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WO2025162379A1 - Polypeptide et vecteur viral contenant un gène codant pour un polypeptide - Google Patents

Polypeptide et vecteur viral contenant un gène codant pour un 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
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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
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English (en)
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
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Filing date
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Publication of WO2025162379A1 publication Critical patent/WO2025162379A1/fr
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

La présente invention concerne un polypeptide TCP et un vecteur viral contenant un gène codant pour le polypeptide TCP. Le vecteur viral contient en outre une molécule de signalisation d'activation de lymphocytes T, et peut cibler, activer et transduire efficacement les lymphocytes T.
PCT/CN2025/075188 2024-02-01 2025-01-26 Polypeptide et vecteur viral contenant un gène codant pour un polypeptide Pending WO2025162379A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170166622A1 (en) * 2015-05-18 2017-06-15 TCR2 Therapeutics Inc. Compositions and methods for tcr reprogramming using fusion proteins
CN110582509A (zh) * 2017-01-31 2019-12-17 诺华股份有限公司 使用具有多特异性的嵌合t细胞受体蛋白治疗癌症
US20200216502A1 (en) * 2017-09-22 2020-07-09 Centre National De La Recherche Scientifique (Cnrs) Mutated Glycoprotein of Vesicular Stomatitis Virus
US20210147871A1 (en) * 2018-04-12 2021-05-20 Umoja Biopharma, Inc. Viral vectors and packaging cell lines
WO2021129559A1 (fr) * 2019-12-24 2021-07-01 南京北恒生物科技有限公司 Protéine de fusion de récepteur de lymphocytes t et son utilisation
CN116497065A (zh) * 2022-01-25 2023-07-28 广东东阳光药业股份有限公司 病毒载体及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170166622A1 (en) * 2015-05-18 2017-06-15 TCR2 Therapeutics Inc. Compositions and methods for tcr reprogramming using fusion proteins
CN110582509A (zh) * 2017-01-31 2019-12-17 诺华股份有限公司 使用具有多特异性的嵌合t细胞受体蛋白治疗癌症
US20200216502A1 (en) * 2017-09-22 2020-07-09 Centre National De La Recherche Scientifique (Cnrs) Mutated Glycoprotein of Vesicular Stomatitis Virus
US20210147871A1 (en) * 2018-04-12 2021-05-20 Umoja Biopharma, Inc. Viral vectors and packaging cell lines
WO2021129559A1 (fr) * 2019-12-24 2021-07-01 南京北恒生物科技有限公司 Protéine de fusion de récepteur de lymphocytes t et son utilisation
CN116497065A (zh) * 2022-01-25 2023-07-28 广东东阳光药业股份有限公司 病毒载体及其应用

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