmaceutical composition containing any described engineered T cell and/or viral vector provided by the invention will be adopted and will depend on for example treatment degree and target.It will be appreciated by those skilled in the art that the appropriate dosage level for the treatment of will depend in part on the molecule sent, indication, administration route and patient condition (body weight, body surface or organ size) and/or situation (age and general health) and change.In some embodiments, clinician's titration dosage also changes administration route to obtain best therapeutic effect.

给药频率将取决于所用配制物中所述工程化T细胞或所述病毒载体的药物动力学参数。临床医生典型地给予药物组合物直到达到实现所需效果的剂量。药物组合物因此可作为单次剂量给予，或随时间以作为两次或多次剂量(可含有或不含有相同量的所需分子)给予，或通过植入装置或导管以连续输液的方式给予。The frequency of administration will depend on the pharmacokinetic parameters of the engineered T cells or the viral vector in the formulation used. Clinicians typically administer the pharmaceutical composition until the dosage is achieved to achieve the desired effect. The pharmaceutical composition can therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or administered as a continuous infusion via an implantable device or catheter.

“给予”：药物组合物的给予途径是本领域常规的，例如经口、经鼻、通过静脉内、皮下、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉或病灶内途径注射，还可以通过持续释放系统或通过植入装置进行给予。"Administering": The administration routes of the pharmaceutical composition are conventional in the art, such as oral, nasal, intravenous, subcutaneous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional injection, and can also be administered by sustained release system or by implantation device.

“和/或”：应理解为是指一种或两种替代方案。“And/or”: should be understood to mean one or two alternatives.

“约”/“大约”：如本文所用，术语“约”是指本技术领域的技术人员容易知道的相应值的常用误差范围，示例性地，包括但不限于指与参考数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度相比变化高达15％、10％、9％、8％、7％、6％、5％、4％、3％、2％或1％的数量、水平、值、数目、频率、百分比、尺寸、大小、量、重量或长度。本文对“约”某一值或参数的提及包括(并描述)针对所述值或参数本身的实施方案。例如，提及“约X”的描述包括“X”的描述。"About"/"approximately": As used herein, the term "about" refers to the usual error range for the corresponding value as readily known to those skilled in the art, including, by way of example, but not limited to, reference to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. Reference herein to "about" a value or parameter includes (and describes) embodiments for the value or parameter itself. For example, a description referring to "about X" includes a description of "X."

“包含”：本文中，除非上下文另外要求，词语“包含”将理解为意味着包含规定的步骤、或元素、或步骤或元素的组，但不排除任何其它步骤、或元素、或步骤或元素的组。在本发明的一些实施例中，术语“包括”、“具有”、“含有”和“包含”同义使用。"Comprising": As used herein, unless the context requires otherwise, the word "comprising" will be understood to mean the inclusion of the specified steps, elements, or groups of steps or elements, but not the exclusion of any other steps, elements, or groups of steps or elements. In some embodiments of the present invention, the terms "including," "having," "containing," and "comprising" are used synonymously.

“实施例”：在本说明书通篇中提及“一些实施方案”、“一些实施例”、“实施方案”、“特定的实施方案”、“相关实施方案”、“某个实施方案”、“另一实施方案”或“其他实施方案”或其组合是指结合实施方案加以描述的特定特征、结构或特性包括在本发明的至少一个实施方案中。因此，本说明书通篇在各个地方出现前述短语不必要全部是指相同的实施方案。此外，特定的特征、结构或特性可以任何适合的方式组合在一个或多个实施方案中。"Embodiments": Reference throughout this specification to "some embodiments," "some embodiments," "embodiments," "specific embodiments," "related embodiments," "an embodiment," "another embodiment," or "other embodiments" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, the various appearances of the foregoing phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

“预防”：如本文所用，“预防”和类似的词，诸如“防止”等，指示用于防止、抑制或降低病症发生或复发的可能性的方法。如本文所用，“预防”和类似的词还包括在发病或复发之前减轻疾病或病症的强度、效果、症状和/或负担。"Prevention": As used herein, "prevention" and similar words, such as "preventing," refer to methods used to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a condition. As used herein, "prevention" and similar words also include lessening the intensity, effects, symptoms, and/or burden of a disease or condition prior to onset or recurrence.

“稳定整合”：又称“稳定转染，是指外源性多核苷酸导入宿主细胞后整合至宿主细胞基因组中，在宿主细胞中长期稳定地表达(Stable Gene Expression)；与之相对的是瞬时转染和瞬时表达(Transient Expression)。"Stable integration": also known as "stable transfection, refers to the integration of exogenous polynucleotides into the host cell genome after introduction into the host cell, and their long-term stable expression in the host cell (Stable Gene Expression); in contrast, transient transfection and transient expression (Transient Expression).

“特异性结合”：如本文所用，术语“特异性结合”是指发生在成对分子种类(例如受体与配体，抗体和抗原)之间的结合。当两个种类的相互作用产生非共价结合的复合物时，发生的结合通常是静电、氢键结合或亲脂性相互作用的结果。在各种实施例中，一个或多个种类之间的特异性结合是直接的。在本发明的一些实施例中，特异性结合的亲和力是背景结合(非特异性结合)的约2倍、背景结合的约5倍、背景结合的约10倍、背景结合的约20倍、背景结合的约50倍、背景结合的约100倍、或背景结合的约1000倍或更多倍。"Specific binding": As used herein, the term "specific binding" refers to the binding that occurs between paired molecular species (e.g., a receptor and a ligand, an antibody and an antigen). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, the specific binding between one or more species is direct. In some embodiments of the invention, the affinity of the specific binding is about 2 times greater than background binding (non-specific binding), about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.

“序列同一性”：一般来讲，“序列同一性”或“序列同源性”分别是指两个多核苷酸或多肽序列的核苷酸与核苷酸或氨基酸与氨基酸的精确对应。通常，用于测定序列同一性的技术包括确定多核苷酸的核苷酸序列和/或确定由此编码的氨基酸序列，以及将这些序列与第二核苷酸或氨基酸序列进行比较。两个或更多个序列(多核苷酸或氨基酸)可通过测定它们的“同一性百分比”来比较。无论是核酸还是氨基酸序列，两个序列的同一性百分比，是两个比对序列之间的精确匹配数目除以较短序列的长度，再乘以100。例如，还可使用可购自美国国立卫生研究院(National Institutes of Health)的高级BLAST计算机程序来比较序列信息，从而确定同一性百分比。BLAST程序基于以下比对方法：Karlin和Altschul，Proc.Natl.Acad.Sci.USA 87：2264-2268(1990)并且讨论于Altschul等人，J.Mol.Biol.215：403-410(1990)；Karlin和Altschul，Proc.Natl.Acad.Sci.USA 90：5873-5877(1993)；和Altschul等人，Nucleic Acids Res.25：3389-3402(1997)中。简而言之，BLAST程序将同一性定义为相同的比对符号(通常是核苷酸或氨基酸)的数目除以两个序列中较短符号的总数。程序可用于确定所比较的整个蛋白质长度上的同一性百分比。"Sequence identity": In general, "sequence identity" or "sequence homology" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity." The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. For example, the advanced BLAST computer program available from the National Institutes of Health can also be used to compare sequence information to determine the percent identity. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of aligned symbols (usually nucleotides or amino acids) that are identical divided by the total number of shorter symbols in the two sequences. The program can be used to determine percent identity over the entire length of the proteins being compared.

“信号肽”：Signal Peptide，有时又被称为信号序列、靶向信号、定位信号、定位序列、转运肽或前导肽，是一种短肽(通常16-30个氨基酸长)(Kapp,Katja；Schrempf,Sabrina；Lemberg,Marius K.；Dobberstein,Bernhard(2013-01-01).)，存在于大多数通往分泌通路的新合成蛋白的N末端(偶尔非经典地存在于C末端或者内部)(Owji,et al.,A comprehensive review of signal peptides:Structure,roles,and applications,European Journal of Cell Biology.97(6):422-441.(2018))(Blobel G,Dobberstein B,et al.,Transfer of proteins across membranes.I.Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma,The Journal of Cell Biology,67(3):835-51.(1975))。"Signal peptide": Signal peptide, sometimes also called signal sequence, targeting signal, localization signal, localization sequence, transit peptide or leader peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), present at the N-terminus of most newly synthesized proteins that enter the secretory pathway (occasionally non-classically present at the C-terminus or internally) (Owji, et al., A comprehensive review of signal peptides: Structure, roles, and applications, European Jour nal of Cell Biology.97(6):422-441.(2018))(Blobel G,Dobberstein B,et al.,Transfer of proteins across membranes.I.Presence of proteolytically processe d and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma, The Journal of Cell Biology, 67(3):835-51.(1975)).

信号肽是存在于新合成的蛋白质的N-末端的短肽，信号肽专用于质膜或分泌途径。信号序列通常在N-末端包含一段短的亲水性、带正电荷的氨基酸、5-15个残基的中心疏水结构域以及具有信号序列切割位点的C-末端区。在真核生物中，信号序列促使新合成的蛋白质易位至内质网，在此该蛋白质被信号肽酶切割，产生成熟蛋白质，然后进入其适当的目的地。信号序列长度和氨基酸组成的多样性使得难以精确预测切割位点。对于本文公开的多肽序列，当提及信号序列时，还设想了不存在信号序列或具有部分信号序列的多肽序列。Signal peptides are short peptides present at the N-terminus of newly synthesized proteins that are specific for the plasma membrane or secretory pathway. Signal sequences typically contain a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a signal sequence cleavage site. In eukaryotes, signal sequences cause newly synthesized proteins to translocate to the endoplasmic reticulum, where the protein is cleaved by a signal peptidase to produce the mature protein, which then enters its appropriate destination. The diversity of signal sequence length and amino acid composition makes it difficult to accurately predict the cleavage site. For the polypeptide sequences disclosed herein, when referring to a signal sequence, polypeptide sequences in which no signal sequence or a partial signal sequence is present are also contemplated.

信号肽的作用是促使细胞转移蛋白质，通常转移到细胞膜上。在原核生物中，信号肽将新合成的蛋白质引导至存在于质膜中的SecYEG蛋白质传导通道。真核生物中存在同源系统，其中信号肽将新合成的蛋白质引导至Sec6L通道，该通道与SecYEG具有结构和序列同源性，但存在于内质网中(Rapoport TA,Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes,Nature.450(7170):663-9(2007).)。SecYEG和Sec6L通道通常称为转运子，通过该通道的转运称为易位。当分泌的蛋白质穿过通道时，跨膜区可能会扩散穿过易位子中的侧门以分配到周围的膜中。The function of a signal peptide is to prompt the cell to transfer proteins, usually to the cell membrane. In prokaryotes, signal peptides direct newly synthesized proteins to the SecYEG protein-conducting channel present in the plasma membrane. A homologous system exists in eukaryotes, in which signal peptides direct newly synthesized proteins to the Sec6L channel, which has structural and sequence homology with SecYEG but is present in the endoplasmic reticulum (Rapoport TA, Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes, Nature. 450(7170):663-9(2007).). SecYEG and Sec6L channels are often referred to as transporters, and transport through these channels is called translocation. When a secreted protein passes through the channel, the transmembrane region may diffuse through the side gate in the translocon to be distributed to the surrounding membrane.

“MOI”：即“Mutiplicity of Infection(MOI)”，是指在感染过程中，被添加到每个细胞的病毒粒子的数量。例如，当一百万个病毒粒子被添加至一百万个细胞时，MOI＝1。"MOI": Multiplicity of Infection (MOI) refers to the number of virus particles added to each cell during infection. For example, if one million virus particles are added to one million cells, the MOI is 1.

“可操作地”：当一个多核苷酸与另一个多核苷酸处于功能关系时，则该核酸是“可操作地连接”的。例如，如果前序列或分泌前导序列的DNA表达为参与多肽分泌的前蛋白，则该DNA与该多肽的DNA可操作地连接；如果启动子或增强子影响编码序列的转录，则该启动子或该增强子与该序列可操作地连接；或者，如果核糖体结合位点被定位以便促进翻译，则该核糖体结合位点与编码序列可操作地连接。一般而言，“可操作地连接”意指所连接的多核苷酸是邻接的，并且就分泌前导序列而言，是邻接的且处于阅读框中。然而，增强子不必是邻接的。连接通过在适当限制性位点处的连接来实现。如果不存在这些位点，则根据常规实践使用合成寡核苷酸衔接子或接头。"Operably": A polynucleotide is "operably linked" when it is in a functional relationship with another polynucleotide. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned so as to promote translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the polynucleotides being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

“转导”：如本文所用，术语“转染”、“转化”和“转导”同义使用，是指外源性核酸转入或引入宿主细胞、包装细胞的过程。“转染的”、“转化的”或“转导的”细胞是已用外源性核酸转染、转化或转导的细胞。该细胞包括原代受试者细胞及其子代。"Transduction": As used herein, the terms "transfection," "transformation," and "transduction" are used synonymously to refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell, packaging cell, or the like. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

将载体如病毒载体等或分离的多核苷酸引入哺乳动物细胞的方法是本领域已知的。所描述的载体可以通过物理、化学或生物学方法转移到免疫效应细胞中。Methods for introducing vectors such as viral vectors or isolated polynucleotides into mammalian cells are known in the art. The described vectors can be transferred to immune effector cells by physical, chemical or biological methods.

将载体或分离的多核苷酸引入免疫效应细胞的物理方法包括磷酸钙沉淀、脂质转染、粒子轰击、显微注射、电穿孔等。用于产生包含载体和/或外源核酸的细胞的方法是本领域众所周知的(参见Sambrook,J.,Fritsch,E.F.and Maniatis,T.(2001)Molecular Cloning:A Laboratory Manual.Cold Spring Harbor Laboratory Press,Cold Spring Harbor.)。本发明的一些实施例中，通过电穿孔将载体引入细胞中。本发明的一些实施例中，通过PEI(Polyethylenimine，聚乙烯亚胺转染试剂)转染试剂将载体引入细胞中。Physical methods for introducing vectors or isolated polynucleotides into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for generating cells containing vectors and/or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments of the present invention, the vector is introduced into the cell by electroporation. In some embodiments of the present invention, the vector is introduced into the cell by PEI (Polyethylenimine, polyethyleneimine transfection reagent) transfection reagent.

将载体或分离的多核苷酸引入免疫效应细胞的生物学方法包括使用DNA和RNA载体。病毒载体已成为将基因插入哺乳动物(例如人类细胞)中最广泛使用的方法。Biological methods for introducing vectors or isolated polynucleotides into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.

将载体或分离的多核苷酸引入免疫效应细胞的化学方法包括胶体分散系统，例如包括大分子复合物、纳米胶囊、微球、珠和基于脂质的系统，例如包括水包油乳液、胶束、混合胶束和脂质体。用作体外递送载体的示例性胶体系统是脂质体。Chemical methods for introducing vectors or isolated polynucleotides into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome.

本文所有出版物、文献和提及的专利特此以引用的方式整体并入，如同将每个未以特定地和单独地指示以引用的方式整体并入本文的单独的出版物、文献或专利。在冲突的情况下，以本申请(包括本文中的任何定义)为准。然而，本文所引用的任何参考文献、文章、出版物、专利、专利出版物和专利申请，并不也不应被视为承认或任何形式的建议或它们构成有效的现有技术或形成世界上任何国家的公知常识的一部分。All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety, just as if each individual publication, document, or patent were not specifically and individually indicated as being incorporated by reference in its entirety. In the event of a conflict, the present application (including any definitions herein) will control. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be taken as an admission or any form of suggestion or that they constitute valid prior art or form part of the common general knowledge in any country in the world.

本文所使用的小节标题仅出于组织性目的并且不解释为限制所描述的主题。The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1：为实施例1中，包装所述对照组慢病毒载体m-CAR和所述靶向慢病毒载体m-TCP-E/G的各组HEK-293T细胞分别表达所述CD19-CAR分子或所述CD19-TCP-E/G分子的出膜表达效率的流式检测结果图；Figure 1 is a flow cytometry result showing the expression efficiency of the CD19-CAR molecule or the CD19-TCP-E/G molecule in HEK-293T cells packaged with the control group lentiviral vector m-CAR and the targeted lentiviral vector m-TCP-E/G in Example 1;

图2：为实施例1中，分别加入所述对照组慢病毒载体m-CAR、所述靶向慢病毒载体m-TCP-E和m-TCP-G的各组Nalm-6细胞培养基中，CD19的表达丰度的流式检测结果图；FIG2 is a flow cytometry result showing the expression abundance of CD19 in the culture medium of Nalm-6 cells in each group to which the control group lentiviral vector m-CAR, the targeted lentiviral vectors m-TCP-E and m-TCP-G were added, respectively, in Example 1;

图3：为实施例2中，所述对照组慢病毒载体m-7-E、所述靶向慢病毒载体m-TCP-E和m-TCP-G分别转导人非活化PBMCs后，所述CD19-TCP-E/G分子在各组PBMCs中的CD3+T细胞中的出膜表达效率流式检测结果图；FIG3 is a graph showing the flow cytometry results of the expression efficiency of the CD19-TCP-E/G molecule in CD3 + T cells in PBMCs of the control group after the lentiviral vector m-7-E, the targeted lentiviral vectors m-TCP-E and m-TCP-G were respectively transduced into non-activated human PBMCs in Example 2;

图4：为实施例3中，所述靶向慢病毒载体m-TCP-G分别活化刺激Donor 1和Donor 2的非活化PBMCs后，各组PBMCs中的CD3+T细胞的增殖曲线图；FIG4 is a graph showing the proliferation curves of CD3 + T cells in PBMCs of each group after the targeted lentiviral vector m-TCP-G activated and stimulated the non-activated PBMCs of Donor 1 and Donor 2, respectively, in Example 3;

图5：为实施例4中，所述靶向慢病毒载体m-TCP-G转导人非活化T细胞制备的CD19-TCP-T细胞体外杀伤Nalm-6细胞的杀伤效率的流式检测结果图；FIG5 is a flow cytometry result showing the killing efficiency of CD19-TCP-T cells prepared by transducing human non-activated T cells with the targeted lentiviral vector m-TCP-G in Example 4 against Nalm-6 cells in vitro;

图6：为实施例5中，3组造模小鼠在Day 0、5和7的活体成像图，以检测所述靶向慢病毒载体m-TCP-G在小鼠体内转导人PBMCs制备的CD19-TCP-T细胞杀伤Nalm-6细胞的杀伤效率；Figure 6 shows in vivo imaging of three groups of model mice on Days 0, 5, and 7 in Example 5, to detect the killing efficiency of CD19-TCP-T cells prepared by transducing human PBMCs with the targeted lentiviral vector m-TCP-G against Nalm-6 cells in mice;

图7：为实施例6中，所述对照组慢病毒载体m-7-G、所述靶向慢病毒载体m-3-G和m-TCP-G分别转导各组人非活化PBMCs，其中CD3+T细胞表达所述CD19-TCP-G分子的效率的流式检测结果图；Figure 7 shows the flow cytometry results of the efficiency of CD3 + T cells expressing the CD19-TCP-G molecule in non-activated human PBMCs of each group, respectively, transduced by the control group lentiviral vector m-7-G, the targeted lentiviral vectors m-3-G, and m-TCP-G in Example 6;

图8：为实施例7中，所述靶向慢病毒载体m-TCP-G和m-86-G分别转导人非活化PBMCs后，其中CD3+T细胞表达所述CD19-TCP-G分子的效率的流式检测结果图；FIG8 is a flow cytometry result showing the efficiency of CD3 + T cells expressing the CD19-TCP-G molecule after the targeted lentiviral vectors m-TCP-G and m-86-G were respectively transduced into non-activated human PBMCs in Example 7;

图9：为实施例8中，所述靶向慢病毒载体m-a33-G转导人非活化PBMCs后，其中CD3+T细胞表达所述CD33-TCP-G分子的效率的流式检测结果图。FIG9 is a flow cytometry result showing the efficiency of CD3 + T cells expressing the CD33-TCP-G molecule after the targeted lentiviral vector m-a33-G transduced human non-activated PBMCs in Example 8. FIG9 is a flow cytometry result showing the efficiency of CD33-TCP-G molecule expression in CD3 + T cells after the targeted lentiviral vector m-a33-G transduced human non-activated PBMCs.

图10：为实施例8中，所述CD33-TCP-T细胞体外杀伤CD33+靶细胞MOLM-13细胞的杀伤效率的流式检测结果图。FIG10 is a flow cytometry result showing the killing efficiency of the CD33-TCP-T cells in killing CD33 + target MOLM-13 cells in vitro in Example 8.

具体实施方式DETAILED DESCRIPTION

下面结合实施例对本发明的构思及产生的技术效果进行清楚、完整的描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的部分实施例，而不是全部实施例；基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均在本发明保护的范围之内。The following is a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments, so that the purpose, features and effects of the present invention are fully understood. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

下列实施例中未注明具体条件的实验方法,按照本领域公知的常规方法和条件,或按照商品说明书选择。本发明中未注明具体成分的试剂和原料均市售可得。In the following examples, the experimental methods without specific conditions are based on conventional methods and conditions known in the art, or are selected according to the product specifications. Reagents and raw materials not specified in the present invention are all commercially available.

实施例1：构建靶向非活化T细胞的慢病毒载体m-TCP-E/GExample 1: Construction of a lentiviral vector m-TCP-E/G targeting non-activated T cells

1.设计膜表达抗CD3抗体×抗CD28抗体结构 1. Design of membrane-expressed anti-CD3 antibody × anti-CD28 antibody structure

本实施例中，编码膜表达抗CD3抗体×抗CD28抗体(膜表达抗CD3×抗CD28双抗)的多核苷酸从5’端至3’端依次为：编码人CD8α信号肽的多核苷酸、编码所述UCHT1-scFv的多核苷酸、编码人CD8α铰链区的多核苷酸、编码人CD8α跨膜区的多核苷酸、编码FT2A肽的多核苷酸、编码人CD8α信号肽的多核苷酸、编码所述15E8-scFv的多核苷酸、编码人CD8α铰链区的多核苷酸、编码人CD8α跨膜区的多核苷酸；In this embodiment, the polynucleotides encoding membrane-expressed anti-CD3 antibody × anti-CD28 antibody (membrane-expressed anti-CD3 × anti-CD28 dual antibody) are as follows from the 5' end to the 3' end: a polynucleotide encoding a human CD8α signal peptide, a polynucleotide encoding the UCHT1-scFv, a polynucleotide encoding a human CD8α hinge region, a polynucleotide encoding a human CD8α transmembrane region, a polynucleotide encoding an FT2A peptide, a polynucleotide encoding a human CD8α signal peptide, a polynucleotide encoding the 15E8-scFv, a polynucleotide encoding a human CD8α hinge region, and a polynucleotide encoding a human CD8α transmembrane region;

(1)所述人CD8α信号肽的氨基酸序列如SEQ ID NO:12所示；(1) The amino acid sequence of the human CD8α signal peptide is shown in SEQ ID NO: 12;

(2)所述UCHT1-scFv的氨基酸序列如SEQ ID NO:13所示；(2) The amino acid sequence of the UCHT1-scFv is shown in SEQ ID NO: 13;

(3)所述人CD8α铰链区的氨基酸序列如SEQ ID NO:14所示；(3) The amino acid sequence of the human CD8α hinge region is shown in SEQ ID NO: 14;

(4)所述人CD8α跨膜区的氨基酸序列如SEQ ID NO:15所示；(4) The amino acid sequence of the human CD8α transmembrane region is shown in SEQ ID NO: 15;

(5)所述FT2A肽的氨基酸序列如SEQ ID NO:16所示；(5) The amino acid sequence of the FT2A peptide is shown in SEQ ID NO: 16;

(6)所述15E8-scFv的氨基酸序列如SEQ ID NO:17所示。(6) The amino acid sequence of the 15E8-scFv is shown in SEQ ID NO:17.

在自切割肽FT2A肽的作用下，所述膜表达的抗CD3抗体，UCHT1-scFv(膜表达UCHT1-scFv)和膜表达的抗CD28抗体，15E8-scFv(膜表达15E8-scFv)分开膜表达在包装细胞的细胞膜上，并随着慢病毒载体的出芽，转移至慢病毒载体的包膜上，使慢病毒载体具备靶向活化非活化T细胞的能力。Under the action of the self-cleaving peptide FT2A peptide, the membrane-expressed anti-CD3 antibody, UCHT1-scFv (membrane-expressed UCHT1-scFv) and the membrane-expressed anti-CD28 antibody, 15E8-scFv (membrane-expressed 15E8-scFv) are separately expressed on the cell membrane of the packaging cell, and as the lentiviral vector buds out, they are transferred to the envelope of the lentiviral vector, thereby enabling the lentiviral vector to have the ability to target and activate non-activated T cells.

2.构建T细胞受体嵌合蛋白2. Construction of T cell receptor chimeric protein

本实施例中，构建靶向CD19的TCP分子(CD19-TCP)；编码所述CD19-TCP分子的多核苷酸从5’端至3’端依次为：编码所述人CD8α信号肽的多核苷酸、编码靶向人CD19的胞外抗原结合区的多核苷酸、编码所述连接肽1的多核苷酸、编码人CD3ε(CD19-TCP-E)或人CD3γ(CD19-TCP-G)的多核苷酸；In this example, a TCP molecule targeting CD19 (CD19-TCP) was constructed; the polynucleotides encoding the CD19-TCP molecule were, from the 5' end to the 3' end, the following: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding the extracellular antigen binding region targeting human CD19, a polynucleotide encoding the connecting peptide 1, and a polynucleotide encoding human CD3ε (CD19-TCP-E) or human CD3γ (CD19-TCP-G);

(1)所述胞外抗原结合区是可特异性结合人CD19的所述FMC63-scFv；所述FMC63-scFv的VH区的氨基酸序列如SEQ ID NO:18所示，所述FMC63-scFv的VL区的氨基酸序列如SEQ ID NO:19所示；所述FMC63-scFv的氨基酸序列如SEQ ID NO:64所示；所述FMC63-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:65-67所示，所述FMC63-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:68-70所示；(1) The extracellular antigen binding region is the FMC63-scFv that can specifically bind to human CD19; the amino acid sequence of the VH region of the FMC63-scFv is shown in SEQ ID NO: 18, the amino acid sequence of the VL region of the FMC63-scFv is shown in SEQ ID NO: 19; the amino acid sequence of the FMC63-scFv is shown in SEQ ID NO: 64; the amino acid sequences of the HCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NO: 65-67, respectively, and the amino acid sequences of the LCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NO: 68-70, respectively;

(2)所述人CD3ε的氨基酸序列如SEQ ID NO:21所示，所述CD3ε不包含其信号肽，仅包含其胞外域、跨膜区和胞内域；(2) The amino acid sequence of human CD3ε is shown in SEQ ID NO: 21, wherein the CD3ε does not contain its signal peptide and only contains its extracellular domain, transmembrane region and intracellular domain;

(3)所述人CD3γ的氨基酸序列如SEQ ID NO:22所示，所述CD3γ不包含其信号肽，仅包含其胞外域、跨膜区和胞内域；(3) The amino acid sequence of human CD3γ is shown in SEQ ID NO: 22, wherein CD3γ does not contain its signal peptide and only contains its extracellular domain, transmembrane region and intracellular domain;

(4)所述连接肽1的氨基酸序列如SEQ ID NO:23所示；(4) The amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO: 23;

所述胞外抗原结合区的C-末端通过所述连接肽1可操作地连接至所述人CD3ε或人CD3γ的N-末端；The C-terminus of the extracellular antigen binding region is operably connected to the N-terminus of the human CD3ε or human CD3γ through the connecting peptide 1;

所述CD19-TCP-E的氨基酸序列如SEQ ID NO:100所示；The amino acid sequence of the CD19-TCP-E is shown in SEQ ID NO: 100;

所述CD19-TCP-G的氨基酸序列如SEQ ID NO:101所示。The amino acid sequence of the CD19-TCP-G is shown in SEQ ID NO:101.

人CD3ε，Uniprot ID:P07766。Human CD3ε, Uniprot ID: P07766.

不包含其信号肽的人CD3ε：
Human CD3ε without its signal peptide:

人CD3γ，Uniprot ID:P09693。Human CD3γ, Uniprot ID: P09693.

不包含其信号肽的人CD3γ：

Human CD3γ without its signal peptide:

3.包装靶向慢病毒载体m-TCP-E/G 3. Packaging of Targeted Lentiviral Vector m-TCP-E/G

A.准备以下4种质粒，包装靶向慢病毒载体m-TCP-E/G：A. Prepare the following four plasmids for packaging the targeted lentiviral vector m-TCP-E/G :

携带编码突变型VSV-G的多核苷酸和编码所述膜表达抗CD3×抗CD28双抗的多核苷酸的包膜质粒(包膜质粒1)、pMDLg/pRRE包装质粒、pRSV-REV包装质粒和携带编码所述CD19-TCP-E/G分子的多核苷酸的主质粒/转移质粒(CD19-TCP-E/G主质粒)；所述包膜质粒1和所述主质粒通过常规分子克隆方法合成；An envelope plasmid (envelope plasmid 1) carrying a polynucleotide encoding a mutant VSV-G and a polynucleotide encoding the membrane-expressed anti-CD3 × anti-CD28 dual antibody, a pMDLg/pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a master plasmid/transfer plasmid (CD19-TCP-E/G master plasmid) carrying a polynucleotide encoding the CD19-TCP-E/G molecule; the envelope plasmid 1 and the master plasmid were synthesized by conventional molecular cloning methods;

所述突变型VSV-G胞外域的氨基酸序列如SEQ ID NO:8所示；相对于SEQ ID NO:1,SEQ ID NO:8包含K47缺失、T214N和T352A；野生型VSV-G胞外域的氨基酸序列如SEQ ID NO:1所示；所述野生型VSV-G全长蛋白(包含VSV-G信号肽)的氨基酸序列如SEQ ID NO:24所示；The amino acid sequence of the mutant VSV-G extracellular domain is shown in SEQ ID NO:8; relative to SEQ ID NO:1, SEQ ID NO:8 includes a K47 deletion, T214N, and T352A; the amino acid sequence of the wild-type VSV-G extracellular domain is shown in SEQ ID NO:1; the amino acid sequence of the wild-type VSV-G full-length protein (including the VSV-G signal peptide) is shown in SEQ ID NO:24;

所述靶向慢病毒载体m-TCP-G包含编码所述CD19-TCP-G分子的多核苷酸；The targeted lentiviral vector m-TCP-G comprises a polynucleotide encoding the CD19-TCP-G molecule;

所述靶向慢病毒载体m-TCP-E包含编码所述CD19-TCP-E分子的多核苷酸；The targeted lentiviral vector m-TCP-E comprises a polynucleotide encoding the CD19-TCP-E molecule;

所述突变型VSV-G因其胞外域的第47位氨基酸赖氨酸缺失，使所述突变型VSV-G特异性结合LDL-R的能力减弱甚至丧失，从而使所述慢病毒载体m-TCP-G或m-TCP-E靶向激活并转导非活化T细胞的靶向性提高；所述突变型VSV-G还包含使其拮抗被补体失活的能力增强或不被补体失活的T214N和T352A突变，更适合被应用于体内靶向转导非活化T细胞；The mutant VSV-G has a lysine deletion at position 47 of its extracellular domain, which weakens or even eliminates its ability to specifically bind to LDL-R, thereby improving the targeting of the lentiviral vector m-TCP-G or m-TCP-E to activate and transduce non-activated T cells. The mutant VSV-G also contains T214N and T352A mutations that enhance its ability to antagonize complement inactivation or prevent complement inactivation, making it more suitable for in vivo targeted transduction of non-activated T cells.

突变型VSV-G(K47缺失、T214N和T352A)

Mutant VSV-G (K47 deletion, T214N, and T352A)

B.包装靶向慢病毒载体m-TCP-E/G：B. Packaging of targeted lentiviral vector m-TCP-E/G :

(a)混合所述4种质粒，通过PEI试剂将所述4种质粒转染进包装细胞系HEK-293T细胞系，包装靶向慢病毒载体m-TCP-G或m-TCP-E；(a) mixing the four plasmids, transfecting the four plasmids into the packaging cell line HEK-293T cell line using a PEI reagent, and packaging the targeted lentiviral vector m-TCP-G or m-TCP-E;

具体步骤为：The specific steps are:

准备HEK-293T细胞培养体系：取56mL的FBS过滤到500mL DMEM/高糖(10％FBS)并添加4mL P/S(双抗，青霉素×链霉素)，摇匀，放置二氧化碳培养箱预热转染中和用。Prepare the HEK-293T cell culture system: filter 56 mL of FBS into 500 mL of DMEM/high glucose (10% FBS) and add 4 mL of P/S (double antibody, penicillin × streptomycin), shake well, and place in a carbon dioxide incubator to preheat for transfection and neutralization.

Day 0，使用10cm培养皿接种HEK-293T细胞4.5×106个，接种约48h后，当细胞汇合度为80-90％时，通过PEI试剂将所述4种质粒转染进包装细胞HEK-293T细胞，包括：On Day 0, 4.5×10 6 HEK-293T cells were seeded in a 10 cm culture dish. About 48 hours after seeding, when the cell confluence reached 80-90%, the four plasmids were transfected into the packaging HEK-293T cells using PEI reagent, including:

将9μg CD19-TCP-E/G主质粒、4μg pMDLg/pRRE包装质粒、2μg pRSV-REV包装质粒和所述2μg包膜质粒1添加到1mL的Opti-MEM培养基中，摇匀后加入64μL的PEI试剂，吹打均匀后静置10分钟，随后加入HEK-293T细胞的培养基中，6小时后更新培养基，转染后的48小时收集培养基上清，使用0.45um的滤膜过滤，50000g离心2.5h，吸弃上清；使用200μL的F12培养基重悬所述慢病毒载体m-TCP-E或m-TCP-G并冻存于-80℃；同时，收集HEK-293T细胞，使用流式细胞术检测各组HEK-293T细胞中CD19-TCP-E/G分子的表达情况，结果如图1所示。9 μg CD19-TCP-E/G main plasmid, 4 μg pMDLg/pRRE packaging plasmid, 2 μg pRSV-REV packaging plasmid and the 2 μg envelope plasmid 1 were added to 1 mL Opti-MEM medium, shaken and added with 64 μL PEI reagent, pipetted evenly and allowed to stand for 10 minutes, then added to the culture medium of HEK-293T cells. The culture medium was renewed after 6 hours, and the culture supernatant was collected 48 hours after transfection, filtered using a 0.45 μm filter membrane, centrifuged at 50,000 g for 2.5 hours, and the supernatant was discarded; the lentiviral vector m-TCP-E or m-TCP-G was resuspended in 200 μL F12 medium and frozen at -80°C; at the same time, HEK-293T cells were collected, and the expression of CD19-TCP-E/G molecules in each group of HEK-293T cells was detected by flow cytometry. The results are shown in Figure 1.

检测FMC-63的流式抗体：商品名称：PE-Labeled Monoclonal Anti-FMC63 Antibody,Mouse IgG1(Y45)(Site-specific conjugation)(0.03％Proclin)DMF Filed，品牌：Acro，货号：#FM3-PY54A2-200 tests。Flow cytometry antibody for detecting FMC-63: Trade name: PE-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) (Site-specific conjugation) (0.03% Proclin) DMF Filed, Brand: Acro, Product Number: #FM3-PY54A2-200 tests.

Opti-MEM alpha减血清培养基：品牌：GIBCO,货号：#SP0272；Opti-MEM alpha Reduced Serum Medium: Brand: GIBCO, Catalog Number: #SP0272;

HEK-293T细胞培养基：DMEM+10％FBS；DMEM：品牌：GIBCO，货号：#C12430500BT；FBS：品牌：EXCELL，货号：#FSP500；HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog Number: #C12430500BT; FBS: Brand: EXCELL, Catalog Number: #FSP500;

F12培养基：品牌：GIBCO，货号：#C11330500BT；F12 culture medium: Brand: GIBCO, catalog number: #C11330500BT;

针式滤器：品牌：SORFA，货号：#622120。Syringe filter: Brand: SORFA, item number: #622120.

4.包装对照组慢病毒载体m-CAR 4. Packaging of the control group lentiviral vector m-CAR

参照上述包装靶向慢病毒载体m-TCP-E/G的方法，包装对照组慢病毒载体m-CAR。Referring to the above-mentioned method for packaging the targeting lentiviral vector m-TCP-E/G, the control group lentiviral vector m-CAR was packaged.

准备以下4种质粒：所述包膜质粒1、pMDLg/pRRE包装质粒、pRSV-REV包装质粒和携带编码CD19-CAR分子的多核苷酸的主质粒；Prepare the following four plasmids: the envelope plasmid 1, the pMDLg/pRRE packaging plasmid, the pRSV-REV packaging plasmid, and a main plasmid carrying a polynucleotide encoding a CD19-CAR molecule;

所述CD19-CAR分子从N-末端至C-末端的结构依次为：胞外抗原结合区、所述人CD8α铰链区、所述人CD8α跨膜区、人4-1BB共刺激信号传导结构域和人CD3ζ胞内信号传导结构域；所述胞外抗原结合区是所述FMC63-scFv；The structure of the CD19-CAR molecule from N-terminus to C-terminus is: an extracellular antigen binding region, the human CD8α hinge region, the human CD8α transmembrane region, a human 4-1BB co-stimulatory signaling domain, and a human CD3ζ intracellular signaling domain; the extracellular antigen binding region is the FMC63-scFv;

编码所述CD19-CAR分子的多核苷酸可操作地连接至编码所述人CD8α信号肽的多核苷酸，所述人CD8α信号肽位于所述CD19-CAR分子的N-末端；The polynucleotide encoding the CD19-CAR molecule is operably linked to the polynucleotide encoding the human CD8α signal peptide, and the human CD8α signal peptide is located at the N-terminus of the CD19-CAR molecule;

(1)所述4-1BB共刺激结构域的氨基酸序列如SEQ ID NO:49所示；(1) The amino acid sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO: 49;

(2)所述CD3ζ的胞内信号传导结构域的氨基酸序列如SEQ ID NO:50所示。(2) The amino acid sequence of the intracellular signal transduction domain of CD3ζ is shown in SEQ ID NO:50.

参照上述包装靶向慢病毒载体m-TCP-E/G的方法，包装所述对照组慢病毒载体m-CAR并冻存于-80℃；同时收集包装细胞HEK-293细胞，使用流式细胞术检测HEK-293T细胞中，所述CD19-CAR分子的表达情况，结果如图1所示。Referring to the above-mentioned method for packaging the targeted lentiviral vector m-TCP-E/G, the control group lentiviral vector m-CAR was packaged and frozen at -80°C; at the same time, the packaging cell HEK-293 cells were collected, and the expression of the CD19-CAR molecule in the HEK-293T cells was detected by flow cytometry. The results are shown in Figure 1.

由图1可知，在包装所述靶向慢病毒载体m-TCP-E、m-TCP-G和所述对照组慢病毒载体m-CAR的过程中，所述CD19-CAR分子在包装细胞HEK-293T细胞中大量出膜表达(阳性率为约74.76％)，而所述CD19-TCP-E分子(阳性率为约4.21％)、CD19-TCP-G分子(阳性率为约1.12％)在包装细胞HEK-293T细胞中极少出膜表达；因此，根据慢病毒载体在包装细胞中出芽的机制可合理推测，所述CD19-TCP-E/G分子在慢病毒载体的病毒包膜的含量相对于CD19-CAR分子也将显著减少，进而可有效减少使用所述靶向慢病毒载体m-TCP-E/G转导T细胞时的假转导；并使所述靶向慢病毒载体m-TCP-E/G结合癌细胞表面抗原和/或转导癌细胞的能力相对于所述对照组慢病毒载体m-CAR显著降低。As shown in Figure 1, during the packaging of the targeted lentiviral vectors m-TCP-E, m-TCP-G and the control lentiviral vector m-CAR, the CD19-CAR molecule was expressed in large quantities outside the membrane in the packaging cell HEK-293T cells (positive rate of about 74.76%), while the CD19-TCP-E molecule (positive rate of about 4.21%) and CD19-TCP-G molecule (positive rate of about 1.12%) were rarely expressed outside the membrane in the packaging cell HEK-293T cells; therefore, based on the mechanism of budding of the lentiviral vector in the packaging cells, it can be reasonably inferred that the content of the CD19-TCP-E/G molecule in the viral envelope of the lentiviral vector will also be significantly reduced relative to the CD19-CAR molecule, thereby effectively reducing false transduction when the targeted lentiviral vector m-TCP-E/G is used to transduce T cells; and the ability of the targeted lentiviral vector m-TCP-E/G to bind to cancer cell surface antigens and/or transduce cancer cells is significantly reduced relative to the control lentiviral vector m-CAR.

5.检测靶向慢病毒载体m-TCP-E/G和对照组慢病毒载体m-CAR转导Nalm-6 细胞的转导效率 5. Detection of the transduction efficiency of Nalm-6 cells transduced with the targeted lentiviral vector m-TCP-E/G and the control lentiviral vector m-CAR

Day 0，按照MOI＝5，分别将所述靶向慢病毒载体m-TCP-E、m-TCP-G和所述对照组慢病毒载体m-CAR加入Nalm-6细胞培养体系中(1640培养基+10％FBS)，分别转导2×105个CD19+Nalm-6细胞(人B淋巴白血病细胞)；Day 2，使用流式细胞术分别检测各组Nalm-6细胞中，CD19的表达情况，结果如图2所示。On Day 0, the targeted lentiviral vectors m-TCP-E and m-TCP-G and the control group lentiviral vector m-CAR were added to the Nalm-6 cell culture system (1640 medium + 10% FBS) at an MOI of 5, and 2×10 5 CD19 + Nalm-6 cells (human B lymphoid leukemia cells) were transduced respectively; on Day 2, the expression of CD19 in Nalm-6 cells in each group was detected by flow cytometry, and the results are shown in Figure 2.

由图2可知，相对于空白对照组的阳性率97.03％，加入所述对照组慢病毒载体m-CAR的Nalm-6细胞培养体系中，CD19的表达量显著降低(阳性率为34.69％)，这证明了所述对照组慢病毒载体m-CAR可通过其病毒包膜包含的所述FMC63-scFv有效结合Nalm-6细胞的表面抗原CD19，进而使所述Nalm-6细胞表面CD19的表达量减少；而加入所述靶向慢病毒载体m-TCP-E或m-TCP-G的Nalm-6细胞培养体系中，CD19的阳性率分别维持在97.07％或97.61％，这证明了所述靶向慢病毒载体m-TCP-E/G的病毒包膜不包含或几乎不包含所述FMC63-scFv，从而难以特异性结合Nalm-6细胞表面抗原CD19，可有效保持Nalm-6细胞的表面抗原CD19的表达丰度。这证明了，本发明提供的TCP分子仅在T细胞中特异性表达，不在或几乎不在T细胞以外的细胞，如包装细胞HEK-293T细胞中表达。As shown in Figure 2, compared with the positive rate of 97.03% in the blank control group, the expression level of CD19 in the Nalm-6 cell culture system to which the control group lentiviral vector m-CAR was added was significantly reduced (positive rate 34.69%), which proves that the control group lentiviral vector m-CAR can effectively bind to the surface antigen CD19 of Nalm-6 cells through the FMC63-scFv contained in its viral envelope, thereby reducing the expression level of CD19 on the surface of the Nalm-6 cells; and in the Nalm-6 cell culture system to which the targeting lentiviral vector m-TCP-E or m-TCP-G was added, the positive rate of CD19 was maintained at 97.07% or 97.61%, respectively, which proves that the viral envelope of the targeting lentiviral vector m-TCP-E/G does not contain or almost does not contain the FMC63-scFv, making it difficult to specifically bind to the Nalm-6 cell surface antigen CD19, and can effectively maintain the expression abundance of the surface antigen CD19 of Nalm-6 cells. This proves that the TCP molecule provided by the present invention is specifically expressed only in T cells, and is not expressed or hardly expressed in cells other than T cells, such as packaging cells HEK-293T cells.

检测CD19的流式抗体：CD19-FITC抗体(品牌：BD，货号：#555412)。Flow cytometry antibody for detecting CD19: CD19-FITC antibody (brand: BD, catalog number: #555412).

实施例2：靶向慢病毒载体m-TCP-E/G和对照组慢病毒载体m-7-E转导非活化T细胞后,TCP分子出膜表达效率检测Example 2: Detection of TCP molecule expression efficiency after transduction of non-activated T cells with the targeted lentiviral vector m-TCP-E/G and the control lentiviral vector m-7-E

1.包装对照组慢病毒载体m-7-E 1. Packaging control group lentiviral vector m-7-E

准备以下4种质粒，包装对照组慢病毒载体m-7-E：Prepare the following four plasmids to package the control lentiviral vector m-7-E:

携带编码所述突变型VSV-G的多核苷酸和编码膜表达抗CD7抗体的多核苷酸的包膜质粒(包膜质粒2)、pMDLg/pRRE包装质粒、pRSV-REV包装质粒和所述CD19-TCP-E主质粒；所述包膜质粒2通过常规分子克隆方法合成；An envelope plasmid (envelope plasmid 2) carrying a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding a membrane-expressed anti-CD7 antibody, a pMDLg/pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the CD19-TCP-E main plasmid; the envelope plasmid 2 is synthesized by conventional molecular cloning methods;

所述对照组慢病毒载体m-7-E(a)包含编码所述CD19-TCP-E分子的多核苷酸；和(b)病毒包膜包含膜表达抗CD7抗体；The control group lentiviral vector m-7-E (a) comprises a polynucleotide encoding the CD19-TCP-E molecule; and (b) the viral envelope comprises a membrane-expressed anti-CD7 antibody;

编码所述膜表达抗CD7抗体的多核苷酸从5’端至3’端的结构依次为：编码所述人CD8α信号肽的多核苷酸、编码特异性结合人CD7的scFv(源自TH-69的scFv，TH69-scFv)的多核苷酸、编码所述人CD8α铰链区的多核苷酸、编码所述人CD8α跨膜区的多核苷酸；The structure of the polynucleotide encoding the membrane-expressed anti-CD7 antibody from the 5' end to the 3' end is: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding a scFv that specifically binds to human CD7 (scFv derived from TH-69, TH69-scFv), a polynucleotide encoding the human CD8α hinge region, and a polynucleotide encoding the human CD8α transmembrane region;

所述TH69-scFv的重链可变区(VH区)通过连接肽2连接所述TH69-scFv的轻链可变区(VL区)；The heavy chain variable region (VH region) of the TH69-scFv is connected to the light chain variable region (VL region) of the TH69-scFv via connecting peptide 2;

(1)所述TH69-scFv的VH区的氨基酸序列如SEQ ID NO:25所示；所述TH69-scFv的VL区的氨基酸序列如SEQ ID NO:26所示；所述TH69-scFv的氨基酸序列如SEQ ID NO:71所示，所述TH69-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:72-74所示，所述TH69-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:75-77所示；(1) The amino acid sequence of the VH region of the TH69-scFv is shown in SEQ ID NO: 25; the amino acid sequence of the VL region of the TH69-scFv is shown in SEQ ID NO: 26; the amino acid sequence of the TH69-scFv is shown in SEQ ID NO: 71, the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 72-74, respectively, and the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 75-77, respectively;

(2)所述(G4S)3/连接肽2的氨基酸序列如SEQ ID NO:20所示；(2) The amino acid sequence of the (G 4 S) 3 /connector peptide 2 is shown in SEQ ID NO: 20;

参照实施例1中包装所述靶向慢病毒载体m-TCP-E/G的包装方法，同批次包装所述对照组慢病毒载体m-7-E和所述靶向慢病毒载体m-TCP-E和m-TCP-G。Referring to the packaging method of the targeted lentiviral vector m-TCP-E/G in Example 1, the control lentiviral vector m-7-E and the targeted lentiviral vectors m-TCP-E and m-TCP-G were packaged in the same batch.

2.靶向慢病毒载体m-TCP-E/G和m-7-E转导非活化T细胞 2. Transduction of non-activated T cells with targeted lentiviral vectors m-TCP-E/G and m-7-E

Day 0，分别取3组1×106个健康人非活化PBMCs细胞重悬于200μL的PBMCs培养基中，所述PBMCs培养基包括XVT培养基、终浓度为20ng/mL的IL-7和终浓度为20ng/mL的IL-15；按照MOI＝5，分别将所述靶向慢病毒载体m-TCP-G、m-TCP-E和所述对照组慢病毒载体m-7-E加入3组所述人非活化PBMCs培养基中，混匀，放置于5％CO2、37℃培养箱培养；On Day 0, 1×10 6 healthy human non-activated PBMCs were collected from three groups and resuspended in 200 μL of PBMC culture medium, which included XVT medium, IL-7 at a final concentration of 20 ng/mL, and IL-15 at a final concentration of 20 ng/mL. The targeted lentiviral vectors m-TCP-G and m-TCP-E, as well as the control lentiviral vector m-7-E, were added to the non-activated PBMC culture medium of the three groups at an MOI of 5. The cells were mixed and cultured in a 5% CO 2 , 37°C incubator.

Day 5，使用流式细胞术分别检测3组PBMCs中，所述CD19-TCP-E/G分子的表达情况，结果如图3所示。On Day 5, flow cytometry was used to detect the expression of CD19-TCP-E/G molecules in the three groups of PBMCs. The results are shown in Figure 3.

由图3可知，3组PBMCs培养基中，所述对照组慢病毒载体m-7-E、所述靶向慢病毒载体m-TCP-E和m-TCP-G分别转导人非活化PBMCs中的CD3+T细胞递送的所述CD19-TCP-E/G基因的表达效率分别为约5.25％、12.65％和34.81％；As shown in Figure 3, in the culture medium of the three groups of PBMCs, the expression efficiency of the CD19-TCP-E/G gene delivered by the control lentiviral vector m-7-E, the targeted lentiviral vectors m-TCP-E and m-TCP-G to transduce CD3 + T cells in human non-activated PBMCs was approximately 5.25%, 12.65% and 34.81%, respectively;

首先，相对于所述对照组慢病毒载体m-7-E，所述靶向慢病毒载体m-TCP-G和m-TCP-E将编码所述CD19-TCP-E/G分子的多核苷酸稳定整合至T细胞基因组后，所述CD19-TCP-E/G分子在T细胞中出膜表达的效率显著提高；这可能是因为相对于不能有效活化刺激非活化T细胞的抗CD7抗体，T细胞活化初级信号分子抗CD3抗体和次级信号分子抗CD28抗体可有效活化刺激人非活化PBMCs中的非活化T细胞，而活化的T细胞组装和表达TCR/CD3复合体和所述CD19-TCP-E/G分子(可能并入内源性TCR/CD3复合体，和/或与内源性TCR/CD3复合体发生功能性相互作用)的效率优于显著非活化T细胞。First, compared with the control group lentiviral vector m-7-E, the targeted lentiviral vectors m-TCP-G and m-TCP-E stably integrated the polynucleotide encoding the CD19-TCP-E/G molecule into the T cell genome, and the efficiency of the CD19-TCP-E/G molecule in T cell membrane expression was significantly improved; this may be because, compared with the anti-CD7 antibody that cannot effectively activate and stimulate non-activated T cells, the T cell activation primary signal molecule anti-CD3 antibody and the secondary signal molecule anti-CD28 antibody can effectively activate and stimulate non-activated T cells in human non-activated PBMCs, and the efficiency of activated T cells in assembling and expressing TCR/CD3 complexes and the CD19-TCP-E/G molecules (which may be incorporated into endogenous TCR/CD3 complexes and/or functionally interact with endogenous TCR/CD3 complexes) is better than that of significantly non-activated T cells.

其次，由图3可知，相对于所述靶向慢病毒载体m-TCP-E，所述靶向慢病毒载体m-TCP-G转导人非活化T细胞后，所述CD19-TCP-G分子(阳性率为约34.81％)出膜表达的效率比起所述CD19-TCP-E分子(阳性率为约12.65％)显著更高。Secondly, as shown in Figure 3, compared with the targeted lentiviral vector m-TCP-E, after the targeted lentiviral vector m-TCP-G transduced human non-activated T cells, the efficiency of the CD19-TCP-G molecule (positive rate of approximately 34.81%) membrane expression was significantly higher than that of the CD19-TCP-E molecule (positive rate of approximately 12.65%).

这可能是因为本实施例所使用的膜表达抗CD3抗体，所述UCHT1-scFv为抗CD3ε抗体；在包装所述靶向慢病毒载体m-TCP-E的过程中，UCHT1-scFv在包装细胞HEK-293T细胞中可特异性结合所述CD19-TCP-E分子所包含的CD3ε，从而使可出膜表达在HEK-293T细胞细胞膜、并随出芽转移至所述靶向慢病毒载体m-TCP-E的包膜上的可供(avai lable)特异性结合T细胞表面抗原CD3ε的UCHT1-scFv的含量减少，进而使所述靶向慢病毒载体m-TCP-E靶向和活化刺激非活化T细胞的能力降低，最终导致转导效率的降低和/或所述CD19-TCP-E分子在活化程度相对较低的T细胞中出膜表达的效率降低；This may be because the membrane-expressed anti-CD3 antibody used in this example, and the UCHT1-scFv is an anti-CD3ε antibody; during the packaging process of the targeted lentiviral vector m-TCP-E, UCHT1-scFv can specifically bind to CD3ε contained in the CD19-TCP-E molecule in the packaging cell HEK-293T cells, thereby reducing the content of UCHT1-scFv that can be expressed on the cell membrane of the HEK-293T cells and transferred to the envelope of the targeted lentiviral vector m-TCP-E with budding, thereby reducing the ability of the targeted lentiviral vector m-TCP-E to target and activate and stimulate non-activated T cells, ultimately leading to a decrease in transduction efficiency and/or a decrease in the efficiency of membrane expression of the CD19-TCP-E molecule in T cells with relatively low activation levels;

而在包装所述靶向慢病毒载体m-TCP-G的过程中，因为所述CD19-TCP-G分子包含的TCR/CD3复合体亚基为CD3γ，抗CD3ε抗体UCHT1-scFv并不能特异性结合CD3γ，因此也不会导致所述靶向慢病毒载体m-TCP-G的包膜中的可供特异性T细胞表面抗原CD3ε的UCHT1-scFv的含量减少，进而也不会影响所述靶向慢病毒载体m-TCP-G靶向和活化刺激非活化T细胞的能力，最终不会导致转导效率的降低和/或所述CD19-TCP-G分子在活化程度相对较高的T细胞中出膜表达的效率降低。During the packaging of the targeted lentiviral vector m-TCP-G, because the TCR/CD3 complex subunit contained in the CD19-TCP-G molecule is CD3γ, the anti-CD3ε antibody UCHT1-scFv cannot specifically bind to CD3γ, and therefore will not lead to a reduction in the content of UCHT1-scFv available for the specific T cell surface antigen CD3ε in the envelope of the targeted lentiviral vector m-TCP-G, and thus will not affect the ability of the targeted lentiviral vector m-TCP-G to target and activate and stimulate non-activated T cells, and ultimately will not lead to a decrease in transduction efficiency and/or a decrease in the efficiency of the CD19-TCP-G molecule to express outside the membrane in T cells with a relatively high degree of activation.

因此，在使用抗CD3抗体或其抗原结合片段作为T细胞活化初级信号分子，并同时使用本发明提供的TCP分子时，为最大程度地优化所构建的靶向慢病毒载体的转导效率，所使用的抗CD3抗体或其抗原结合片段应不能特异性结合TCP分子所包含的TSP多肽。Therefore, when using an anti-CD3 antibody or an antigen-binding fragment thereof as a primary signal molecule for T cell activation and simultaneously using the TCP molecule provided by the present invention, in order to maximize the transduction efficiency of the constructed targeted lentiviral vector, the anti-CD3 antibody or antigen-binding fragment thereof used should not specifically bind to the TSP polypeptide contained in the TCP molecule.

XVT培养基：商品名称：PRIME-XV T cell CDM，品牌：IRVINE(FUJIFILM)，货号：#91154；XVT medium: Trade name: PRIME-XV T cell CDM, brand: IRVINE (FUJIFILM), catalog number: #91154;

IL-7：商品名称：IL-7Protein,Human,Recombinant，品牌：义翘神州，货号：#11821-HNAE；IL-7: Trade Name: IL-7 Protein, Human, Recombinant, Brand: Sino Biological, Catalog Number: #11821-HNAE;

IL-15：商品名称：IL-15Protein,Human,Recombinant(His Tag)，品牌：义翘神州，货号：#10360-H07E；IL-15: Trade Name: IL-15 Protein, Human, Recombinant (His Tag), Brand: Sino Biological, Catalog Number: #10360-H07E;

检测CD3的流式抗体：商品名称：FITC Mouse Anti-Human CD3；品牌：BIOLEGEND，货号:#555339。Flow cytometry antibody for detecting CD3: Trade name: FITC Mouse Anti-Human CD3; Brand: BIOLEGEND, Item number: #555339.

实施例3：靶向慢病毒载体m-TCP-G可有效活化刺激非活化T细胞Example 3: Targeted lentiviral vector m-TCP-G can effectively activate and stimulate non-activated T cells

在Day 0，分别从Donor 1(健康人)和Donor 2(健康人)各取两组2×105个人非活化PBMCs细胞，重悬于200μL所述人非活化PBMCs培养基中；On Day 0, two groups of 2×10 5 human non-activated PBMCs were obtained from Donor 1 (healthy person) and Donor 2 (healthy person), respectively, and resuspended in 200 μL of the human non-activated PBMCs culture medium;

Day 0，按照MOI＝5，向Donor 1和Donor 2的其中1组人非活化PBMCs培养基中分别加入所述靶向慢病毒载体m-TCP-G，混合，持续计数并记录2组细胞中CD3+T细胞的增殖情况，结果如图4所示。On Day 0, the targeted lentiviral vector m-TCP-G was added to the culture medium of one of the non-activated human PBMCs groups of Donor 1 and Donor 2 at an MOI of 5, mixed, and the proliferation of CD3 + T cells in the two groups of cells was continuously counted and recorded. The results are shown in Figure 4.

由图4可知，所述靶向慢病毒载体m-TCP-G可有效活化刺激Donor 1和Donor 2的非活化PBMCs中的非活化T细胞。As shown in Figure 4, the targeted lentiviral vector m-TCP-G can effectively activate and stimulate the non-activated T cells in the non-activated PBMCs of Donor 1 and Donor 2.

实施例4：CD19-TCP-T细胞体外杀伤靶细胞Example 4: CD19-TCP-T cells kill target cells in vitro

Day 0，分别取两组5×106个健康人Donor 1的非活化PBMCs重悬于200μL的所述PBMCs培养基中，其中一组为对照组，另一组按照MOI＝5，加入所述靶向慢病毒载体m-TCP-G，制备CD19-TCP-T细胞(实验组)；On Day 0, two groups of 5 × 10 6 non-activated PBMCs from healthy donor 1 were resuspended in 200 μL of the PBMCs culture medium. One group served as the control group, and the other group was injected with the targeted lentiviral vector m-TCP-G at an MOI of 5 to prepare CD19-TCP-T cells (experimental group).

Day 1，向对照组和实验组分别加入1mL的DPBS清洗一次；Day 1: Add 1 mL of DPBS to each of the control and experimental groups for washing.

Day 5，分别取对照组和实验组的细胞计数各3×105个，按照效靶比E:T＝3:1，向2组细胞分别加入1×105个靶细胞Nalm-6细胞，混合；On Day 5, 3×10 5 cells were counted from the control group and the experimental group, and 1×10 5 target Nalm-6 cells were added to each group of cells at an effector-target ratio of E:T=3:1 and mixed.

Day 7，使用流式细胞术分别检测对照组和实验组杀伤靶细胞Nalm-6细胞的杀伤情况，结果如图5所示。On Day 7, flow cytometry was used to detect the killing of target cells Nalm-6 in the control group and the experimental group, respectively. The results are shown in Figure 5.

由图5可知，所述靶向慢病毒载体m-TCP-G转导T细胞后制备所得的CD19-TCP-T细胞可高效杀伤靶细胞Nalm-6细胞。As shown in FIG5 , the CD19-TCP-T cells prepared by transducing T cells with the targeted lentiviral vector m-TCP-G can efficiently kill the target Nalm-6 cells.

实施例5：靶向慢病毒载体m-TCP-G体内杀伤癌细胞Example 5: Targeted lentiviral vector m-TCP-G kills cancer cells in vivo

Day 0，取12只4～8周周龄的NKG免疫缺陷雌性小鼠(购自赛业生物)，分为3组，每组各4只小鼠，分别为对照组1、对照组2和实验组；On Day 0, 12 NKG immunodeficient female mice aged 4 to 8 weeks (purchased from Saiye Bio) were divided into three groups, with 4 mice in each group, namely control group 1, control group 2, and experimental group;

Day 0，向各组小鼠尾静脉注射5×105个Nalm-6细胞，所述Nalm-6细胞携带Luciferase(荧光素酶)；并向各组小鼠腹腔注射200μL的浓度为15mg/mL的荧光素钠盐(品牌：翌圣生物，货号：#40901ES10)，分别对三组小鼠进行活体成像；On Day 0, 5 × 10 5 Nalm-6 cells carrying luciferase were injected into the tail vein of each group of mice. 200 μL of 15 mg/mL luciferin sodium salt (brand: Yisheng Bio, catalog number: #40901ES10) was injected into the peritoneal cavity of each group of mice. In vivo imaging was performed on the three groups of mice.

4小时后，分别向对照组2和实验组的小鼠尾静脉注射1×107个人非活化PBMCs；并单独向实验组小鼠注射1×106TU的所述靶向慢病毒载体m-TCP-G病毒上清液；Four hours later, 1×10 7 human non-activated PBMCs were injected into the tail vein of mice in control group 2 and experimental group, respectively; and 1×10 6 TU of the viral supernatant of the targeted lentiviral vector m-TCP-G was injected into mice in the experimental group alone;

Day 5和Day 7，分别对3组小鼠进行活体成像，结果如图6所示。On Day 5 and Day 7, live imaging was performed on the three groups of mice, and the results are shown in Figure 6.

由图6可知，所述靶向慢病毒载体m-TCP-G在实验组小鼠体内可转导人PBMCs细胞制备CD19-TCP-T细胞，高效杀伤Nalm-6细胞。As shown in Figure 6, the targeted lentiviral vector m-TCP-G can transduce human PBMCs cells to prepare CD19-TCP-T cells in the experimental group mice, and effectively kill Nalm-6 cells.

实施例6：靶向慢病毒载体m-TCP-G和m-3-G和对照组慢病毒载体m-7-G分别转导人非活化PBMCsExample 6: Targeted lentiviral vectors m-TCP-G and m-3-G and control lentiviral vector m-7-G were used to transduce human non-activated PBMCs

1.包装慢病毒载体m-3-G 1. Packaging of Lentiviral Vector m-3-G

参照实施例1中包装所述靶向慢病毒载体m-TCP-E/G的方法，同批次包装所述对照组慢病毒载体m-7-G、靶向慢病毒载体m-3-G和m-TCP-G；Referring to the method for packaging the targeted lentiviral vector m-TCP-E/G in Example 1, the control lentiviral vector m-7-G, targeted lentiviral vectors m-3-G and m-TCP-G were packaged in the same batch;

其中，所述靶向慢病毒载体m-3-G：(a)的包膜包含的T细胞靶向分子为T细胞活化初级信号分子，所述膜表达UCHT1-scFv，且不包含T细胞活化次级信号分子以及其他T细胞靶向分子；(b)包含编码所述CD19-TCP-G的多核苷酸；具体地，将所述包膜质粒1替换为包含编码所述突变型VSV-G的多核苷酸和编码所述膜表达UCHT1-scFv的多核苷酸的包膜质粒(包膜质粒3)。Among them, the targeted lentiviral vector m-3-G: (a) the envelope contains a T cell targeting molecule that is a primary signal molecule for T cell activation, the membrane expresses UCHT1-scFv, and does not contain secondary signal molecules for T cell activation and other T cell targeting molecules; (b) contains a polynucleotide encoding the CD19-TCP-G; specifically, the envelope plasmid 1 is replaced with an envelope plasmid (envelope plasmid 3) containing a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding the membrane-expressed UCHT1-scFv.

Day 0，按照MOI＝5，使用所述对照组慢病毒载体m-7-G、所述靶向慢病毒载体m-3-G和m-TCP-G分别转导1×106个健康人Donor 1的非活化PBMCs；Day 0: At an MOI of 5, 1×10 6 non-activated PBMCs from healthy donor 1 were transduced using the control lentiviral vector m-7-G, the targeted lentiviral vector m-3-G, and m-TCP-G, respectively.

Day 5，使用流式细胞术分别检测3组PBMCs的CD3+T细胞中，所述CD19-TCP-G分子所包含的抗原结合区，所述FMC63-scFv的表达情况，结果如图7所示。On Day 5, flow cytometry was used to detect the antigen binding region contained in the CD19-TCP-G molecule and the expression of the FMC63-scFv in the CD3 + T cells of the three groups of PBMCs. The results are shown in Figure 7.

由图7可知，首先，相较于所述对照组慢病毒载体m-7-G，所述靶向慢病毒载体m-3-G和m-TCP-G转导人非活化PBMCs，其中CD3+T细胞表达所述CD19-TCP-G分子的效率更高；这可能是因为膜表达抗CD7抗体不可有效活化刺激非活化T细胞，这对于在T细胞中限定表达、表达效率可能更依赖于T细胞的活化程度的TCP分子来说，病毒包膜至少包含T细胞活化初级信号分子的慢病毒载体的转导效率相对更高。As can be seen from Figure 7, first, compared with the control group lentiviral vector m-7-G, the targeted lentiviral vectors m-3-G and m-TCP-G transduced human non-activated PBMCs, where CD3 + T cells expressed the CD19-TCP-G molecule more efficiently; this may be because membrane-expressed anti-CD7 antibodies cannot effectively activate and stimulate non-activated T cells. For TCP molecules whose expression is limited in T cells and whose expression efficiency may be more dependent on the degree of T cell activation, the transduction efficiency of lentiviral vectors whose viral envelope contains at least primary signal molecules for T cell activation is relatively higher.

其次，相较于所述靶向慢病毒载体m-3-G，所述靶向慢病毒载体m-TCP-G转导人非活化PBMCs，其中CD3+T细胞表达所述CD19-TCP-G分子的效率显著更高；Secondly, compared with the targeted lentiviral vector m-3-G, the targeted lentiviral vector m-TCP-G transduced human non-activated PBMCs, in which CD3 + T cells expressed the CD19-TCP-G molecule at a significantly higher efficiency;

这可能是因为所述靶向慢病毒载体m-3-G的包膜仅包含模拟T细胞活化信号的抗原特异性的第一信号，即T细胞活化初级信号分子的膜表达抗CD3抗体，而缺失模拟第二信号，T细胞活化共刺激/次级信号，如抗CD28抗体等T细胞活化次级信号分子；故相较于包膜同时包含T细胞活化初级和次级信号分子的所述靶向慢病毒载体m-TCP-G，所述靶向慢病毒载体m-3-G活化刺激非活化T细胞的能力相对较低，因此导致所述TCP分子在相对未充分活化的T细胞中的出膜表达效率较低。This may be because the envelope of the targeted lentiviral vector m-3-G only contains the antigen-specific first signal that simulates the T cell activation signal, that is, the membrane-expressed anti-CD3 antibody of the T cell activation primary signal molecule, but lacks the T cell activation secondary signal molecules such as anti-CD28 antibodies that simulate the second signal, T cell activation co-stimulatory/secondary signal; therefore, compared with the targeted lentiviral vector m-TCP-G whose envelope contains both T cell activation primary and secondary signal molecules, the ability of the targeted lentiviral vector m-3-G to activate and stimulate non-activated T cells is relatively low, resulting in a low efficiency of membrane expression of the TCP molecule in relatively underactivated T cells.

实施例7：包装包膜包含抗CD3抗体和CD86的靶向慢病毒载体m-86-GExample 7: Packaging of a targeted lentiviral vector m-86-G containing an anti-CD3 antibody and CD86

1.设计膜表达抗CD3抗体×CD86结构 1. Design of membrane-expressed anti-CD3 antibody × CD86 construct

本实施例中，编码膜表达抗CD3抗体×CD86(膜表达抗CD3抗体×CD86)的多核苷酸从5’端至3’端依次为：编码所述人CD8α信号肽的多核苷酸、编码所述UCHT1-scFv的多核苷酸、编码所述人CD8α铰链区的多核苷酸、编码所述人CD8α跨膜区的多核苷酸、编码所述FT2A肽的多核苷酸、编码人CD86信号肽的多核苷酸、编码人CD86胞外域的多核苷酸、编码人CD86跨膜区的多核苷酸；In this embodiment, the polynucleotides encoding membrane-expressed anti-CD3 antibody × CD86 (membrane-expressed anti-CD3 antibody × CD86) are, from 5' to 3' end, the following: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding the UCHT1-scFv, a polynucleotide encoding the human CD8α hinge region, a polynucleotide encoding the human CD8α transmembrane region, a polynucleotide encoding the FT2A peptide, a polynucleotide encoding the human CD86 signal peptide, a polynucleotide encoding the human CD86 extracellular domain, and a polynucleotide encoding the human CD86 transmembrane region;

人CD86，Uniprot ID:P42081；Human CD86, Uniprot ID: P42081;

(1)所述人CD86信号肽的氨基酸序列如SEQ ID NO:30所示；(1) The amino acid sequence of the human CD86 signal peptide is shown in SEQ ID NO: 30;

(2)所述人CD86胞外域的氨基酸序列如SEQ ID NO:31所示；(2) The amino acid sequence of the human CD86 extracellular domain is shown in SEQ ID NO: 31;

(3)所述人CD86跨膜区的氨基酸序列如SEQ ID NO:32所示。(3) The amino acid sequence of the human CD86 transmembrane region is shown in SEQ ID NO:32.

同批次包装所述靶向慢病毒载体m-TCP-G和m-86-G：The targeted lentiviral vectors m-TCP-G and m-86-G were packaged in the same batch:

参照实施例1中，包装所述靶向慢病毒载体m-TCP-E/G的方法，包装包膜包含所述膜表达抗CD3抗体×CD86的靶向慢病毒载体m-86-G；具体地，将所述包膜质粒1替换为携带编码所述突变型VSV-G的多核苷酸和编码所述膜表达抗CD3抗体×CD86的多核苷酸的包膜质粒(包膜质粒4)。Referring to the method for packaging the targeted lentiviral vector m-TCP-E/G in Example 1, the packaging envelope comprises the targeted lentiviral vector m-86-G expressing the membrane-expressing anti-CD3 antibody × CD86; specifically, the envelope plasmid 1 is replaced with an envelope plasmid (envelope plasmid 4) carrying a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding the membrane-expressing anti-CD3 antibody × CD86.

2.靶向慢病毒载体m-86-G转导人非活化PBMCs 2. Transduction of human non-activated PBMCs with the targeted lentiviral vector m-86-G

Day 0，参照实施例1中，所述靶向慢病毒载体m-TCP-E/G转导人非活化PBMCs的方法，按照MOI＝5，使用所述靶向慢病毒载体m-86-G和m-TCP-G分别转导1×106个健康人Donor 1的人非活化PBMCs；Day 0: Referring to the method for transducing non-activated human PBMCs with the targeted lentiviral vector m-TCP-E/G in Example 1, 1×10 6 non-activated human PBMCs from healthy donor 1 were transduced using the targeted lentiviral vectors m-86-G and m-TCP-G, respectively, at an MOI of 5.

Day 5，使用流式细胞术检测各组PBMCs的CD3+T细胞中，所述CD19-TCP-G分子的表达情况，结果如图8所示。On Day 5, flow cytometry was used to detect the expression of the CD19-TCP-G molecule in the CD3 + T cells of PBMCs in each group. The results are shown in FIG8 .

由图8可知，所述靶向慢病毒载体m-TCP-G和m-86-G分别转导人非活化PBMCs，其中CD3+T细胞表达所述CD19-TCP-G的效率分别为约12.66％和16.15％；所述靶向慢病毒载体m-TCP-G和m-86-G均可有效转导人非活化PBMCs中的CD3+T细胞，表达所述CD19-TCP-G。As can be seen from Figure 8, the targeted lentiviral vectors m-TCP-G and m-86-G respectively transduced human non-activated PBMCs, wherein the efficiency of CD3 + T cells expressing the CD19-TCP-G was approximately 12.66% and 16.15%, respectively; the targeted lentiviral vectors m-TCP-G and m-86-G can both effectively transduce CD3 + T cells in human non-activated PBMCs to express the CD19-TCP-G.

实施例8：构建包含编码靶向CD33的TCP分子的多核苷酸的靶向慢病毒载体Example 8: Construction of a targeted lentiviral vector containing a polynucleotide encoding a TCP molecule targeting CD33

1.构建包含靶向CD33的TCP 1. Construction of TCP containing CD33 targeting

参照实施例1中构建的所述CD19-TCP-G，构建靶向人CD33(Uniprot ID:P20138)的TCP分子(CD33-TCP-G)；具体地，将所述FMC63-scFv替换为靶向人CD33的抗原结合区；Referring to the CD19-TCP-G constructed in Example 1, a TCP molecule targeting human CD33 (Uniprot ID: P20138) (CD33-TCP-G) was constructed; specifically, the FMC63-scFv was replaced with the antigen binding region targeting human CD33;

编码所述CD33-TCP-G分子的多核苷酸从5’端至3’端依次为：编码所述人CD8α信号肽的多核苷酸、编码靶向人CD33的胞外抗原结合区的多核苷酸、编码所述连接肽1的多核苷酸、编码所述人CD3γ(CD33-TCP-G)的多核苷酸；The polynucleotides encoding the CD33-TCP-G molecule are, from the 5' end to the 3' end, the following: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding the extracellular antigen binding region targeting human CD33, a polynucleotide encoding the connecting peptide 1, and a polynucleotide encoding the human CD3γ (CD33-TCP-G);

所述靶向人CD33的抗原结合区是源自抗人CD33单克隆抗体Gemtuzumab的scFv(Gemtuzumab-scFv)；所述Gemtuzumab-scFv的氨基酸序列如SEQ ID NO:78所示，所述Gemtuzumab-scFv的VL区的氨基酸序列如SEQ ID NO:59所示；所述Gemtuzumab-scFv的VH区的氨基酸序列如SEQ ID NO:60所示；所述Gemtuzumab-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:79-81所示；所述Gemtuzumab-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:82-84所示。The antigen binding region targeting human CD33 is a scFv (Gemtuzumab-scFv) derived from the anti-human CD33 monoclonal antibody Gemtuzumab; the amino acid sequence of the Gemtuzumab-scFv is shown in SEQ ID NO: 78, the amino acid sequence of the VL region of the Gemtuzumab-scFv is shown in SEQ ID NO: 59; the amino acid sequence of the VH region of the Gemtuzumab-scFv is shown in SEQ ID NO: 60; the amino acid sequences of the HCDR1-3 regions of the Gemtuzumab-scFv are shown in SEQ ID NO: 79-81, respectively; the amino acid sequences of the LCDR1-3 regions of the Gemtuzumab-scFv are shown in SEQ ID NO: 82-84, respectively.

2.包装靶向慢病毒载体m-a33-G 2. Packaging of the Targeted Lentiviral Vector m-a33-G

参照实施例1中，包装所述靶向慢病毒载体m-TCP-E/G的方法，包装m-a33-G。Referring to the method for packaging the targeted lentiviral vector m-TCP-E/G in Example 1, m-a33-G was packaged.

3.使用所述靶向慢病毒载体m-a33-G转导非活化T细胞，制备TCP-T细 胞 3. Use the targeted lentiviral vector m-a33-G to transduce non-activated T cells to prepare TCP-T cells .

参照实施例2中，所述靶向慢病毒载体m-TCP-E/G转导人非活化PBMCs的方法，Day 0，使用所述靶向慢病毒载体m-a33-G转导Donor 1(健康人)的非活化PBMCs，制备CD33-TCP-T细胞；Referring to the method for transducing human non-activated PBMCs with the targeted lentiviral vector m-TCP-E/G in Example 2, on Day 0, non-activated PBMCs of Donor 1 (a healthy individual) were transduced with the targeted lentiviral vector m-a33-G to prepare CD33-TCP-T cells.

Day 5，使用流式细胞术检测所述CD33-TCP-G分子在CD3+T细胞中的出膜表达的效率，结果如图9所示。On Day 5, flow cytometry was used to detect the efficiency of the membrane expression of the CD33-TCP-G molecule in CD3 + T cells. The results are shown in FIG9 .

由图9可知，所述靶向载体m-a33-G可有效转导人非活化PBMCs中的CD3+T细胞，所述CD33-TCP-G分子的出膜表达效率为约27.84％。As shown in FIG9 , the targeting vector m-a33-G can effectively transduce CD3 + T cells in non-activated human PBMCs, and the cell membrane expression efficiency of the CD33-TCP-G molecule is approximately 27.84%.

4.CD33-TCP-T细胞体外杀伤靶细胞 4. CD33-TCP-T cells kill target cells in vitro

参照实施例4中，所述CD19-TCP-T细胞杀伤靶细胞Nalm-6细胞的方法，使用所述CD33-TCP-T细胞在体外杀伤CD33+靶细胞MOLM-13细胞(人急性髓性白血病细胞)，Day 7，使用流式细胞术检测杀伤效率，结果如图10所示；Referring to the method for killing target Nalm-6 cells by the CD19-TCP-T cells in Example 4, the CD33-TCP-T cells were used to kill CD33 + target MOLM-13 cells (human acute myeloid leukemia cells) in vitro. On Day 7, the killing efficiency was detected by flow cytometry. The results are shown in FIG10 .

由图10可知，所述CD33-TCP-T细胞在体外可有效杀伤CD33+的MOLM-13细胞。As shown in FIG10 , the CD33 − TCP-T cells can effectively kill CD33 + MOLM-13 cells in vitro.

流式检测使用的抗体：Antibodies used in flow cytometry:

检测CD33：FITC-CD33；品牌：BD，货号：#561818；Detection of CD33: FITC-CD33; Brand: BD, Catalog No.: #561818;

检测CD3：品牌：ACRO，货号：#CD3-HP2E3-200tests。Detection CD3: Brand: ACRO, Product Number: #CD3-HP2E3-200tests.

Claims (99)

一种病毒载体，其特征在于，A viral vector, characterized in that (a)所述病毒载体包含编码T细胞受体嵌合蛋白(T Cell Receptor Chimeric Protein，TCP)的多核苷酸；和(a) the viral vector comprises a polynucleotide encoding a T cell receptor chimeric protein (TCP); and (b)所述病毒载体表面包含T细胞活化信号分子；(b) the surface of the viral vector contains T cell activation signal molecules; 其中，所述TCP包含：Wherein, the TCP includes: (i)TCR/CD3复合体亚基相关多肽(TCR/CD3 Complex Subunit Related Peptide，TSP),所述TSP包含(1)TCR/CD3复合体亚基、(2)TCR/CD3复合体亚基功能性片段、(3)TCR/CD3复合体亚基变体和(4)TCR/CD3复合体亚基功能性片段的变体中的至少一种；以及(i) a TCR/CD3 complex subunit related peptide (TSP), wherein the TSP comprises at least one of (1) a TCR/CD3 complex subunit, (2) a functional fragment of a TCR/CD3 complex subunit, (3) a TCR/CD3 complex subunit variant, and (4) a variant of a functional fragment of a TCR/CD3 complex subunit; and (ii)抗原结合区。(ii) Antigen binding region. 根据权利要求1所述的病毒载体，其特征在于，所述病毒载体为慢病毒载体(LVV)或逆转录病毒载体(RVV)。The viral vector according to claim 1, characterized in that the viral vector is a lentiviral vector (LVV) or a retroviral vector (RVV). 根据权利要求1或2所述的病毒载体，其特征在于，所述TCR/CD3复合体亚基选自TCRα(TCRA)、TCRβ(TCRB)、TCRγ(TCRG)、TCRδ(TCRD)、CD3γ(CD3G)、CD3δ(CD3D)、CD3ζ(CD3Z)和CD3ε(CD3E)中的至少一种。The viral vector according to claim 1 or 2, characterized in that the TCR/CD3 complex subunit is selected from at least one of TCRα (TCRA), TCRβ (TCRB), TCRγ (TCRG), TCRδ (TCRD), CD3γ (CD3G), CD3δ (CD3D), CD3ζ (CD3Z) and CD3ε (CD3E). 根据权利要求1-3中任一项所述的病毒载体，其特征在于，所述TCR/CD3复合体亚基功能性片段包含所述TCR/CD3复合体亚基的胞外区、跨膜区、胞内区、可变区和恒定区中的至少一种。The viral vector according to any one of claims 1 to 3, characterized in that the TCR/CD3 complex subunit functional fragment comprises at least one of the extracellular region, transmembrane region, intracellular region, variable region and constant region of the TCR/CD3 complex subunit. 根据权利要求4所述的病毒载体，其特征在于，所述TSP包含CD3γ或其功能性片段；The viral vector according to claim 4, wherein the TSP comprises CD3γ or a functional fragment thereof; 优选地，所述TSP包含CD3γ的跨膜区和胞内区；Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3γ; 更优选地，所述TSP包含CD3γ的跨膜区和胞内区以及至少一种以下蛋白的胞外区：TCRα、TCRβ、TCRγ、TCRδ、CD3ε、CD3ζ和CD3δ；More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3γ and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3ζ and CD3δ; 更进一步优选地，所述TSP包含CD3γ的跨膜区和胞内区以及(a)CD3δ的胞外区、(b)CD3ζ的胞外区或(c)CD3ε的胞外区；More preferably, the TSP comprises the transmembrane region and intracellular region of CD3γ and the extracellular region of (a) CD3δ, (b) CD3ζ or (c) CD3ε; 最优选地，所述CD3δ的胞外区、CD3ζ的胞外区或CD3ε的胞外区的C-末端位于所述CD3γ的跨膜区的N-末端方向，所述CD3γ的跨膜区的C-末端位于所述CD3γ的胞内区的N-末端方向。Most preferably, the C-terminus of the extracellular region of CD3δ, the extracellular region of CD3ζ or the extracellular region of CD3ε is located towards the N-terminus of the transmembrane region of CD3γ, and the C-terminus of the transmembrane region of CD3γ is located towards the N-terminus of the intracellular region of CD3γ. 根据权利要求4所述的病毒载体，其特征在于，所述TSP包含CD3ε或其功能性片段。The viral vector according to claim 4, wherein the TSP comprises CD3ε or a functional fragment thereof. 优选地，所述TSP包含CD3ε的跨膜区和胞内区；Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ε; 更优选地，所述TSP包含CD3ε的跨膜区和胞内区以及至少一种以下蛋白的胞外区：TCRα、TCRβ、TCRγ、TCRδ、CD3γ、CD3ζ和CD3δ；More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ε and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3ζ and CD3δ; 更进一步优选地，所述TSP包含CD3ε的跨膜区和胞内区；以及(a)CD3γ的胞外区、(b)CD3ζ的胞外区或(c)CD3δ的胞外区；More preferably, the TSP comprises the transmembrane region and intracellular region of CD3ε; and (a) the extracellular region of CD3γ, (b) the extracellular region of CD3ζ, or (c) the extracellular region of CD3δ; 最优选地，所述CD3γ的胞外区、CD3ζ的胞外区或CD3δ的胞外区的C-末端位于所述CD3ε的跨膜区的N-末端方向，所述CD3ε的跨膜区的C-末端位于所述CD3ε的胞内区的N-末端方向。Most preferably, the C-terminus of the extracellular region of CD3γ, the extracellular region of CD3ζ or the extracellular region of CD3δ is located in the N-terminal direction of the transmembrane region of CD3ε, and the C-terminus of the transmembrane region of CD3ε is located in the N-terminal direction of the intracellular region of CD3ε. 根据权利要求4所述的病毒载体，其特征在于，所述TSP包含TCRα或其功能性片段和TCRβ或其功能性片段；The viral vector according to claim 4, wherein the TSP comprises TCRα or a functional fragment thereof and TCRβ or a functional fragment thereof; 优选地，所述TSP包含TCRα的恒定区和TCRβ的恒定区。Preferably, the TSP comprises the constant region of TCRα and the constant region of TCRβ. 根据权利要求7所述的病毒载体，其特征在于，所述TCRα的恒定区发生突变，所述突变包括所述TCRα恒定区包含半胱氨酸替换；The viral vector according to claim 7, wherein the constant region of the TCRα is mutated, and the mutation includes a cysteine substitution in the TCRα constant region; 优选地，所述TCRα恒定区源自人或小鼠的TCRα恒定区，所述人的TCRα恒定区包含如SEQ ID NO:39所示的氨基酸序列，所述小鼠的TCRα恒定区包含如SEQ ID NO:40所示的氨基酸序列；Preferably, the TCRα constant region is derived from a human or mouse TCRα constant region, the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 39, and the mouse TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 40; 所述突变包括所述人的TCRα恒定区的第47位氨基酸苏氨酸T替换为半胱氨酸C(人的TCRα恒定区变体1)或所述小鼠的TCRα恒定区的第47位氨基酸苏氨酸T替换为半胱氨酸C(小鼠的TCRα恒定区变体1)；The mutation includes replacing the 47th amino acid Threonine T of the human TCRα constant region with Cysteine C (human TCRα constant region variant 1) or replacing the 47th amino acid Threonine T of the mouse TCRα constant region with Cysteine C (mouse TCRα constant region variant 1); 所述人的TCRα恒定区变体1包含如SEQ ID NO:41所示的氨基酸序列，所述小鼠的TCRα恒定区变体1包含如SEQ ID NO:42所示的氨基酸序列。The human TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:41, and the mouse TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:42. 根据权利要求7或8所述的病毒载体，其特征在于，所述TCRβ恒定区发生突变，所述突变包括所述TCRβ恒定区包含半胱氨酸替换；The viral vector according to claim 7 or 8, characterized in that the TCRβ constant region is mutated, and the mutation includes a cysteine substitution in the TCRβ constant region; 优选地，所述TCRβ恒定区源自人的TCRβ恒定区，所述人的TCRβ恒定区包含如SEQ ID NO:43(hTRBC1)或SEQ ID NO:44(hTRBC2)所示的氨基酸序列，所述突变包括所述人的TCRβ恒定区的第56位氨基酸丝氨酸S替换为半胱氨酸C(人的TCRβ恒定区变体1)，所述人的TCRβ的恒定区变体1包含如SEQ ID NO:45或SEQ ID NO:46所示的氨基酸序列。Preferably, the TCRβ constant region is derived from a human TCRβ constant region, and the human TCRβ constant region comprises the amino acid sequence shown in SEQ ID NO: 43 (hTRBC1) or SEQ ID NO: 44 (hTRBC2), and the mutation includes replacing the 56th amino acid serine S of the human TCRβ constant region with cysteine C (human TCRβ constant region variant 1), and the human TCRβ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46. 根据权利要求7-9中任一项所述的病毒载体，其特征在于，所述TCRα恒定区发生突变，所述突变包括所述TCRα恒定区的至少一个不带电氨基酸被替换为疏水氨基酸；The viral vector according to any one of claims 7 to 9, characterized in that the TCRα constant region is mutated, and the mutation comprises replacing at least one uncharged amino acid in the TCRα constant region with a hydrophobic amino acid; 优选地，所述TCRα恒定区源自人的TCRα恒定区，所述人的TCRα恒定区包含如SEQ ID NO:39所示的氨基酸序列，所述突变包括所述人的TCRα恒定区的第115位氨基酸、118位氨基酸、第119位氨基酸中的至少一个氨基酸被疏水氨基酸替换；Preferably, the TCRα constant region is derived from a human TCRα constant region, the human TCRα constant region comprising the amino acid sequence as shown in SEQ ID NO: 39, and the mutation comprises replacement of at least one of amino acids 115, 118, and 119 of the human TCRα constant region with a hydrophobic amino acid; 更优选地，所述突变包括所述人的TCRα恒定区包含至少一种以下突变：第115位氨基酸丝氨酸S替换为亮氨酸L(S115T)、第118位氨基酸甘氨酸G替换为缬氨酸V(G118V)、第119位氨基酸苯丙氨酸F替换为亮氨酸L(F119L)；More preferably, the mutations include at least one of the following mutations in the human TCRα constant region: substitution of amino acid serine S at position 115 with leucine L (S115T), substitution of amino acid glycine G at position 118 with valine V (G118V), and substitution of amino acid phenylalanine F at position 119 with leucine L (F119L); 更进一步优选地，所述突变包括所述人的TCRα恒定区的第115位氨基酸丝氨酸S替换为亮氨酸L、第118位氨基酸甘氨酸G替换为缬氨酸V和第119位氨基酸苯丙氨酸F替换为亮氨酸L(人的TCRα恒定区变体2)，所述人的TCRα恒定区变体2包含如SEQ ID NO:47所示的氨基酸序列。Further preferably, the mutation includes replacing the 115th amino acid serine S of the human TCRα constant region with leucine L, replacing the 118th amino acid glycine G with valine V, and replacing the 119th amino acid phenylalanine F with leucine L (human TCRα constant region variant 2), and the human TCRα constant region variant 2 comprises the amino acid sequence shown in SEQ ID NO:47. 根据权利要求7-10中任一项所述的病毒载体，其特征在于，所述TCRα恒定区源自人的TCRα恒定区，所述人的TCRα恒定区包含如SEQ ID NO:39所示的氨基酸序列；所述人的TCRα恒定区发生突变，所述突变包括所述人的TCRα恒定区的第47位氨基酸苏氨酸T替换为半胱氨酸C、第115位氨基酸丝氨酸S替换为亮氨酸L、第118位氨基酸甘氨酸G替换为缬氨酸V和第119位氨基酸苯丙氨酸F替换为亮氨酸L(人的TCRα恒定区变体3)，所述人的TCRα恒定区变体3包含如SEQ ID NO:48所示的氨基酸序列；The viral vector according to any one of claims 7 to 10, characterized in that the TCRα constant region is derived from a human TCRα constant region, the human TCRα constant region comprising the amino acid sequence shown in SEQ ID NO: 39; the human TCRα constant region undergoes a mutation, the mutation comprising substitution of amino acid threonine T at position 47 of the human TCRα constant region with cysteine C, amino acid serine S at position 115 with leucine L, amino acid glycine G at position 118 with valine V, and amino acid phenylalanine F at position 119 with leucine L (human TCRα constant region variant 3), the human TCRα constant region variant 3 comprising the amino acid sequence shown in SEQ ID NO: 48; 优选地，所述TCRα恒定区和所述TCRβ恒定区源自人的TCRα恒定区和人的TCRβ恒定区；所述人的TCRα恒定区包含如SEQ ID NO:39所示的氨基酸序列，所述人的TCRβ恒定区包含如SEQ ID NO:43或SEQ ID NO:44所示的氨基酸序列；所述人的TCRα恒定区和所述人的TCRβ恒定区发生突变，所述突变包括所述人的TCRα恒定区的第47位氨基酸替换为半胱氨酸、第115位氨基酸丝氨酸S替换为亮氨酸L、第118位氨基酸甘氨酸G替换为缬氨酸V和第119位氨基酸苯丙氨酸F替换为亮氨酸L(人的TCRα恒定区变体3)，以及所述人的TCRβ恒定区的第56位氨基酸丝氨酸S替换为半胱氨酸C(人的TCRβ恒定区变体1)；所述人的TCRα恒定区变体3包含如SEQ ID NO:48所示的氨基酸序列，所述人的TCRβ的恒定区变体1包含如SEQ ID NO:45或SEQ ID NO:46所示的氨基酸序列。Preferably, the TCRα constant region and the TCRβ constant region are derived from human TCRα constant region and human TCRβ constant region; the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO:39, and the human TCRβ constant region comprises the amino acid sequence shown in SEQ ID NO:43 or SEQ ID NO:44; the human TCRα constant region and the human TCRβ constant region are mutated, and the mutation includes the replacement of the 47th amino acid of the human TCRα constant region with cysteine and the 115th amino acid serine S The human TCRα constant region variant 3 comprises the amino acid sequence shown in SEQ ID NO: 48, the human TCRβ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46. 根据权利要求1-11中任一项所述的病毒载体，其特征在于，所述TSP还可包含所述TCR/CD3复合体亚基的铰链区。The viral vector according to any one of claims 1 to 11, characterized in that the TSP further comprises the hinge region of the TCR/CD3 complex subunit. 根据权利要求1-12中任一项所述的病毒载体，其特征在于，所述抗原结合区通过连接肽可操作地连接至所述TSP的N-末端。The viral vector according to any one of claims 1 to 12, characterized in that the antigen binding region is operably linked to the N-terminus of the TSP via a connecting peptide. 根据权利要求13所述的病毒载体，其特征在于，所述连接肽包括柔性连接肽。The viral vector according to claim 13, wherein the connecting peptide comprises a flexible connecting peptide. 根据权利要求14所述的病毒载体，其特征在于，所述柔性连接肽选自(G4S)n连接肽和氨基酸序列如SEQ ID NO:23所示的连接肽1；其中，n＝1至4；The viral vector according to claim 14, wherein the flexible connecting peptide is selected from a (G 4 S) n connecting peptide and a connecting peptide 1 whose amino acid sequence is shown in SEQ ID NO: 23; wherein n=1 to 4; 优选地，所述柔性连接肽为连接肽1或(G4S)3连接肽(连接肽2)。Preferably, the flexible connecting peptide is connecting peptide 1 or (G 4 S) 3 connecting peptide (connecting peptide 2). 根据权利要求1-15中任一项所述的病毒载体，其特征在于，所述抗原结合区结合疾病相关抗原。The viral vector according to any one of claims 1 to 15, wherein the antigen binding region binds to a disease-associated antigen. 根据权利要求16所述的病毒载体，其特征在于，所述疾病选自癌症和自身免疫性疾病；所述癌症包括血液癌和实体癌。The viral vector according to claim 16, characterized in that the disease is selected from cancer and autoimmune diseases; and the cancer includes blood cancer and solid cancer. 根据权利要求17所述的病毒载体，其特征在于，所述疾病相关抗原选自：The viral vector according to claim 17, wherein the disease-associated antigen is selected from: TSHR、CD2、CD3、CD4、CD5、CD7、CD8、CD14、CD15、CD19、CD20、CD21、CD23、CD24、CD25、CD28、CD37、CD38、CD40、CD40L、CD44、CD46、CD47、CD52、CD54、CD56、CD70、CD73、CD80、CD97、CD123、CD22、CD126、CD138、DR4、DR5、TAC、TEM1/CD248、VEGF、GUCY2C、EGP40、EGP-2、EGP-4、CDL33、IFNAR1、DLL3、kappa轻链、TIM3、tEGFR、IL-22Ra、IL-2、ErbB3、ErbB4、MUC16、MAGE-3、MAGE-A6、NKG2DL、BAFF-R、CD30、CD171、CS-1、CLL-1、CD33、EGFRvⅢ、GD2、GD3、BCMA、GPRC5D、Tn Ag、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、uPAR、GCC(鸟苷酸环化酶C)、EPCAM、Nectin4、B7H3、KIT、IL-13Ra2、间皮素、IL-1Ra、PSCA、PRSS21、VEGFR2、Lewis-Y、CD24、PDGFR-β、SSEA-4、CD20、AFP、Folate受体α、Her2/neu/ERBB2、MUC1、EGFR、CS1、CD138、NCAM、Claudin18.2、Prostase、PAP、ELF2M、Ephrin B2、IGF-Ⅰ受体、CAIX、LMP2、gploo、bcr-abl、酪氨酸酶、EphA2、Fucosyl GM1、sLe、GM3、TGS5、HMWMAA、o-乙酰基-GD2、Folate受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD179a、ALK、多聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-1a、MAGE-A1、豆英蛋白、HPV E6/E7、MAGE-A4、MART-1、WT-1、ETV6-AML、精子蛋白17、XAGE1、Tie2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、前列腺特异性蛋白、存活蛋白和端粒酶、PCTA-1/Galectin 8、MelanA/MARTI、Ras突变体，hTERT、肉瘤易位断点、ML-IAP、TMPRSS2-ETS融合基因/ERG、NA17、PAX3、雄激素受体、CyclinB1、MYCN、RhoC、TRP-2、CYP1B 1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠道羧酸酯酶、mut hsp70-2、CD79A、CD79B、ASGPR、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLLI、PD1、PDL1、PDL2、TGFβ、APRIL、MSLN和NKG2D中的至少一种；TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38 , CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138 , DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM 3. tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, uPAR, GCC (guanylate cyclase C), EPCAM, Nectin4, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Le wis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2/neu/ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-Ⅰ receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6 K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6/E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1/ at least one of Galectin 8, MelanA/MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2-ETS fusion gene/ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79A, CD79B, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, and NKG2D; 优选地，所述疾病相关抗原选自CD19、CD20、CD33、MSLN、BCMA、CEA、uPAR、DLL3、GCC、Nectin4、HER2、Claudin18.2和GUCY2C中的至少一种。Preferably, the disease-associated antigen is selected from at least one of CD19, CD20, CD33, MSLN, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2 and GUCY2C. 根据权利要求1-18中任一项所述的病毒载体，其特征在于，所述抗原结合区包含抗体或其抗原结合片段和/或配体或其受体结合片段，所述抗体或其抗原结合片段选自免疫球蛋白(全长抗体)、半抗体、Fab、Fab'、F(ab')2、Fv片段、单链可变区片段(scFv)、二硫键稳定性抗体(dsFv)、抗体的重链可变区(VH)或轻链可变区(VL)、由VH和CH1结构域组成的Fd片段、线性抗体和单域抗体(纳米抗体VHH)中的至少一种。The viral vector according to any one of claims 1 to 18, characterized in that the antigen-binding region comprises an antibody or an antigen-binding fragment thereof and/or a ligand or a receptor-binding fragment thereof, and the antibody or antigen-binding fragment thereof is selected from at least one of an immunoglobulin (full-length antibody), a half antibody, Fab, Fab', F(ab') 2 , an Fv fragment, a single-chain variable region fragment (scFv), a disulfide-stabilized antibody (dsFv), an antibody heavy chain variable region (VH) or a light chain variable region (VL), an Fd fragment consisting of a VH and a CH1 domain, a linear antibody, and a single-domain antibody (Nanobody VHH). 根据权利要求1-19中任一项所述的病毒载体，其特征在于，所述TCP不包含信号肽。The viral vector according to any one of claims 1 to 19, wherein the TCP does not comprise a signal peptide. 根据权利要求1-19中任一项所述的病毒载体，其特征在于，所述TCP包含信号肽。The viral vector according to any one of claims 1 to 19, wherein the TCP comprises a signal peptide. 根据权利要求21所述的病毒载体，其特征在于，所述信号肽选自以下信号肽：CD8α信号肽、CD28信号肽、IgG信号肽、HLA-A信号肽、CD3γ信号肽、CD3δ信号肽、CD3ζ信号肽和CD3ε信号肽。The viral vector according to claim 21, characterized in that the signal peptide is selected from the following signal peptides: CD8α signal peptide, CD28 signal peptide, IgG signal peptide, HLA-A signal peptide, CD3γ signal peptide, CD3δ signal peptide, CD3ζ signal peptide and CD3ε signal peptide. 根据权利要求22所述的病毒载体，其特征在于，所述信号肽为CD8α信号肽。The viral vector according to claim 22, characterized in that the signal peptide is a CD8α signal peptide. 据权利要求1-23中任一项所述的病毒载体，其特征在于，所述TCP还包含共刺激信号传导结构域；The viral vector according to any one of claims 1 to 23, wherein the TCP further comprises a co-stimulatory signaling domain; 优选地，所述共刺激信号传导结构域源自至少一种以下蛋白的共刺激信号传导结构域：Preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of at least one of the following proteins: CD28、4-1BB、CD27、CD2、CD3、CD7、CD8、CD8α、CD8β、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcαRlγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-l、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD40和MyD88；优选的，所述共刺激信号传导结构域源自4-1BB和/或CD28的共刺激信号传导结构域；CD28, 4-1BB, CD27, CD2, CD3, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαR1γ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88; preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of 4-1BB and/or CD28; 更优选地，所述共刺激信号传导结构域源自4-1BB和CD28的共刺激信号传导结构域。More preferably, the costimulatory signaling domain is derived from the costimulatory signaling domains of 4-1BB and CD28. 根据权利要求1-24中任一项所述的病毒载体，其特征在于，所述TCP在T细胞中表达时(a)并入内源性TCR/CD3复合体或TCR/CD3复合体亚基或其功能性片段中；或(b)与内源性TCR/CD3复合体或内源性TCR/CD3复合体亚基或其功能性片段发生功能性相互作用。The viral vector according to any one of claims 1 to 24, characterized in that when the TCP is expressed in T cells, it is (a) incorporated into the endogenous TCR/CD3 complex or TCR/CD3 complex subunit or a functional fragment thereof; or (b) functionally interacts with the endogenous TCR/CD3 complex or an endogenous TCR/CD3 complex subunit or a functional fragment thereof. 根据权利要求1-25中任一项所述的病毒载体，其特征在于，所述TCP在T细胞以外的细胞中不出膜表达或所述TCP在T细胞以外的细胞中出膜表达的效率相对于所述TCP在T细胞中出膜表达的效率降低。The viral vector according to any one of claims 1 to 25, characterized in that the TCP is not expressed outside the membrane in cells other than T cells or the efficiency of the membrane expression of the TCP in cells other than T cells is reduced relative to the efficiency of the membrane expression of the TCP in T cells. 根据权利要求1-26中任一项所述的病毒载体，其特征在于，所述T细胞活化信号分子包括T细胞活化初级信号分子。The viral vector according to any one of claims 1 to 26, characterized in that the T cell activation signal molecule comprises a T cell activation primary signal molecule. 根据权利要求27所述的病毒载体，其特征在于，所述T细胞活化初级信号分子结合TCR/CD3复合体亚基和TCR/CD3复合体亚基功能性片段中的至少一种；所述TCR/CD3复合体亚基选自CD3ε、CD3γ、CD3δ、CD3ζ、TCRγ、TCRδ、TCRα和TCRβ中的至少一种。The viral vector according to claim 27, characterized in that the T cell activation primary signal molecule binds to at least one of a TCR/CD3 complex subunit and a TCR/CD3 complex subunit functional fragment; the TCR/CD3 complex subunit is selected from at least one of CD3ε, CD3γ, CD3δ, CD3ζ, TCRγ, TCRδ, TCRα and TCRβ. 根据权利要求28所述的病毒载体，其特征在于，所述TCR/CD3复合体亚基是人的TCR/CD3复合体亚基。The viral vector according to claim 28, wherein the TCR/CD3 complex subunit is a human TCR/CD3 complex subunit. 根据权利要求28或29所述的病毒载体，其特征在于，所述T细胞活化初级信号分子包括抗CD3抗体或其抗原结合片段。The viral vector according to claim 28 or 29, characterized in that the T cell activation primary signal molecule comprises an anti-CD3 antibody or an antigen-binding fragment thereof. 根据权利要求30所述的病毒载体，其特征在于，所述抗CD3抗体选自OKT3、UCHT1、YTH12.5和TR66中的至少一种。The viral vector according to claim 30, characterized in that the anti-CD3 antibody is selected from at least one of OKT3, UCHT1, YTH12.5 and TR66. 根据权利要求30所述的病毒载体，其特征在于，所述抗CD3抗体是SP34。The viral vector according to claim 30, characterized in that the anti-CD3 antibody is SP34. 根据权利要求30-32中任一项所述的病毒载体，其特征在于，所述抗CD3抗体或其抗原结合片段不可结合所述TSP。The viral vector according to any one of claims 30 to 32, wherein the anti-CD3 antibody or antigen-binding fragment thereof cannot bind to the TSP. 根据权利要求30-33中任一项所述的病毒载体，其特征在于，所述抗CD3抗体是抗CD3ε抗体，所述TSP不是CD3ε、或其功能性片段、或其变体、或其变体的功能性片段。The viral vector according to any one of claims 30 to 33, characterized in that the anti-CD3 antibody is an anti-CD3ε antibody, and the TSP is not CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof. 根据权利要求34所述的病毒载体，其特征在于，所述抗CD3ε抗体或其抗原结合片段是UCHT1或其抗原结合片段；The viral vector according to claim 34, wherein the anti-CD3ε antibody or antigen-binding fragment thereof is UCHT1 or an antigen-binding fragment thereof; 优选地，所述UCHT1的抗原结合片段是scFv(UCHT1-scFv)；Preferably, the antigen-binding fragment of UCHT1 is a scFv (UCHT1-scFv); 更优选地，所述UCHT1-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:81-83所示，所述UCHT1-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:84-86所示。More preferably, the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NO: 81-83, respectively, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NO: 84-86, respectively. 根据权利要求34所述的病毒载体，其特征在于，所述抗CD3ε抗体或其抗原结合片段是OKT3或其抗原结合片段；The viral vector according to claim 34, wherein the anti-CD3ε antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof; 优选地，所述OKT3的抗原结合片段是scFv(OKT3-scFv)；Preferably, the antigen-binding fragment of OKT3 is scFv (OKT3-scFv); 更优选地，所述OKT3-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:104-106所示，所述OKT3-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:107-109所示。More preferably, the amino acid sequences of the HCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NO: 104-106, respectively, and the amino acid sequences of the LCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NO: 107-109, respectively. 根据权利要求34所述的病毒载体，其特征在于，所述抗CD3ε抗体或其抗原结合片段是SP34或其抗原结合片段；The viral vector according to claim 34, wherein the anti-CD3ε antibody or antigen-binding fragment thereof is SP34 or an antigen-binding fragment thereof; 优选地，所述SP34的抗原结合片段是scFv(SP34-scFv)；Preferably, the antigen-binding fragment of SP34 is scFv (SP34-scFv); 更优选地，所述SP34-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:113-115所示，所述SP34-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:116-118所示。More preferably, the amino acid sequences of the HCDR1-3 regions of the SP34-scFv are shown as SEQ ID NO: 113-115, respectively, and the amino acid sequences of the LCDR1-3 regions of the SP34-scFv are shown as SEQ ID NO: 116-118, respectively. 根据权利要求34-37中任一项所述病毒载体，其特征在于，所述TSP是：The viral vector according to any one of claims 34 to 37, wherein the TSP is: (a)CD3γ、或其功能性片段、或其变体、或其变体的功能性片段；(a) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; (b)CD3δ、或其功能性片段、或其变体、或其变体的功能性片段；或(b) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or (c)CD3ζ，或其功能性片段、或其变体、或其变体的功能性片段。(c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof. 根据权利要求30-33中任一项所述的病毒载体，其特征在于，所述抗CD3抗体是抗CD3γ抗体，所述TSP不是CD3γ、或其功能性片段、或其变体、或其变体的功能性片段。The viral vector according to any one of claims 30 to 33, characterized in that the anti-CD3 antibody is an anti-CD3γ antibody, and the TSP is not CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof. 根据权利要求39所述病毒载体，其特征在于，所述TSP是：The viral vector according to claim 39, wherein the TSP is: (a)CD3ε、或其功能性片段、或其变体、或其变体的功能性片段；(a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; (b)CD3δ、或其功能性片段、或其变体、或其变体的功能性片段；或(b) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or (c)CD3ζ，或其功能性片段、或其变体、或其变体的功能性片段。(c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof. 根据权利要求30-33中任一项所述的病毒载体，其特征在于，所述抗CD3抗体是抗CD3δ抗体，所述TSP不是CD3δ、或其功能性片段、或其变体、或其变体的功能性片段。The viral vector according to any one of claims 30 to 33, characterized in that the anti-CD3 antibody is an anti-CD3δ antibody, and the TSP is not CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof. 根据权利要求41所述的病毒载体，其特征在于，所述抗CD3δ抗体或其抗原结合片段是TR66或其抗原结合片段；The viral vector according to claim 41, wherein the anti-CD3δ antibody or antigen-binding fragment thereof is TR66 or an antigen-binding fragment thereof; 优选地，所述TR66的抗原结合片段是scFv(TR66-scFv)。Preferably, the antigen-binding fragment of TR66 is scFv (TR66-scFv). 根据权利要求41或42所述病毒载体，其特征在于，所述TSP是：The viral vector according to claim 41 or 42, wherein the TSP is: (a)CD3ε、或其功能性片段、或其变体、或其变体的功能性片段；(a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; (b)CD3γ、或其功能性片段、或其变体、或其变体的功能性片段；或(b) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or (c)CD3ζ，或其功能性片段、或其变体、或其变体的功能性片段。(c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof. 根据权利要求30-33中任一项所述的病毒载体，其特征在于，所述抗CD3抗体是抗CD3ζ抗体，所述TSP不是CD3ζ、或其功能性片段、或其变体、或其变体的功能性片段。The viral vector according to any one of claims 30 to 33, characterized in that the anti-CD3 antibody is an anti-CD3ζ antibody, and the TSP is not CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof. 根据权利要求44所述的病毒载体，其特征在于，所述抗CD3ζ抗体或其抗原结合片段是YTH12.5或其抗原结合片段；The viral vector according to claim 44, wherein the anti-CD3ζ antibody or antigen-binding fragment thereof is YTH12.5 or an antigen-binding fragment thereof; 优选地，所述YTH12.5的抗原结合片段是scFv(YTH12.5-scFv)。Preferably, the antigen-binding fragment of YTH12.5 is scFv (YTH12.5-scFv). 根据权利要求44或45所述病毒载体，其特征在于，所述TSP是：The viral vector according to claim 44 or 45, characterized in that the TSP is: (a)CD3ε、或其功能性片段、或其变体、或其变体的功能性片段；(a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; (b)CD3γ、或其功能性片段、或其变体、或其变体的功能性片段；或(b) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or (c)CD3δ，或其功能性片段、或其变体、或其变体的功能性片段。(c) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof. 根据权利要求1-46中任一项所述的病毒载体，其特征在于，所述T细胞活化信号分子还包括T细胞活化次级信号分子。The viral vector according to any one of claims 1-46, characterized in that the T cell activation signal molecule further includes a T cell activation secondary signal molecule. 根据权利要求47所述的病毒载体，其特征在于，所述T细胞活化次级信号分子结合CD28。The viral vector according to claim 47, wherein the T cell activation secondary signal molecule binds to CD28. 根据权利要求48所述的病毒载体，其特征在于，所述CD28是人的CD28。The viral vector according to claim 48, wherein the CD28 is human CD28. 根据权利要求48或49所述的病毒载体，其特征在于，所述T细胞活化次级信号分子选自抗CD28抗体或其抗原结合片段和CD28配体或其受体结合片段中的至少一种。The viral vector according to claim 48 or 49, characterized in that the T cell activation secondary signal molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof. 根据权利要求50所述的病毒载体，其特征在于，所述CD28配体或其受体结合片段包括CD80或其受体结合片段和CD86或其受体结合片段。The viral vector according to claim 50, characterized in that the CD28 ligand or its receptor binding fragment comprises CD80 or its receptor binding fragment and CD86 or its receptor binding fragment. 根据权利要求51所述的病毒载体，其特征在于，所述T细胞活化次级信号分子包括抗CD28抗体或其抗原结合片段；The viral vector according to claim 51, wherein the T cell activation secondary signal molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof; 优选地，所述抗CD28抗体或其抗原结合片段是源自15E8的scFv(15E8-scFv)，所述15E8-scFv的氨基酸序列如SEQ ID NO:17所示；所述15E8-scFv的HCDR1-3区的氨基酸序列分别如SEQ ID NO:93-95所示，所述15E8-scFv的LCDR1-3区的氨基酸序列分别如SEQ ID NO:96-98所示。Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is a scFv derived from 15E8 (15E8-scFv), and the amino acid sequence of the 15E8-scFv is shown in SEQ ID NO: 17; the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO: 93-95, respectively, and the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO: 96-98, respectively. 根据权利要求48-52中任一项所述的病毒载体，其特征在于，所述T细胞活化次级信号分子还包括选自ICOS配体(ICOSL)或其受体结合片段、4-1BB配体(4-1BBL)或其受体结合片段和OX40配体(OX40L)或其受体结合片段中的至少一种配体或其受体结合片段。The viral vector according to any one of claims 48 to 52, characterized in that the T cell activation secondary signal molecule further comprises at least one ligand or its receptor binding fragment selected from ICOS ligand (ICOSL) or its receptor binding fragment, 4-1BB ligand (4-1BBL) or its receptor binding fragment, and OX40 ligand (OX40L) or its receptor binding fragment. 根据权利要求1-53中任一项所述的病毒载体，其特征在于，所述T细胞活化信号分子直接地或间接地与跨膜多肽(Transmembrane Polypeptide)相连，展示在所述病毒载体的表面。The viral vector according to any one of claims 1-53 is characterized in that the T cell activation signal molecule is directly or indirectly connected to a transmembrane polypeptide and displayed on the surface of the viral vector. 根据权利要求54所述的病毒载体，其特征在于，所述跨膜多肽选自以下蛋白的跨膜区：The viral vector according to claim 54, wherein the transmembrane polypeptide is selected from the transmembrane regions of the following proteins: CD2、CD3、CD4、CD5、CD7、CD8、CD8α、CD8β、CD9、CD16、CD22、CD27、CD28、CD28H、CD30、CD33、CD37、CD40、CD45、CD64、CD80、CD86、CD84、CD154、CD166、CD226、CD244、4-1BB、OX40、ICOS、ICAM-1、CTLA-4、PD-1、LAG-3、GITR、HVEM、DAP10、DAP12、TIM-1、LIGHT、ICOS、OX40、2B4、BTLA、DNAM-1、DR3、FcERIγ、IL7、IL12、IL15、SLAM、KIR2DL4、KIR2DS1、KIR2DS2、NKG2C、NKG2D和CS1；CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD 37. CD40, CD45, CD64, CD80, CD86, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; 优选地，所述跨膜多肽是CD8α跨膜区。Preferably, the transmembrane polypeptide is the CD8α transmembrane region. 根据权利要求54或55所述的病毒载体，其特征在于，所述T细胞活化信号分子通过连接结构域，间接地与所述跨膜多肽相连，展示在所述病毒载体的表面；The viral vector according to claim 54 or 55, characterized in that the T cell activation signaling molecule is indirectly connected to the transmembrane polypeptide through a linker domain and is displayed on the surface of the viral vector; 优选地，所述连接结构域选自：Preferably, the linker domain is selected from: (a)免疫球蛋白铰链区，所述免疫球蛋白铰链区选自野生型或经修饰的IgG1、IgG2、IgG3、IgG4、IgA和IgD铰链区；(a) an immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge region; (b)铰链区，所述铰链区选自以下蛋白的野生型或经修饰的铰链区：CD28、CD7、CD8、CD8α、CD8β、CD3、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154；(b) a hinge region selected from the wild-type or modified hinge region of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (c)Fc结构域的全部或一部分，所述Fc结构域选自CH1结构域、CH2结构域和CH3结构域中的一种或多种；(c) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; (d)Ⅱ型C-凝集素的茎区，所述Ⅱ型C-凝集素选自CD23、CD69、CD72、CD94、NKG2A和NKG2D的茎区；和(d) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and (e)柔性连接肽；(e) flexible linker peptide; 更优选地，所述连接结构域是CD8α铰链区。More preferably, the connecting domain is the CD8α hinge region. 根据权利要求1-56中任一项所述的病毒载体，其特征在于，所述病毒载体表面包含糖蛋白，所述糖蛋白选自水疱性口炎病毒属毒株的包膜糖蛋白及其变体、狒狒内源性逆转录病毒BaEV的包膜糖蛋白及其变体、猫内源性逆转录病毒的包膜糖蛋白RD114及其变体和长臂猿白血病病毒的包膜糖蛋白GALV及其变体。The viral vector according to any one of claims 1 to 56, characterized in that the surface of the viral vector comprises a glycoprotein, and the glycoprotein is selected from the envelope glycoprotein of the vesicular stomatitis virus strain and its variants, the envelope glycoprotein of the baboon endogenous retrovirus BaEV and its variants, the envelope glycoprotein RD114 of the feline endogenous retrovirus and its variants, and the envelope glycoprotein GALV of the gibbon ape leukemia virus and its variants. [根据细则26改正 18.02.2025]
根据权利要求57所述的病毒载体，其特征在于，所述跨膜多肽是所述糖蛋白，所述糖蛋白直接地或间接地连接至所述T细胞活化信号分子；[Corrected 18.02.2025 in accordance with Rule 26]
The viral vector according to claim 57, wherein the transmembrane polypeptide is the glycoprotein, and the glycoprotein is directly or indirectly linked to the T cell activation signal molecule; 优选地，所述糖蛋白通过多肽连接子(Polypeptide Linker)，间接地连接至所述T细胞活化信号分子。Preferably, the glycoprotein is indirectly connected to the T cell activation signal molecule through a polypeptide linker. 根据权利要求58所述的病毒载体，其特征在于，所述T细胞活化初级信号分子直接地或间接地连接至所述T细胞活化次级信号分子；The viral vector according to claim 58, wherein the T cell activation primary signal molecule is directly or indirectly linked to the T cell activation secondary signal molecule; 优选地，所述T细胞活化初级信号分子通过多肽连接子，间接地连接至所述T细胞活化次级信号分子。Preferably, the T cell activation primary signal molecule is indirectly linked to the T cell activation secondary signal molecule via a polypeptide linker. 根据权利要求58或59所述的病毒载体，其特征在于，所述多肽连接子是柔性连接肽；The viral vector according to claim 58 or 59, wherein the polypeptide linker is a flexible connecting peptide; 优选地，所述柔性连接肽选自(G4S)n连接肽、连接肽1：GSTSGSGKPGSGEGSTKG(SEQ ID NO:23)和连接肽3：GSSGGSGGGGSGGGGSGGGGSSG(SEQ ID NO:63)；其中，n＝1至4。Preferably, the flexible connecting peptide is selected from (G 4 S) n connecting peptide, connecting peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23) and connecting peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 63); wherein n=1 to 4. 根据权利要求58-60中任一项所述的病毒载体，其特征在于，The viral vector according to any one of claims 58 to 60, characterized in that (a)所述糖蛋白通过(G4S)n连接肽，间接地连接至所述抗CD3抗体或其抗原结合片段，所述抗CD3抗体或其抗原结合片段通过(G4S)n连接肽，间接地连接至(i)所述抗CD28抗体或其抗原结合片段、(ii)CD80胞外域(iii)或CD86胞外域；和/或(a) the glycoprotein is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a ( G4S ) n linker peptide, and the anti-CD3 antibody or antigen-binding fragment thereof is indirectly linked to ( i ) the anti-CD28 antibody or antigen-binding fragment thereof, (ii) the CD80 extracellular domain, (iii) or the CD86 extracellular domain via a (G4S)n linker peptide; and/or (b)所述糖蛋白通过(G4S)n连接肽，间接地连接至(i)所述抗CD28抗体或其抗原结合片段、(ii)CD80胞外域(iii)或CD86胞外域；所述抗CD28抗体或其抗原结合片段、CD80胞外域或CD86胞外域通过(G4S)n连接肽，间接地连接至所述抗CD3抗体或其抗原结合片段；(b) the glycoprotein is indirectly linked to (i) the anti-CD28 antibody or antigen-binding fragment thereof, (ii) the CD80 extracellular domain, (iii) the CD86 extracellular domain via a ( G4S ) n connecting peptide; the anti-CD28 antibody or antigen-binding fragment thereof, the CD80 extracellular domain, or the CD86 extracellular domain is indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof via a ( G4S ) n connecting peptide; 优选地，n＝3；Preferably, n=3; 优选地，所述抗CD3抗体或其抗原结合片段是所述UCHT1-scFv；Preferably, the anti-CD3 antibody or antigen-binding fragment thereof is the UCHT1-scFv; 优选地，所述抗CD28抗体或其抗原结合片段是所述15E8-scFv。Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is the 15E8-scFv. 根据权利要求57-61中任一项所述的病毒载体，其特征在于，所述糖蛋白选自水疱性口炎病毒属毒株的包膜糖蛋白及其变体。The viral vector according to any one of claims 57 to 61, characterized in that the glycoprotein is selected from the envelope glycoprotein of a vesicular stomatitis virus strain and its variants. 根据权利要求62所述的病毒载体，其特征在于，所述水疱性口炎病毒属毒株的包膜糖蛋白及其变体包括以下包膜糖蛋白及其变体：水疱性口炎病毒属Indiana毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Cocal毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Maraba毒株的包膜糖蛋及其变体、水疱性口炎病毒属Morreton毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Alagoas毒株的包膜糖蛋白及其变体、水疱性口炎病毒属New Jersey毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Carajas毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Chandipura毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Eptesicus毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Isfahan毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Jurona毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Malpais毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Perinet毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Piry毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Radi毒株的包膜糖蛋白及其变体、水疱性口炎病毒属Rhinolopus毒株的包膜糖蛋白及其变体和水疱性口炎病毒属Yug Bogdanovac毒株的包膜糖蛋白及其变体。The viral vector according to claim 62 is characterized in that the envelope glycoprotein and variants thereof of the vesicular stomatitis virus strain include the following envelope glycoproteins and variants thereof: envelope glycoprotein and variants of the Indiana strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants of the Cocal strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants of the Maraba strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants of the Morreton strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants of the Alagoas strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants of the New Jersey strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants of the Carajas strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants of the Chandipur strain of the vesicular stomatitis virus genus a strain and its variants, the envelope glycoprotein of the Eptesicus strain and its variants, the envelope glycoprotein of the Isfahan strain and its variants, the envelope glycoprotein of the Jurona strain and its variants, the envelope glycoprotein of the Malpais strain and its variants, the envelope glycoprotein of the Perinet strain and its variants, the envelope glycoprotein of the Piry strain and its variants, the envelope glycoprotein of the Radi strain and its variants, the envelope glycoprotein of the Rhinolopus strain and its variants, and the envelope glycoprotein of the Yug Bogdanovac strain and its variants. 根据权利要求63所述的病毒载体，其特征在于，所述糖蛋白为水疱性口炎病毒属Indiana毒株或Cocal毒株的包膜糖蛋白或其变体，所述糖蛋白的胞外域包含如SEQ ID NO:1或SEQ ID NO:2所示、或与如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列。The viral vector according to claim 63 is characterized in that the glycoprotein is the envelope glycoprotein of the Indiana strain or Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or has at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. 根据权利要求57-64中任一项所述的病毒载体，其特征在于，所述糖蛋白发生第一突变，使所述糖蛋白结合糖蛋白受体的能力相对于发生所述第一突变前降低或丧失。The viral vector according to any one of claims 57 to 64, characterized in that the glycoprotein undergoes a first mutation, which reduces or loses the ability of the glycoprotein to bind to a glycoprotein receptor relative to before the first mutation occurs. 根据权利要求65所述的病毒载体，其特征在于，所述糖蛋白选自水疱性口炎病毒属毒株的包膜糖蛋白及其变体。The viral vector according to claim 65, characterized in that the glycoprotein is selected from the envelope glycoprotein of a vesicular stomatitis virus strain and its variants. 根据权利要求66所述的病毒载体，其特征在于，所述糖蛋白为水疱性口炎病毒属Indiana毒株或Cocal毒株的包膜糖蛋白或其变体，所述糖蛋白受体为低密度脂蛋白受体LDL-R；所述糖蛋白的胞外域包含如SEQ ID NO:1或SEQ ID NO:2所示、或与如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列。The viral vector according to claim 66 is characterized in that the glycoprotein is the envelope glycoprotein of the Indiana strain or Cocal strain of the vesicular stomatitis virus or a variant thereof, and the glycoprotein receptor is the low-density lipoprotein receptor LDL-R; the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or has at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. 根据权利要求67所述的病毒载体，其特征在于，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：The viral vector according to claim 67, wherein the first mutation comprises a mutation in the amino acid sequence comprising at least one of the following amino acids: (a)位于SEQ ID NO:1或SEQ ID NO:2的第8位氨基酸的替换或缺失、第9位氨基酸的替换或缺失、第10位氨基酸的替换或缺失、第47位氨基酸的替换或缺失、第50位氨基酸的替换或缺失、第51位氨基酸的替换或缺失、第183位氨基酸的替换或缺失、第179位氨基酸的替换或缺失、第180位氨基酸的替换或缺失、第182位氨基酸的替换或缺失、第184位氨基酸的替换或缺失、第209位氨基酸的替换或缺失、第347位氨基酸的替换或缺失、第350位氨基酸的替换或缺失、第352位氨基酸的替换或缺失、第353位氨基酸的替换或缺失、第354位氨基酸的替换、第1-18位氨基酸缺失、第19-36位氨基酸缺失、第37-51位氨基酸缺失、第314-384位氨基酸缺失、第321-374位氨基酸缺失、第331-364位氨基酸缺失、第344-354位氨基酸缺失、第345-353位氨基酸缺失；和(a) substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 209 of SEQ ID NO: 1 or SEQ ID NO: 2. 347, substitution or deletion of amino acid 350, substitution or deletion of amino acid 352, substitution or deletion of amino acid 353, substitution of amino acid 354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; and (b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第8位氨基酸的替换或缺失、第9位氨基酸的替换或缺失、第10位氨基酸的替换或缺失、第47位氨基酸的替换或缺失、第50位氨基酸的替换或缺失、第51位氨基酸的替换或缺失、第183位氨基酸的替换或缺失、第179位氨基酸的替换或缺失、第180位氨基酸的替换或缺失、第182位氨基酸的替换或缺失、第184位氨基酸的替换或缺失、第209位氨基酸的替换或缺失、第347位氨基酸的替换或缺失、第350位氨基酸的替换或缺失、第352位氨基酸的替换或缺失、第353位氨基酸的替换或缺失、第354位氨基酸的替换、第1-18位氨基酸缺失、第19-36位氨基酸缺失、第37-51位氨基酸缺失、第314-384位氨基酸缺失、第321-374位氨基酸缺失、第331-364位氨基酸缺失、第344-354位氨基酸缺失、第345-353位氨基酸缺失。(b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 185, substitution or deletion of amino acid at position 186, substitution or deletion of amino acid at position 187, substitution or deletion of amino acid at position 188, substitution or deletion of amino acid at position 189, substitution or deletion of amino acid at position 190, substitution or deletion of amino acid at position 191, substitution or deletion of amino acid at position 192, substitution or deletion of amino acid at position 193, substitution or deletion of amino acid at position 194, substitution or deletion of amino acid at position 195, substitution or deletion of amino acid at position 196, substitution or deletion of amino acid at position 197, substitution or deletion of amino acid at position 198, substitution or deletion of amino acid at position 199, substitution or deletion of amino acid at position 200, substitution or deletion of amino acid at position 201, substitution or deletion of amino acid at position 202, substitution or deletion of amino acid at position 203, substitution or deletion of amino acid at position 204 The present invention also includes substitution or deletion of the amino acid at position 350, substitution or deletion of the amino acid at position 352, substitution or deletion of the amino acid at position 353, substitution of the amino acid at position 354, deletion of amino acids at positions 1-18, deletion of amino acids at positions 19-36, deletion of amino acids at positions 37-51, deletion of amino acids at positions 314-384, deletion of amino acids at positions 321-374, deletion of amino acids at positions 331-364, deletion of amino acids at positions 344-354, and deletion of amino acids at positions 345-353. 根据权利要求68所述的病毒载体，其特征在于，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：The viral vector according to claim 68, wherein the first mutation comprises a mutation in the amino acid sequence comprising at least one of the following amino acids: (a)位于SEQ ID NO:1或SEQ ID NO:2的第331-364位氨基酸缺失、第344-354位氨基酸缺失、K47的替换、K47的缺失、R354的替换；和(a) a deletion of amino acids 331-364, a deletion of amino acids 344-354, a substitution of K47, a deletion of K47, or a substitution of R354 in SEQ ID NO: 1 or SEQ ID NO: 2; and (b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第331-364位氨基酸缺失、第344-354位氨基酸缺失、K47的替换、K47的缺失、R354的替换；(b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, and substitution of R354 are located at positions equivalent to SEQ ID NO: 1 or SEQ ID NO: 2; 优选地，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a)位于SEQ ID NO:1或SEQ ID NO:2的第331-第364位氨基酸缺失、第344-354位氨基酸缺失、K47Q、R354Q、K47缺失；和(a) a deletion of amino acids 331 to 364, a deletion of amino acids 344 to 354, K47Q, R354Q, or K47 in SEQ ID NO: 1 or SEQ ID NO: 2; and (b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第331-第364位氨基酸缺失、第344-354位氨基酸缺失、K47Q、R354Q、K47缺失。(b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, amino acid deletions at positions 331 to 364, amino acid deletions at positions 344 to 354, K47Q, R354Q, and K47 deletions are located at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2. 根据权利要求69所述的病毒载体，其特征在于，所述第一突变包括所述氨基酸序列包含以下氨基酸的突变：The viral vector according to claim 69, wherein the first mutation comprises a mutation in which the amino acid sequence comprises the following amino acids: (a)位于SEQ ID NO:1或SEQ ID NO:2的第47位氨基酸赖氨酸缺失；或(a) the amino acid lysine at position 47 of SEQ ID NO: 1 or SEQ ID NO: 2 is missing; or (b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第47位氨基酸赖氨酸缺失。(b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, the lysine residue at position 47 corresponding to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is missing. 根据权利要求67或68所述的病毒载体，其特征在于，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：The viral vector according to claim 67 or 68, wherein the first mutation comprises a mutation in the amino acid sequence comprising at least one of the following amino acids: (a)位于SEQ ID NO:1的K47的替换、K47缺失、I182的替换、R354的替换、Y209的替换；(a) Substitution of K47, deletion of K47, substitution of I182, substitution of R354, and substitution of Y209 in SEQ ID NO: 1; (b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的K47的替换、K47缺失、I182的替换、R354的替换、Y209的替换；(b) After optimal global alignment with SEQ ID NO: 1, the substitutions at K47, K47 deletion, I182 substitution, R354 substitution, and Y209 substitution are equivalent to those in SEQ ID NO: 1; (c)位于SEQ ID NO:2的K47的替换、K47缺失、V182的替换、R354的替换、Y209的替换；和(c) substitution of K47, deletion of K47, substitution of V182, substitution of R354, substitution of Y209 at SEQ ID NO: 2; and (d)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的K47的替换、K47缺失、V182的替换、R354的替换、Y209的替换；(d) After optimal global alignment with SEQ ID NO: 2, the sequence includes substitutions at K47, deletion of K47, substitutions at V182, substitutions at R354, and substitutions at Y209, which are equivalent to those in SEQ ID NO: 2; 优选地，所述第一突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a)位于SEQ ID NO:1的K47Q或K47A、K47缺失、I182E或I182D、R354Q或R354A、Y209Q；(a) K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q in SEQ ID NO: 1; (b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的K47Q或K47A、K47缺失、I182E或I182D、R354Q或R354A、Y209Q；(b) After optimal global alignment with SEQ ID NO: 1, the residues located at K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, and Y209Q are equivalent to those in SEQ ID NO: 1; (c)位于SEQ ID NO:2的K47Q或K47A、K47缺失、V182E或V182D、R354Q或R354A、Y209Q；和(c) K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q at SEQ ID NO: 2; and (d)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的K47Q或K47A、K47缺失、V182E或V182D、R354Q或R354A、Y209Q。(d) After optimal global alignment with SEQ ID NO: 2, it is located at K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q equivalent to SEQ ID NO: 2. 根据权利要求66所述的病毒载体，其特征在于，所述糖蛋白的胞外域包含如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:33或SEQ ID NO:34所示的氨基酸序列。The viral vector according to claim 66, characterized in that the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 33 or SEQ ID NO: 34. 根据权利要求57-72中任一项所述的病毒载体，其特征在于，所述糖蛋白发生第二突变，使所述糖蛋白拮抗被补体失活的能力相对于发生所述第二突变前增强或不被补体失活。The viral vector according to any one of claims 57 to 72, characterized in that the glycoprotein undergoes a second mutation, so that the ability of the glycoprotein to antagonize inactivation by complement is enhanced or not inactivated by complement relative to before the second mutation occurs. 根据权利要求73所述的病毒载体，其特征在于，所述糖蛋白为水疱性口炎病毒属Indiana毒株或Cocal毒株的包膜糖蛋白或其变体，所述糖蛋白的胞外域包含如SEQ ID NO:1或SEQ ID NO:2所示、或与如SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％的同一性的氨基酸序列。The viral vector according to claim 73 is characterized in that the glycoprotein is the envelope glycoprotein of the Indiana strain or Cocal strain of the vesicular stomatitis virus or a variant thereof, and the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. 根据权利要求74所述的病毒载体，其特征在于，所述第二突变包括所述氨基酸序列包含至少一个以下氨基酸的突变：The viral vector according to claim 74, wherein the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a)位于SEQ ID NO:1或SEQ ID NO:2的第214位氨基酸；(a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第214位氨基酸；(b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at the amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c)位于SEQ ID NO:1或SEQ ID NO:2的第352位氨基酸；(c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第352位氨基酸；(d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e)位于SEQ ID NO:1或SEQ ID NO:2的第50位氨基酸；(e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第50位氨基酸；(f) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 50th amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (g)位于SEQ ID NO:1或SEQ ID NO:2的第146位氨基酸；和(g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第146位氨基酸；(h) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at amino acid position 146 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; 优选地，所述氨基酸的突变选自氨基酸的缺失、插入和替换中的至少一种；Preferably, the amino acid mutation is selected from at least one of deletion, insertion and substitution of amino acids; 更优选地，所述第二突变包括所述氨基酸序列包含至少一个以下氨基酸的替换：More preferably, the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids: (a)位于SEQ ID NO:1或SEQ ID NO:2的第214位氨基酸；(a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第214位氨基酸；(b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at the amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c)位于SEQ ID NO:1或SEQ ID NO:2的第352位氨基酸；(c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第352位氨基酸；(d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e)位于SEQ ID NO:1或SEQ ID NO:2的第50位氨基酸；(e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第50位氨基酸；(f) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 50th amino acid position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (g)位于SEQ ID NO:1或SEQ ID NO:2的第146位氨基酸；和(g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h)与SEQ ID NO:1或SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:1或SEQ ID NO:2的第146位氨基酸。(h) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the 146th amino acid position equivalent to SEQ ID NO: 1 or SEQ ID NO: 2. 根据权利要求75所述的病毒载体，其特征在于，所述第二突变包括如SEQ ID NO:1所示或与如SEQ ID NO:1所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％的同一性的氨基酸序列包含至少一个以下位点突变：The viral vector of claim 75, wherein the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 1, comprising at least one of the following site mutations: (a)位于SEQ ID NO:1的T214的替换、T352的替换、K50的替换、S146的替换；和(a) substitution of T214, substitution of T352, substitution of K50, and substitution of S146 in SEQ ID NO: 1; and (b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的T214的替换、T352的替换、K50的替换、S146的替换；(b) After optimal global alignment with SEQ ID NO: 1, the substitutions at T214, T352, K50, and S146 are equivalent to those in SEQ ID NO: 1; 优选地，所述第二突变包括所述氨基酸序列包含至少一个以下位点突变：Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence: (a)位于SEQ ID NO:1的T214N、T352A、K50T、S146T；和(a) T214N, T352A, K50T, S146T located in SEQ ID NO: 1; and (b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的T214N、T352A、K50T、S146T。(b) After optimal global alignment with SEQ ID NO:1, T214N, T352A, K50T, and S146T are located equivalent to SEQ ID NO:1. 根据权利要求76所述的病毒载体，其特征在于，所述第二突变包括所述氨基酸序列包含任一种以下位点突变的组合：The viral vector according to claim 76, wherein the second mutation comprises a combination of any one of the following site mutations in the amino acid sequence: (a)位于SEQ ID NO:1的(1)T214和T352的替换；或(2)T214、T352、K50和S146的替换；和(a) substitution of (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1; and (b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的(1)T214和T352的替换；或(2)T214、T352、K50和S146的替换；(b) substitutions at positions equivalent to (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1 after optimal global alignment with SEQ ID NO: 1; 优选地，所述第二突变包括所述氨基酸序列包含任一种以下位点突变的组合：Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a)位于SEQ ID NO:1的(1)T214N和T352A；或(2)T214N、T352A、K50T和S146T；和(a) (1) T214N and T352A; or (2) T214N, T352A, K50T, and S146T located in SEQ ID NO: 1; and (b)与SEQ ID NO:1最佳全局比对后，位于相当于SEQ ID NO:1的(1)T214N和T352A；或(2)T214N、T352A、K50T和S146T。(b) After optimal global alignment with SEQ ID NO:1, located at (1) T214N and T352A; or (2) T214N, T352A, K50T and S146T equivalent to SEQ ID NO:1. 根据权利要求75所述的病毒载体，其特征在于，所述第二突变包括如SEQ ID NO:2所示或与如SEQ ID NO:2所示的氨基酸序列具有至少约50％、约60％、约70％、约80％、约90％、约91％、约92％、约93、约94％、约95％、约96％、约97％、约98％或约99％的同一性的氨基酸序列包含至少一个以下位点突变：The viral vector of claim 75, wherein the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 2, comprising at least one mutation at the following sites: (a)位于SEQ ID NO:2的K214的替换、T352的替换、K50的替换、S146的替换；和(a) substitution of K214, substitution of T352, substitution of K50, and substitution of S146 in SEQ ID NO: 2; and (b)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的K214的替换、T352的替换、K50的替换、S146的替换；(b) After optimal global alignment with SEQ ID NO: 2, the substitutions at K214, T352, K50, and S146 are equivalent to those in SEQ ID NO: 2; 优选地，所述第二突变包括所述氨基酸序列包含至少一个以下位点突变：Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence: (a)位于SEQ ID NO:2的K214N、T352A、K50T、S146T；和(a) K214N, T352A, K50T, S146T located in SEQ ID NO: 2; and (b)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的K214N、T352A、K50T、S146T。(b) After optimal global alignment with SEQ ID NO: 2, K214N, T352A, K50T, and S146T are located equivalent to SEQ ID NO: 2. 根据权利要求78所述的病毒载体，其特征在于，所述第二突变包括所述氨基酸序列包含任一种以下位点突变的组合：The viral vector according to claim 78, wherein the second mutation comprises a combination of any one of the following site mutations in the amino acid sequence: (a)位于SEQ ID NO:2的(1)K214和T352的替换；或(2)K214、T352、K50和S146的替换；和(a) substitution of (1) K214 and T352; or (2) K214, T352, K50, and S146 at SEQ ID NO:2; and (b)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的(1)K214和T352的替换；或(2)K214、T352、K50和S146的替换；(b) substitutions at positions corresponding to (1) K214 and T352 of SEQ ID NO: 2 after optimal global alignment with SEQ ID NO: 2; or (2) substitutions at positions corresponding to K214, T352, K50, and S146 of SEQ ID NO: 2; 优选地，所述第二突变包括所述氨基酸序列包含任一种以下位点突变的组合：Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a)位于SEQ ID NO:2的(1)K214N和T352A；或(2)K214N、T352A、K50T和S146T；和(a) (1) K214N and T352A; or (2) K214N, T352A, K50T, and S146T at SEQ ID NO:2; and (b)与SEQ ID NO:2最佳全局比对后，位于相当于SEQ ID NO:2的(1)K214N和T352A；或(2)K214N、T352A、K50T和S146T。(b) After optimal global alignment with SEQ ID NO:2, located at (1) K214N and T352A; or (2) K214N, T352A, K50T and S146T equivalent to SEQ ID NO:2. 根据权利要求73所述的病毒载体，其特征在于，所述糖蛋白的胞外域包含如SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37或SEQ ID NO:38所示的氨基酸序列。The viral vector according to claim 73 is characterized in that the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 or SEQ ID NO: 38. 一种转导T细胞的方法，其特征在于，包括使用权利要求1-80中任一项所述的病毒载体与T细胞接触。A method for transducing T cells, comprising contacting the viral vector according to any one of claims 1 to 80 with T cells. 根据权利要求81所述的方法，其特征在于，所述T细胞选自活化的T细胞和非活化T细胞中的至少一种。The method according to claim 81, characterized in that the T cells are selected from at least one of activated T cells and non-activated T cells. 根据权利要求81或82所述的方法，其特征在于，所述接触发生在受试者体内和/或体外；The method according to claim 81 or 82, characterized in that the contacting occurs in vivo and/or in vitro of the subject; 所述受试者是被给予经所述转导T细胞的方法转导的T细胞和/或权利要求1-80中任一项所述的病毒载体的个体。The subject is an individual who has been administered T cells transduced by the method for transducing T cells and/or the viral vector according to any one of claims 1 to 80. 一种工程化T细胞，其特征在于，所述工程化T细胞由权利要求1-80中任一项所述的病毒载体接触T细胞制备所得。An engineered T cell, characterized in that the engineered T cell is prepared by contacting T cells with the viral vector described in any one of claims 1-80. 根据权利要求84所述的工程化T细胞，其特征在于，所述T细胞选自活化的T细胞和非活化T细胞中的至少一种。The engineered T cell according to claim 84, wherein the T cell is selected from at least one of an activated T cell and a non-activated T cell. 根据权利要求84或85所述的工程化T细胞，其特征在于，所述接触发生在受试者体内和/或体外；The engineered T cell according to claim 84 or 85, wherein the contact occurs in vivo and/or in vitro in a subject; 所述受试者是被给予所述工程化T细胞和/或权利要求1-80中任一项所述的病毒载体的个体。The subject is an individual administered the engineered T cells and/or the viral vector of any one of claims 1-80. 根据权利要86所述的工程化T细胞，其特征在于，所述给予选自经口、鼻、静脉内、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉、病灶内、持续释放系统和植入装置进行给予中的至少一种。The engineered T cells according to claim 86 are characterized in that the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implant device administration. 一种组合物，其特征在于，所述组合物包含药学上可接受的赋形剂或载体以及(a)权利要求1-80中任一项所述的病毒载体或(b)权利要求84-87中任一项所述的工程化T细胞。A composition, characterized in that the composition comprises a pharmaceutically acceptable excipient or carrier and (a) the viral vector according to any one of claims 1-80 or (b) the engineered T cell according to any one of claims 84-87. 权利要求1-80中任一项所述的病毒载体、权利要求84-87中任一项所述的工程化T细胞或权利要求88所述的组合物在制备预防和/或治疗癌症的药物中的应用。Use of the viral vector of any one of claims 1-80, the engineered T cell of any one of claims 84-87, or the composition of claim 88 in the preparation of a medicament for preventing and/or treating cancer. 根据权利要求89所述的应用，其特征在于，所述疾病选自癌症和自身免疫性疾病中的至少一种；所述癌症包括实体癌和血液癌。The use according to claim 89 is characterized in that the disease is selected from at least one of cancer and autoimmune diseases; and the cancer includes solid cancer and blood cancer. 根据权利要求90所述的应用，其特征在于，所述癌症为血液癌。The use according to claim 90, characterized in that the cancer is blood cancer. 根据权利要求91所述的应用，其特征在于，所述血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤、高级别B细胞淋巴瘤和多发性骨髓瘤。The use according to claim 91, characterized in that the blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin's lymphoma, high-grade B-cell lymphoma and multiple myeloma. 根据权利要求91所述的应用，其特征在于，所述血液癌选自CD19+血液癌和CD33+血液癌；The use according to claim 91, wherein the blood cancer is selected from CD19 + blood cancer and CD33 + blood cancer; 优选地，Preferably, 所述CD19+血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤和高级别B细胞淋巴瘤；The CD19 + blood cancer is selected from the group consisting of marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, and high-grade B-cell lymphoma; 所述CD33+血液癌选自多发性骨髓瘤(MM)、急性髓性白血病(AML)、慢性髓性白血病(CML)和急性单核细胞白血病(AMoL)。The CD33 + blood cancer is selected from the group consisting of multiple myeloma (MM), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and acute monocytic leukemia (AMoL). 一种治疗受试者患有的癌症或杀伤受试者癌细胞的方法，其特征在于，包括向所述受试者给予权利要求1-80中任一项所述的病毒载体、权利要求84-87中任一项所述的工程化T细胞或权利要求88所述的组合物。A method for treating cancer in a subject or killing cancer cells in a subject, characterized by comprising administering to the subject the viral vector of any one of claims 1-80, the engineered T cell of any one of claims 84-87, or the composition of claim 88. 根据权利要求94所述的方法，其特征在于，所述给予选自经口、鼻、静脉内、腹膜内、脑内(脑实质内)、脑室内、肌肉内、眼内、动脉内、门静脉、病灶内、持续释放系统和植入装置中的至少一种。The method according to claim 94 is characterized in that the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implant device. 根据权利要求94或95所述的方法，其特征在于，所述癌症包括实体癌和血液癌。The method according to claim 94 or 95, characterized in that the cancer includes solid cancer and blood cancer. 根据权利要求96所述的方法，其特征在于，所述癌症是血液癌。The method of claim 96, wherein the cancer is a blood cancer. 根据权利要求97所述的方法，其特征在于，所述血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤、高级别B细胞淋巴瘤和多发性骨髓瘤。The method of claim 97, wherein the blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, high-grade B-cell lymphoma, and multiple myeloma. 根据权利要求97所述的方法，其特征在于，所述血液癌选自CD19+血液癌和CD33+血液癌；The method of claim 97, wherein the blood cancer is selected from CD19 + blood cancer and CD33 + blood cancer; 优选地，Preferably, 所述CD19+血液癌选自边缘区淋巴瘤、弥漫性大B细胞淋巴瘤、套细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性纵隔淋巴瘤B细胞淋巴瘤、小淋巴细胞淋巴瘤、B细胞幼淋巴细胞白血病、滤泡性淋巴瘤、伯基特淋巴瘤、原发性眼内淋巴瘤、慢性淋巴细胞白血病、急性淋巴细胞白血病、毛细胞白血病、前体B淋巴细胞白血病、非霍奇金淋巴瘤和高级别B细胞淋巴瘤；The CD19 + blood cancer is selected from the group consisting of marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, and high-grade B-cell lymphoma; 所述CD33+血液癌选自多发性骨髓瘤(MM)、急性髓性白血病(AML)、慢性髓性白血病(CML)和急性单核细胞白血病(AMoL)。The CD33 + blood cancer is selected from the group consisting of multiple myeloma (MM), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and acute monocytic leukemia (AMoL).
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