Polypeptide and viral vector containing gene encoding polypeptide

Abstract

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

Description

A polypeptide and a viral vector containing the polypeptide gene Technical Field

[0001] The present invention relates to the field of cell therapy, and in particular to a polypeptide and a viral vector comprising the polypeptide gene. Background Art

[0002] In the field of genetic engineering and cell therapy, lipid nanoparticles (LNPs), virus-like particles (VLPs), adenovirus, adeno-associated virus, retroviral vector (RVV) and lentiviral vector (LVV) are commonly used gene vectors.

[0003] 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.

[0004] However, during the packaging of LVV or RVV, the CAR molecule can be expressed on the cell membrane of the packaging cell under the action of the signal peptide, and buds out from the packaging cell as the packaged LVV or RVV buds out, becoming part of the envelope of the LVV or RVV.

[0005] When transducing host cells such as T cells, the envelope of the LVV or RVV fuses with the host cell membrane, and the CAR molecules contained in its envelope also become part of the host cell membrane. This transfer of CAR molecules between the viral vector envelope and the host cell membrane is called "pseudotransduction".

[0006] In pseudotransduction, the CAR molecule transferred from the viral envelope to the host cell membrane is only detectable for a short period of time and is then degraded by the host cell shortly thereafter.

[0007] Therefore, false transduction seriously affects the accuracy of detecting the positive rate of LVV or RVV transduction of host cells such as T cells to prepare engineered immune cells such as CAR-T cells, which is not conducive to accurately calculating the number of engineered immune cells such as CAR-T cells required for cell therapy, especially in vitro CAR-T cell therapy, and affects the efficacy.

[0008] In addition, LVV or RVV can also transduce target cells such as cancer cells through the antigen binding region of the CAR molecule contained in its envelope, seriously affecting the efficacy of CAR-T cell therapy.

[0009] Therefore, a polypeptide comprising an antigen-binding region, but unlike a CAR molecule, not distributed or distributed in very small amounts on the envelope of LVV or RVV, and an LVV or RVV comprising the polypeptide gene and capable of effectively targeting and stimulating non-activated T cells for activation, are the key to reducing false transduction in traditional in vitro CAR-T cell therapy, reducing the ability of LVV or RVV to transduce target cells such as cancer cells in and outside the patient's body, improving the targeting and transduction efficiency of transduced T cells, and thus improving the efficacy of cell therapy. Summary of the Invention

[0010] In view of this, in order to solve at least one of the above technical problems, the present invention provides a viral vector in one aspect,

[0011] (a) the viral vector comprises a polynucleotide encoding a T cell receptor chimeric protein (TCP); and

[0012] (b) the surface of the viral vector contains T cell activation signal molecules;

[0013] Wherein, the TCP includes:

[0014] (i) a TCR / CD3 complex subunit related peptide (TSP), wherein the TSP comprises at least one of (1) a TCR / CD3 complex subunit, (2) a functional fragment of a TCR / CD3 complex subunit, (3) a TCR / CD3 complex subunit variant, and (4) a variant of a functional fragment of a TCR / CD3 complex subunit; and

[0015] (ii) Antigen binding region.

[0016] In some embodiments of the present invention, the viral vector is a lentiviral vector (LVV) or a retroviral vector (RVV).

[0017] In some embodiments of the present invention, the TCR / CD3 complex subunit is selected from at least one of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ζ and CD3ε.

[0018] In some embodiments of the present invention, the TCR / CD3 complex subunit functional fragment comprises at least one of the extracellular region, transmembrane region, intracellular region, variable region and constant region of the TCR / CD3 complex subunit.

[0019] In some embodiments of the present invention, the TSP comprises CD3γ or a functional fragment thereof;

[0020] Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3γ;

[0021] More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3γ and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3ζ and CD3δ;

[0022] Still more preferably, the TSP comprises the transmembrane region and intracellular region of CD3γ and the extracellular region of (a) CD3δ, (b) CD3ζ or (c) CD3ε;

[0023] Most preferably, the C-terminus of the extracellular region of CD3δ, the extracellular region of CD3ζ or the extracellular region of CD3ε is located towards the N-terminus of the transmembrane region of CD3γ, and the C-terminus of the transmembrane region of CD3γ is located towards the N-terminus of the intracellular region of CD3γ.

[0024] In some embodiments of the present invention, the TSP comprises CD3ε or a functional fragment thereof;

[0025] Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ε;

[0026] More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ε and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3ζ and CD3δ;

[0027] More preferably, the TSP comprises the transmembrane region and intracellular region of CD3ε; and (a) the extracellular region of CD3γ, (b) the extracellular region of CD3ζ, or (c) the extracellular region of CD3δ;

[0028] Most preferably, the C-terminus of the extracellular region of CD3γ, the extracellular region of CD3ζ or the extracellular region of CD3δ is located in the N-terminal direction of the transmembrane region of CD3ε, and the C-terminus of the transmembrane region of CD3ε is located in the N-terminal direction of the intracellular region of CD3ε.

[0029] In some embodiments of the present invention, the TSP comprises CD3δ or a functional fragment thereof;

[0030] Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3δ;

[0031] More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3δ and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3ζ and CD3γ;

[0032] More preferably, the TSP comprises the transmembrane region and intracellular region of CD3δ; and (a) the extracellular region of CD3γ, (b) the extracellular region of CD3ζ, or (c) the extracellular region of CD3ε;

[0033] Most preferably, the C-terminus of the extracellular region of CD3γ, CD3ζ or CD3ε is located towards the N-terminus of the transmembrane region of CD3δ, and the C-terminus of the transmembrane region of CD3δ is located towards the N-terminus of the intracellular region of CD3δ.

[0034] In some embodiments of the present invention, the TSP comprises CD3ζ or a functional fragment thereof;

[0035] Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ζ;

[0036] More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ζ and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3δ and CD3γ;

[0037] More preferably, the TSP comprises the transmembrane region and intracellular region of CD3ζ; and (a) the extracellular region of CD3γ, (b) the extracellular region of CD3δ, or (c) the extracellular region of CD3ε;

[0038] Most preferably, the C-terminus of the extracellular region of CD3γ, CD3δ or CD3ε is located in the direction of the N-terminus of the transmembrane region of CD3ζ, and the C-terminus of the transmembrane region of CD3ζ is located in the direction of the N-terminus of the intracellular region of CD3ζ.

[0039] In some embodiments of the present invention, the TSP comprises TCRα or a functional fragment thereof and TCRβ or a functional fragment thereof;

[0040] Preferably, the TSP comprises the constant region of TCRα and the constant region of TCRβ.

[0041] In some embodiments of the present invention, the constant region of the TCRα is mutated, and the mutation includes a cysteine ​​substitution in the TCRα constant region; the mutation can enhance disulfide bond-based interchain interactions;

[0042] Preferably, the TCRα constant region is derived from a human or mouse TCRα constant region, the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 39, and the mouse TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 40;

[0043] The mutation includes replacing the 47th amino acid Threonine T of the human TCRα constant region with Cysteine ​​C (human TCRα constant region variant 1) or replacing the 47th amino acid Threonine T of the mouse TCRα constant region with Cysteine ​​C (mouse TCRα constant region variant 1);

[0044] The human TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:41, and the mouse TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:42.

[0045] In some embodiments of the present invention, the TCRβ constant region is mutated, and the mutation includes a cysteine ​​substitution in the TCRβ constant region; the mutation can enhance disulfide bond-based interchain interactions;

[0046] Preferably, the TCRβ constant region is derived from a human TCRβ constant region, and the human TCRβ constant region comprises the amino acid sequence shown in SEQ ID NO: 43 (hTRBC1) or SEQ ID NO: 44 (hTRBC2), and the mutation includes replacing the 56th amino acid serine S of the human TCRβ constant region with cysteine ​​C (human TCRβ constant region variant 1), and the human TCRβ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46.

[0047] In some embodiments of the present invention, the TCRα constant region undergoes a mutation, wherein the mutation comprises replacing at least one uncharged amino acid in the TCRα constant region with a hydrophobic amino acid; the mutation increases the hydrophobicity of the TCRα transmembrane region, offsetting the instability caused by the positive charge carried by the TCRα transmembrane region, thereby enabling the TCRα and its dimer formed with TCRβ to be more stably expressed on the T cell membrane, thereby achieving better function;

[0048] Preferably, the TCRα constant region is derived from a human TCRα constant region, the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 39, and the mutation comprises replacement of at least one of amino acids 115, 118, and 119 of the human TCRα constant region with a hydrophobic amino acid;

[0049] More preferably, the mutations include at least one of the following mutations in the human TCRα constant region: substitution of amino acid serine S at position 115 with leucine L, substitution of amino acid glycine G at position 118 with valine V, substitution of amino acid phenylalanine F at position 119 with leucine L;

[0050] Further preferably, the mutation includes replacing the 115th amino acid serine S of the human TCRα constant region with leucine L, replacing the 118th amino acid glycine G with valine V, and replacing the 119th amino acid phenylalanine F with leucine L (human TCRα constant region variant 2), and the human TCRα constant region variant 2 comprises the amino acid sequence shown in SEQ ID NO:47.

[0051] In some embodiments of the present invention, the TCRα constant region is derived from a human TCRα constant region, the human TCRα constant region comprising the amino acid sequence as shown in SEQ ID NO: 39; the human TCRα constant region is mutated, the mutation comprising substitution of amino acid threonine T at position 47 of the human TCRα constant region with cysteine ​​C, amino acid serine S at position 115 with leucine L, amino acid glycine G at position 118 with valine V, and amino acid phenylalanine F at position 119 with leucine L (human TCRα constant region variant 3), the human TCRα constant region variant 3 comprising the amino acid sequence as shown in SEQ ID NO: 48;

[0052] Preferably, the TCRα constant region and the TCRβ constant region are derived from a human TCRα constant region and a human TCRβ constant region; the human TCRα constant region comprises the amino acid sequence as shown in SEQ ID NO: 39, and the human TCRβ constant region comprises the amino acid sequence as shown in SEQ ID NO: 43 or SEQ ID NO: 44; the human TCRα constant region and the human TCRβ constant region are mutated, and the mutations include substitution of amino acid 47 of the human TCRα constant region with cysteine, substitution of amino acid serine S at position 115 with leucine L, substitution of amino acid glycine G at position 118 with valine V, and substitution of amino acid phenylalanine F at position 119 with leucine L (human TCRα constant region variant 3), and substitution of amino acid serine S at position 56 of the human TCRβ constant region with cysteine ​​C (human TCRβ constant region variant 1); the human TCRα constant region variant 3 comprises the amino acid sequence as shown in SEQ ID NO: 48, and the human TCRβ constant region variant 1 comprises the amino acid sequence as shown in SEQ ID NO: 45 or SEQ ID The amino acid sequence shown in NO:46.

[0053] In some embodiments of the present invention, any of the aforementioned TSPs may further comprise the hinge region of the TCR / CD3 complex subunit.

[0054] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of any of the aforementioned TSPs via a linker peptide.

[0055] In some embodiments of the present invention, the connecting peptide includes a flexible connecting peptide.

[0056] In some embodiments of the present invention, the flexible connecting peptide is selected from (G4S) n A connecting peptide and a connecting peptide 1 comprising the amino acid sequence shown in SEQ ID NO: 23; wherein n=1 to 4;

[0057] Preferably, the flexible connecting peptide is the connecting peptide 1, (G4S)3 connecting peptide (connecting peptide 2) or connecting peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 63).

[0058] In some embodiments of the present invention, the flexible connecting peptide is the connecting peptide 1.

[0059] In some embodiments of the present invention, the antigen binding region binds to a disease-associated antigen.

[0060] In some embodiments of the present invention, the disease is selected from cancer and autoimmune diseases; the cancer includes blood cancer and solid cancer.

[0061] In some embodiments of the present invention, the disease-associated antigen is selected from:

[0062] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38, CD40, CD40 L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD 248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB 4. MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, uPAR, GCC (guanylate cyclase C), EPCAM, Nectin4, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut At least one of hsp70-2, CD79A, CD79B, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, and NKG2D.

[0063] In some embodiments of the present invention, the disease-associated antigen is selected from at least one of CD19, CD20, CD33, MSLN, CD79B, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2 and GUCY2C.

[0064] In some embodiments of the present invention, the disease-associated antigen is a human disease-associated antigen.

[0065] In some embodiments of the present invention, the antigen binding region comprises an antibody or an antigen binding fragment thereof and / or a ligand or a receptor binding fragment thereof, and the antibody or antigen binding fragment thereof is selected from at least one of an immunoglobulin (full-length antibody), a half antibody, Fab, Fab', F(ab')2, an Fv fragment, a single-chain variable region fragment (scFv), a disulfide bond-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).

[0066] In some embodiments of the present invention, any of the aforementioned TCRα constant regions or variants thereof can be operably linked to the VH region or the VL region; any of the aforementioned TCRβ constant regions or variants thereof can be operably linked to the VH region or the VL region.

[0067] In some embodiments of the present invention, when any of the aforementioned TCRα constant regions or variants thereof are operably linked to the VH region, any of the aforementioned TCRβ constant regions or variants thereof are operably linked to the VL region; when any of the aforementioned TCRα constant regions or variants thereof are operably linked to the VL region, any of the aforementioned TCRβ constant regions or variants thereof are operably linked to the VH region.

[0068] In some embodiments of the present invention, the VH region and VL region are derived from the same antibody or antigen-binding fragment thereof, or ligand or receptor-binding fragment thereof.

[0069] In some embodiments of the present invention, the antigen binding region is selected from at least one of the following antigen binding regions:

[0070] (a) an antigen-binding region that binds to human CD19, wherein the antigen-binding region is an FMC-63-derived scFv (FMC63-scFv), the amino acid sequence of the VH region of the FMC63-scFv being shown in SEQ ID NO: 18, and the amino acid sequence of the VL region of the FMC63-scFv being shown in SEQ ID NO: 19;

[0071] (b) an antigen-binding region that binds to human asialoglycoprotein receptor (ASGPR), wherein the antigen-binding region is an anti-human ASGPR scFv (anti-ASGPR-scFv); the amino acid sequence of the VL region of the anti-ASGPR-scFv is shown in SEQ ID NO: 51; the amino acid sequence of the anti-ASGPR-scFv is shown in SEQ ID NO: 52;

[0072] (c) an antigen-binding region that binds to human HER2, wherein the antigen-binding region is derived from the anti-human HER2 antibody Pertuzumab scFv (Pertuzumab-scFv); the amino acid sequence of the VL region of the Pertuzumab-scFv is shown in SEQ ID NO: 53; and the amino acid sequence of the VH region of the Pertuzumab-scFv is shown in SEQ ID NO: 54;

[0073] (d) an antigen-binding region that binds to human MESOTHELIN (MSLN), the antigen-binding region being a scFv derived from the anti-human MSLN antibody PE38 (PE38-scFv); the amino acid sequence of the VH region of the PE38-scFv is shown in SEQ ID NO: 55; the amino acid sequence of the VL region of the PE38-scFv is shown in SEQ ID NO: 56;

[0074] (e) an antigen-binding region that binds to human CD8, wherein the antigen-binding region is an anti-human CD8 scFv (anti-CD8-scFv); the amino acid sequence of the VH region of the anti-CD8-scFv is shown in SEQ ID NO: 57; and the amino acid sequence of the VL region of the anti-CD8-scFv is shown in SEQ ID NO: 58;

[0075] (f) an antigen-binding region that binds to human CD33, wherein the antigen-binding region is derived from the anti-human CD33 antibody Gemtuzumab scFv (Gemtuzumab-scFv); the amino acid sequence of the VL region of the Gemtuzumab-scFv is shown in SEQ ID NO: 59; the amino acid sequence of the VH region of the Gemtuzumab-scFv is shown in SEQ ID NO: 60; and

[0076] (g) an antigen-binding region that binds to human CD79B, wherein the antigen-binding region is derived from the scFv (SN8-scFv) of the anti-human CD79B antibody SN-8; the amino acid sequence of the VH region of the anti-SN8-scFv is shown in SEQ ID NO:61; and the amino acid sequence of the VL region of the SN8-scFv is shown in SEQ ID NO:62.

[0077] In some embodiments of the present invention, any of the aforementioned T cell receptor chimeric proteins does not comprise a signal peptide.

[0078] In some embodiments of the present invention, any of the aforementioned T cell receptor chimeric proteins comprises a signal peptide.

[0079] In some embodiments of the present invention, the signal peptide is not particularly limited as long as it can mediate the membrane expression of TCP.

[0080] In some embodiments of the present invention, the signal peptide is selected from the following signal peptides: CD8α signal peptide, CD28 signal peptide, IgG signal peptide, HLA-A signal peptide, CD3γ signal peptide, CD3δ signal peptide, CD3ζ signal peptide and CD3ε signal peptide.

[0081] In some embodiments of the present invention, the signal peptide is a human CD8α signal peptide.

[0082] In some embodiments of the present invention, the amino acid sequence of the human CD8α signal peptide is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:12.

[0083] In some embodiments of the present invention, the T cell receptor chimeric protein further comprises a co-stimulatory signaling domain.

[0084] In some embodiments of the present invention, the costimulatory signaling domain is derived from the costimulatory signaling domain of at least one of the following proteins:

[0085] CD28, 4-1BB, CD27, CD2, CD3, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRl γ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88.

[0086] In some embodiments of the present invention, the costimulatory signaling domain is derived from the costimulatory signaling domain of human 4-1BB and / or CD28.

[0087] In some embodiments of the present invention, the amino acid sequence of the costimulatory signaling domain of human 4-1BB is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49.

[0088] In some embodiments of the present invention, any of the aforementioned T cell receptor chimeric proteins, when expressed in T cells, (a) is incorporated into an endogenous TCR / CD3 complex or a TCR / CD3 complex subunit or a functional fragment thereof; or (b) functionally interacts with an endogenous TCR / CD3 complex or an endogenous TCR / CD3 complex subunit or a functional fragment thereof.

[0089] In some embodiments of the present invention, any of the aforementioned TCPs is not expressed on the membrane in cells other than T cells, or the efficiency of membrane expression of the TCP on cells other than T cells is lower than the efficiency of membrane expression of the TCP on T cells.

[0090] In some embodiments of the present invention, the T cell activation signaling molecule includes a T cell activation primary signaling molecule.

[0091] Non-activated T cells are T cells that are not proliferated, differentiated, in a resting state, do not recognize antigens, and have not been activated by T cell activation signaling molecules such as primary and secondary T cell activation signaling molecules, such as T cells in the G0 phase of the cell cycle, resting / quiescent T cells, or immature T cells. T cells. Resting T cells, also known as quiescent T cells or naive T cells, are T cells that are not mitotically active or have not been exposed to cognate antigens presented on antigen-presenting cells, such as macrophages or dendritic cells.

[0092] T cell activation primary signaling molecules bind to T cell surface proteins and participate in T cell receptor (TCR)-mediated T cell activation.

[0093] In some embodiments of the present invention, the T cell activation primary signal molecule is involved in converting TCR into active PTK (protein tyrosine kinase), which can phosphorylate a series of substrates to generate a large number of downstream signals. When these signals are properly integrated (together with signals from other co-receptors), they lead to T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0094] In some embodiments of the present invention, the T cell activation primary signal molecule binds to at least one of a TCR / CD3 complex subunit and a functional fragment of a TCR / CD3 complex subunit; the TCR / CD3 complex subunit is selected from at least one of CD3ε, CD3γ, CD3δ, CD3ζ, TCRγ, TCRδ, TCRα and TCRβ.

[0095] In some embodiments of the present invention, the TCR / CD3 complex subunit is a human TCR / CD3 complex subunit.

[0096] 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 cell) activation.

[0097] In some embodiments of the present invention, the T cell activation primary signal molecule comprises an anti-CD3 antibody or an antigen-binding fragment thereof.

[0098] In some embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof specifically binds to human CD3.

[0099] In some embodiments of the present invention, the anti-CD3 antibody is selected from at least one of OKT3, UCHT1, YTH12.5 and TR66.

[0100] In some embodiments of the present invention, the anti-CD3 antibody is SP34.

[0101] In some embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof cannot bind to the TSP.

[0102] In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3ε antibody, and the TSP is not CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

[0103] In some embodiments of the present invention, the anti-CD3ε antibody or antigen-binding fragment thereof specifically binds to human CD3ε (Uniprot ID: P07766).

[0104] In some embodiments of the present invention, the anti-CD3ε antibody or antigen-binding fragment thereof is UCHT1 or an antigen-binding fragment thereof;

[0105] Preferably, the antigen-binding fragment of UCHT1 is a scFv (UCHT1-scFv);

[0106] More preferably, the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NOs: 87-89, respectively, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NOs: 90-92, respectively.

[0107] In some embodiments of the present invention, the anti-CD3ε antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof;

[0108] Preferably, the antigen-binding fragment of OKT3 is scFv (OKT3-scFv);

[0109] More preferably, the amino acid sequences of the HCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NOs: 104-106, respectively, and the amino acid sequences of the LCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NOs: 107-109, respectively;

[0110] More preferably, the amino acid sequence of the VH region of the OKT3-scFv is shown in SEQ ID NO: 119, and the amino acid sequence of the VL region of the OKT3-scFv is shown in SEQ ID NO: 120.

[0111] In some embodiments of the present invention, the anti-CD3ε antibody or antigen-binding fragment thereof is SP34 or an antigen-binding fragment thereof;

[0112] Preferably, the antigen-binding fragment of SP34 is scFv (SP34-scFv);

[0113] More preferably, the amino acid sequences of the HCDR1-3 regions of the SP34-scFv are shown as SEQ ID NOs: 113-115, respectively, and the amino acid sequences of the LCDR1-3 regions of the SP34-scFv are shown as SEQ ID NOs: 116-118, respectively;

[0114] More preferably, the amino acid sequence of the VH region of the SP34-scFv is shown in SEQ ID NO: 111, and the amino acid sequence of the VL region of the SP34-scFv is shown in SEQ ID NO: 112.

[0115] In some embodiments of the present invention, when the anti-CD3 antibody or antigen-binding fragment thereof is the anti-CD3ε antibody or antigen-binding fragment thereof, the TSP is:

[0116] (a) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;

[0117] (b) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or

[0118] (c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

[0119] In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3γ antibody, and the TSP is not CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

[0120] In some embodiments of the present invention, when the anti-CD3 antibody or antigen-binding fragment thereof is the anti-CD3γ antibody or antigen-binding fragment thereof, the TSP is:

[0121] (a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;

[0122] (b) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or

[0123] (c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

[0124] In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3δ antibody, and the TSP is not CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

[0125] In some embodiments of the present invention, the anti-CD3δ antibody or antigen-binding fragment thereof is TR66 or an antigen-binding fragment thereof;

[0126] Preferably, the antigen-binding fragment of TR66 is scFv (TR66-scFv).

[0127] In some embodiments of the present invention, when the anti-CD3 antibody or antigen-binding fragment thereof is the anti-CD3δ antibody or antigen-binding fragment thereof, the TSP is:

[0128] (a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;

[0129] (b) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or

[0130] (c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

[0131] In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3ζ antibody, and the TSP is not CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

[0132] In some embodiments of the present invention, the anti-CD3ζ antibody or antigen-binding fragment thereof is YTH12.5 or an antigen-binding fragment thereof;

[0133] Preferably, the antigen-binding fragment of YTH12.5 is scFv (YTH12.5-scFv).

[0134] In some embodiments of the present invention, when the anti-CD3 antibody or antigen-binding fragment thereof is the anti-CD3ζ antibody or antigen-binding fragment thereof, the TSP is:

[0135] (a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof;

[0136] (b) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or

[0137] (c) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

[0138] In some embodiments of the present invention, the T cell activation primary signal molecule includes at least one of an anti-TCRα antibody or an antigen-binding fragment thereof, an anti-TCRβ antibody or an antigen-binding fragment thereof, an anti-TCRγ antibody or an antigen-binding fragment thereof, and an anti-TCRδ antibody or an antigen-binding fragment thereof.

[0139] In some embodiments of the present invention, when the T cell activation primary signal molecule comprises an anti-TCRα antibody or an antigen-binding fragment thereof, the TSP is not TCRα, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

[0140] In some embodiments of the present invention, when the T cell activation primary signal molecule comprises an anti-TCRβ antibody or an antigen-binding fragment thereof, the TSP is not TCRβ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

[0141] In some embodiments of the present invention, when the T cell activation primary signal molecule comprises an anti-TCRγ antibody or an antigen-binding fragment thereof, the TSP is not TCRγ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

[0142] In some embodiments of the present invention, when the T cell activation primary signal molecule comprises an anti-TCRδ antibody or an antigen-binding fragment thereof, the TSP is not TCRδ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof.

[0143] In some embodiments of the present invention, the T cell activation signal molecule further includes a T cell activation secondary signal molecule.

[0144] T cell activation secondary signal molecules (Secondary Signal), also known as co-stimulatory signal molecules (Co-Stimulatory Signal), bind to other T cell surface receptors and provide additional signals necessary for avoiding anergy and effective T cell activation (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0145] In some embodiments of the present invention, the T cell activation secondary signaling molecule binds to CD28.

[0146] In some embodiments of the present invention, the CD28 is human CD28 (Uniprot ID: P10747).

[0147] Although other cell surface receptors (co-stimulatory receptors) can also enhance activation signals through the TCR, CD28-mediated co-stimulation is more potent than other co-stimulatory receptors (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0148] In some embodiments of the present invention, the T cell activation secondary signal molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof.

[0149] In some embodiments of the present invention, the CD28 ligand or its receptor binding fragment includes CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.

[0150] In some embodiments of the present invention, the CD80 and CD86 are human CD80 and CD86.

[0151] In some embodiments of the present invention, the amino acid sequence of human CD80 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:85.

[0152] In some embodiments of the present invention,

[0153] (1) The amino acid sequence of the human CD80 signal peptide is shown in SEQ ID NO: 27;

[0154] (2) The amino acid sequence of the human CD80 extracellular domain is shown in SEQ ID NO: 28;

[0155] (3) The amino acid sequence of the human CD80 transmembrane region is shown in SEQ ID NO: 29.

[0156] In some embodiments of the present invention, the amino acid sequence of human CD86 is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:86.

[0157] In some embodiments of the present invention, the T cell activation secondary signaling molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof;

[0158] Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is a scFv derived from 15E8 (15E8-scFv), the amino acid sequence of which 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 NOs: 93-95, respectively, and the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 96-98, respectively.

[0159] In some embodiments of the present invention, the anti-CD28 antibody or antigen-binding fragment thereof is selected from at least one of CD28.2, 10F3 and TGN1412.

[0160] In some embodiments of the present invention, the T cell activation secondary signal molecule includes 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, and 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.

[0161] Unlike CD28, which is stably expressed on both non-activated and activated T cells, ICOS is inducibly expressed on activated T cells (Hutloff A, Dittrich AM, Beier KC, Eljaschewitsch B, Kraft R, et al. ICOS is an inducible T-cell costimulator structurally and functionally related to CD28. Nature 1999; 397: 263-6. [PubMed: 9930702])(Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619). Lack of ICOS results in impaired immune responses similar to, but less severe than, the CD28 knockout model, suggesting that the two molecules may function in similar pathways (Coyle AJ, Lehar S, Lloyd C, Tian J, Delaney T, et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity 2000; 13: 95-105. [PubMed: 10933398])(Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0162] Co-stimulatory receptors outside the CD28 family, 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).

[0163] In some embodiments of the present invention, the T cell activation secondary signaling molecule can also bind to at least one of CD27, HVEM, LIGHT, CD40, DR3, GITR, CD30, TIM1, SLAM, CD2 and CD226.

[0164] In some embodiments of the present invention, the T cell activation secondary signaling molecule may also be selected from at least one of B7-H2, CD70, LIGHT, HVEM, CD40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155 and CD112.

[0165] In some embodiments of the present invention, the T cell activation signaling molecule is directly or indirectly linked to a transmembrane polypeptide and displayed on the surface of the viral vector.

[0166] In some embodiments of the present invention, the transmembrane polypeptide is selected from the transmembrane regions of the following proteins:

[0167] CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD 37. CD40, CD45, CD64, CD80, CD86, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1;

[0168] Preferably, the transmembrane polypeptide is the CD8α transmembrane region.

[0169] In some embodiments of the present invention, the transmembrane polypeptide is the human CD8α transmembrane region.

[0170] In some embodiments of the present invention, the human CD8α transmembrane 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:15.

[0171] In some embodiments of the present invention, the T cell activation signaling molecule is indirectly linked to the transmembrane polypeptide via a linker domain and displayed on the surface of the viral vector;

[0172] Preferably, the linker domain is selected from:

[0173] (a) an immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge region;

[0174] (b) a hinge region selected from the wild-type or modified hinge region of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;

[0175] (c) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain;

[0176] (d) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and

[0177] (e) flexible linker peptide;

[0178] More preferably, the connecting domain is the CD8α hinge region.

[0179] In some embodiments of the present invention, the connecting domain is the human CD8α hinge region.

[0180] In some embodiments of the invention, the human CD8α hinge region is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:14.

[0181] In some embodiments of the present invention, the surface of the viral vector comprises a glycoprotein, and the glycoprotein is selected from the envelope glycoprotein of the vesicular stomatitis virus strain and its variants, the envelope glycoprotein of the baboon endogenous retrovirus BaEV and its variants, the envelope glycoprotein RD114 of the feline endogenous retrovirus and its variants, and the envelope glycoprotein GALV of the gibbon ape leukemia virus and its variants.

[0182] In some embodiments of the present invention, the transmembrane polypeptide is the glycoprotein, and the glycoprotein is directly or indirectly linked to the T cell activation signaling molecule;

[0183] Preferably, the glycoprotein is indirectly linked to the T cell activation signal molecule via a polypeptide linker.

[0184] In some embodiments of the present invention, the T cell activation primary signaling molecule is directly or indirectly linked to the T cell activation secondary signaling molecule;

[0185] Preferably, the T cell activation primary signal molecule is indirectly linked to the T cell activation secondary signal molecule via a polypeptide linker.

[0186] In some embodiments of the present invention, the polypeptide linker is a flexible connecting peptide;

[0187] Preferably, the flexible connecting peptide is selected from (G4S) n Connector peptide, Connector peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23) and Connector peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 63); wherein, n=1 to 4.

[0188] In some embodiments of the present invention,

[0189] (a) The glycoprotein is passed through (G4S) n A connecting peptide indirectly connected to the anti-CD3 antibody or antigen-binding fragment thereof, wherein the anti-CD3 antibody or antigen-binding fragment thereof is connected to the anti-CD3 antibody or antigen-binding fragment thereof via (G4S) n a linker peptide indirectly linked to the anti-CD28 antibody or antigen-binding fragment thereof, (ii) the CD80 extracellular domain, (iii) or the CD86 extracellular domain; and / or

[0190] (b) The glycoprotein is passed through (G4S) n A connecting peptide indirectly connected to (i) the anti-CD28 antibody or its antigen-binding fragment, (ii) the CD80 extracellular domain (iii) or the CD86 extracellular domain; the anti-CD28 antibody or its antigen-binding fragment, the CD80 extracellular domain or the CD86 extracellular domain is connected to the anti-CD28 antibody or its antigen-binding fragment, the CD80 extracellular domain or the CD86 extracellular domain via (G4S) n a linker peptide, indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof;

[0191] Preferably, n=3;

[0192] Preferably, the anti-CD3 antibody or antigen-binding fragment thereof is the UCHT1-scFv;

[0193] Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is the 15E8-scFv.

[0194] In some embodiments of the present invention, the glycoprotein is selected from at least one of the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof.

[0195] In some embodiments of the present invention, the envelope glycoprotein and variants thereof of the vesicular stomatitis virus strain include the following envelope glycoproteins and variants thereof: envelope glycoprotein and variants of the Indiana strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants thereof of the Cocal strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants thereof of the Maraba strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants thereof of the Morreton strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants thereof of the Alagoas strain of the vesicular stomatitis virus genus, envelope glycoprotein and variants thereof of the New The envelope glycoprotein of Jersey strain and its variants, the envelope glycoprotein of Carajas strain and its variants, the envelope glycoprotein of Chandipura strain and its variants, the envelope glycoprotein of Eptesicus strain and its variants, the envelope glycoprotein of Isfahan strain and its variants, the envelope glycoprotein of Jurona strain and its variants, the envelope glycoprotein of Malpais strain and its variants, the envelope glycoprotein of Perinet strain and its variants, the envelope glycoprotein of Piry strain and its variants, the envelope glycoprotein of Radi strain and its variants, the envelope glycoprotein of Rhinolopus strain and its variants, and the envelope glycoprotein of Yug Bogdanovac strain and its variants.

[0196] The envelope glycoprotein (VSV-G) of vesicular stomatitis virus strains, such as the Indiana and Cocal strains, can bind to the low-density lipoprotein receptor (LDL-R) that is widely present on the surface of various cells, thus having a wide range of infectivity.

[0197] The extracellular domain of the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 2.

[0198] In some embodiments of the present invention, the amino acid sequence of the full-length protein of wild-type VSV-G (including the VSV-G signal peptide) is shown in SEQ ID NO: 24;

[0199] Wherein, the amino acid sequence shown at positions 1 to 16 of SEQ ID NO: 24:

[0200] MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of the wild-type VSV-G.

[0201] In some embodiments of the present invention, the amino acid sequence of the full-length protein of wild-type Cocal-G (including the Cocal-G signal peptide) is shown in SEQ ID NO: 99;

[0202] Among them, the sequence shown in positions 1 to 17 of SEQ ID NO: 99:

[0203] MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of the wild-type Cocal-G.

[0204] Extracellular domain of wild-type VSV-G:

[0205] Extracellular domain of wild-type Cocal-G:

[0206] Full-length protein of wild-type VSV-G (including its signal peptide):

[0207] Full-length wild-type Cocal-G protein (including its signal peptide):

[0208] 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.

[0209] In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus or a variant thereof;

[0210] 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.

[0211] In some embodiments of the present invention, any of the aforementioned glycoproteins undergoes a first mutation, which reduces or loses the ability of the glycoprotein to bind to a glycoprotein receptor relative to before the first mutation occurs.

[0212] 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.

[0213] 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.

[0214] In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus, or a variant thereof; the glycoprotein receptor is the low-density lipoprotein receptor (LDL-R); the glycoprotein undergoes a first mutation, such that the ability of the glycoprotein to bind to the LDL-R is reduced or lost relative to before the first mutation occurs;

[0215] 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.

[0216] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0217] (a) substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 209, substitution or deletion of amino acid at position 347 in SEQ ID NO: 1 or SEQ ID NO: 2. 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

[0218] (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, substitution or deletion of amino acid 8, substitution or deletion of amino acid 9, substitution or deletion of amino acid 10, substitution or deletion of amino acid 47, substitution or deletion of amino acid 50, substitution or deletion of amino acid 51, substitution or deletion of amino acid 183, substitution or deletion of amino acid 179, substitution or deletion of amino acid 180, substitution or deletion of amino acid 182, substitution or deletion of amino acid 184, substitution or deletion of amino acid 209, substitution or deletion of amino acid 347, substitution or deletion of amino acid 358, substitution or deletion of amino acid 360, substitution or deletion of amino acid 361, substitution or deletion of amino acid 362, substitution or deletion of amino acid 364, substitution or deletion of amino acid 365, substitution or deletion of amino acid 366, substitution or deletion of amino acid 367, substitution or deletion of amino acid 368, substitution or deletion of amino acid 369, substitution or deletion of amino acid 370, substitution or deletion of amino acid 371, substitution or deletion of amino acid 372, substitution or deletion of amino acid 373, substitution or deletion of amino acid 374, substitution or deletion of amino acid 375, substitution or deletion of amino acid 376, substitution or deletion of amino acid 377, substitution or deletion of amino acid 378, substitution or deletion of amino acid 379, substitution or deletion of amino acid 371, substitution or deletion of amino acid 377 The present invention also includes substitution or deletion of the amino acid at position 350, substitution or deletion of the amino acid at position 352, substitution or deletion of the amino acid at position 353, substitution of the amino acid at position 354, deletion of amino acids at positions 1-18, deletion of amino acids at positions 19-36, deletion of amino acids at positions 37-51, deletion of amino acids at positions 314-384, deletion of amino acids at positions 321-374, deletion of amino acids at positions 331-364, deletion of amino acids at positions 344-354, and deletion of amino acids at positions 345-353.

[0219] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0220] (a) substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, or deletion of amino acids 345-353 of SEQ ID NO: 1; and

[0221] (b) After optimal global alignment with SEQ ID NO: 1, the position corresponding to SEQ ID NO:1: substitution or deletion of H8, substitution or deletion of N9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of S183, substitution or deletion of S179, substitution or deletion of N180, substitution or deletion of I182, substitution or deletion of M184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of T350, substitution or deletion of T352, substitution or deletion of E353, substitution of R354, deletion of amino acids at positions 1-18, deletion of amino acids at positions 19-36, deletion of amino acids at positions 37-51, deletion of amino acids at positions 314-384, deletion of amino acids at positions 321-374, deletion of amino acids at positions 331-364, deletion of amino acids at positions 344-354, and deletion of amino acids at positions 345-353.

[0222] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0223] (a) substitution of H8, substitution of N9, substitution of Q10, substitution of K47, deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353 of SEQ ID NO: 1; and

[0224] (b) After optimal global alignment with SEQ ID NO: 1, the following residues are located: substitution of H8, substitution of N9, substitution of Q10, substitution of K47, deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353, corresponding to SEQ ID NO: 1.

[0225] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0226] (a) deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, substitution of R354 in SEQ ID NO: 1 or SEQ ID NO: 2; and

[0227] (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, substitution of R354 at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0228] Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0229] (a) deletion of amino acids 331 to 364, 344 to 354, K47Q, R354Q, or K47 located in SEQ ID NO: 1 or SEQ ID NO: 2; and

[0230] (b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, amino acid deletions at positions 331 to 364, amino acid deletions at positions 344 to 354, K47Q, R354Q, and K47 deletion are located corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0231] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises the following amino acids:

[0232] (a) the amino acid lysine at position 47 of SEQ ID NO: 1 or SEQ ID NO: 2 is deleted; or

[0233] (b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, the lysine residue at amino acid position 47 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 is missing.

[0234] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0235] (a) substitution of K47, deletion of K47, substitution of I182, substitution of R354, and substitution of Y209 in SEQ ID NO: 1;

[0236] (b) after optimal global alignment with SEQ ID NO: 1, substitution at K47, deletion of K47, substitution at I182, substitution at R354, and substitution at Y209 equivalent to SEQ ID NO: 1;

[0237] (c) substitution of K47, deletion of K47, substitution of V182, substitution of R354, substitution of Y209 at SEQ ID NO: 2; and

[0238] (d) after optimal global alignment with SEQ ID NO: 2, a substitution at K47, a deletion of K47, a substitution at V182, a substitution at R354, and a substitution at Y209 corresponding to SEQ ID NO: 2;

[0239] Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0240] (a) K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q located in SEQ ID NO: 1;

[0241] (b) after optimal global alignment with SEQ ID NO: 1, located at K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q equivalent to SEQ ID NO: 1;

[0242] (c) K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q at SEQ ID NO: 2; and

[0243] (d) After optimal global alignment with SEQ ID NO: 2, it is located at K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, and Y209Q corresponding to SEQ ID NO: 2.

[0244] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 33 or SEQ ID NO: 34.

[0245] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:3; relative to SEQ ID NO:1, SEQ ID NO:3 comprises a K47 deletion.

[0246] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:4; relative to SEQ ID NO:1, SEQ ID NO:4 comprises R354Q.

[0247] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:33 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:33; relative to SEQ ID NO:2, SEQ ID NO:33 comprises a K47 deletion.

[0248] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:34 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:34; relative to SEQ ID NO:2, SEQ ID NO:34 comprises R354Q.

[0249] In some embodiments of the present invention, the glycoprotein having any of the aforementioned first mutations still retains the ability to fuse membranes and escape from endosomal / lysosomal regions.

[0250] In some embodiments of the present invention, the glycoprotein further undergoes a second mutation, which enhances the ability of the glycoprotein to antagonize inactivation by complement, or prevents the glycoprotein from being inactivated by complement, compared to before the second mutation.

[0251] In some embodiments of the present invention, the glycoprotein having the second mutation is any of the aforementioned glycoproteins.

[0252] 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.

[0253] 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).

[0254] 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.

[0255] 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.

[0256] In some embodiments of the present invention, the glycoprotein with 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 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 with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0257] In some embodiments of the present invention, the second mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0258] (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0259] (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0260] (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0261] (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0262] (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0263] (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0264] (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0265] (h) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 146 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0266] Preferably, the amino acid mutation includes at least one of amino acid deletion, insertion and substitution.

[0267] More preferably, the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids:

[0268] (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0269] (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0270] (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0271] (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0272] (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0273] (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0274] (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0275] (h) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the amino acid position corresponding to 146 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0276] In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 1, 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 shown in SEQ ID NO: 1, comprising at least one of the following position mutations:

[0277] (a) substitution of T214, substitution of T352, substitution of K50, or substitution of S146 in SEQ ID NO: 1; and

[0278] (b) after optimal global alignment with SEQ ID NO: 1, substitutions at T214, T352, K50, and S146 corresponding to SEQ ID NO: 1;

[0279] Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence:

[0280] (a) T214N, T352A, K50T, S146T at SEQ ID NO: 1; and

[0281] (b) After optimal global alignment with SEQ ID NO: 1, T214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO: 1.

[0282] In some embodiments of the present invention, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

[0283] (a) substitution of (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1; and

[0284] (b) after optimal global alignment with SEQ ID NO: 1, substitutions at positions corresponding to (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1;

[0285] Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

[0286] (a) (1) T214N and T352A; or (2) T214N, T352A, K50T, and S146T at SEQ ID NO: 1; and

[0287] (b) After optimal global alignment with SEQ ID NO: 1, located at (1) T214N and T352A; or (2) T214N, T352A, K50T, and S146T equivalent to SEQ ID NO: 1.

[0288] In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown 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 shown in SEQ ID NO: 2, comprising at least one of the following position mutations:

[0289] (a) substitution of K214, substitution of T352, substitution of K50, substitution of S146 in SEQ ID NO: 2; and

[0290] (b) after optimal global alignment with SEQ ID NO: 2, substitutions at positions corresponding to K214, T352, K50, and S146 of SEQ ID NO: 2;

[0291] Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence:

[0292] (a) K214N, T352A, K50T, S146T at SEQ ID NO: 2; and

[0293] (b) After optimal global alignment with SEQ ID NO: 2, K214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO: 2.

[0294] In some embodiments of the present invention, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

[0295] (a) substitution of (1) K214 and T352; or (2) K214, T352, K50, and S146 of SEQ ID NO: 2; and

[0296] (b) after optimal global alignment with SEQ ID NO: 2, at positions corresponding to (1) substitutions of K214 and T352; or (2) substitutions of K214, T352, K50, and S146 of SEQ ID NO: 2;

[0297] Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

[0298] (a) (1) K214N and T352A; or (2) K214N, T352A, K50T, and S146T at SEQ ID NO: 2; and

[0299] (b) After optimal global alignment with SEQ ID NO: 2, located at (1) K214N and T352A; or (2) K214N, T352A, K50T, and S146T equivalent to SEQ ID NO: 2.

[0300] In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus or a variant thereof;

[0301] the extracellular domain of the glycoprotein comprises an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence 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;

[0302] 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.

[0303] In some embodiments of the present invention, the glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus or a variant thereof;

[0304] the extracellular domain of the glycoprotein comprises an amino acid sequence as set forth in SEQ ID NO:2, or an amino acid sequence 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;

[0305] 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.

[0306] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38.

[0307] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:8 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:8; relative to SEQ ID NO:1, SEQ ID NO:8 comprises K47 deletion, T214N and T352A.

[0308] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:9 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:9; relative to SEQ ID NO:1, SEQ ID NO:9 comprises K47 deletion, T214N, T352A, K50T and S146T.

[0309] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 10 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO: 10; relative to SEQ ID NO: 1, SEQ ID NO: 10 comprises R354Q, T214N and T352A.

[0310] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 11; relative to SEQ ID NO: 1, SEQ ID NO: 11 comprises R354Q, T214N, T352A, K50T and S146T.

[0311] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:35 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:35; relative to SEQ ID NO:2, SEQ ID NO:35 comprises K47 deletion, K214N, T352A, K50T and S146T.

[0312] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:36 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:36; relative to SEQ ID NO:2, SEQ ID NO:36 comprises a K47 deletion, K214N and T352A.

[0313] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:37 or an amino acid sequence having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO:37; relative to SEQ ID NO:2, SEQ ID NO:37 comprises R354Q, K214N, T352A, K50T and S146T.

[0314] In some embodiments of the present invention, the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:38 or an amino acid sequence that is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence as shown in SEQ ID NO:38; relative to SEQ ID NO:2, SEQ ID NO:38 comprises R354Q, K214N and T352A.

[0315] In some embodiments of the present invention, the glycoprotein having any of the aforementioned second mutations still retains the ability to fuse membranes and escape from endosomal / lysosomal regions.

[0316] In some embodiments of the present invention, the glycoprotein having any of the aforementioned first and second mutations still retains the ability to fuse with membranes and escape from endosomal / lysosomes.

[0317] In some embodiments of the present invention, compared with the control vector 1 whose surface does not contain the T cell activation signal molecule, after any of the aforementioned viral vectors contacts with non-activated T cells, the efficiency of the T cell receptor chimeric protein in T cell extracellular expression is higher.

[0318] In some embodiments of the present invention, compared with the control vector 1 whose surface does not contain the T cell activation signal molecule, the TCP-T cells prepared after any of the aforementioned viral vectors contact with non-activated T cells have a higher killing efficiency.

[0319] In some embodiments of the present invention, the control carrier 1 comprises at least one T cell targeting molecule on its surface;

[0320] Preferably, the T cell targeting molecule binds to CD5 or CD7;

[0321] More preferably, the T cell targeting molecule binds to CD7;

[0322] Further preferably, the T cell targeting molecule is selected from at least one of an anti-CD7 antibody or an antigen-binding fragment thereof and a CD7 ligand or a receptor-binding fragment thereof; optionally, the anti-CD7 antibody or an antigen-binding fragment thereof is a scFv (TH69-scFv) derived from the monoclonal antibody TH-69; the amino acid sequence of the TH69-scFv is 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.

[0323] In some embodiments of the present invention, any of the aforementioned control vectors 1 is a vector having the same structure and / or characteristics as any of the aforementioned viral vectors except that it does not contain the protein encoding any of the aforementioned T cell activation signaling molecules.

[0324] In some embodiments of the present invention, the surface of any of the aforementioned viral vectors does not contain or contains only a small amount of the antigen binding region; the "small amount" is relative to the control vector 2 containing a polynucleotide encoding other polypeptides; the other polypeptides contain any of the aforementioned antigen binding regions but do not contain any of the aforementioned TSPs.

[0325] In some embodiments of the present invention, after any of the aforementioned viral vectors contacts T cells, the viral vector does not undergo pseudo-transduction or pseudo-transduction is reduced compared to the control vector 2 comprising a polynucleotide encoding other polypeptides; the other polypeptide comprises any of the aforementioned antigen binding regions but does not comprise any of the aforementioned TSPs.

[0326] In some embodiments of the present invention, relative to a control vector 2 that does not contain a polynucleotide encoding other polypeptides, any of the aforementioned viral vectors (a) has a reduced ability to bind to target cell surface antigens or does not bind to target cell surface antigens; and / or (b) has a reduced ability to transduce target cells or does not transduce target cells; the target cell surface antigen can bind to the antigen binding region, and the other polypeptide comprises any of the aforementioned antigen binding regions but does not comprise any of the aforementioned TSPs.

[0327] In some embodiments of the present invention, the other polypeptide is linked to a signal peptide.

[0328] In some embodiments of the present invention, the other polypeptide comprises a chimeric antigen receptor, wherein the N-terminus of the chimeric antigen receptor is operably linked to the C-terminus of the signal peptide.

[0329] In some embodiments of the present invention, the signal peptide is selected from any one of the aforementioned signal peptides.

[0330] In some embodiments of the present invention, the chimeric antigen receptor is selected from at least one of first-generation, second-generation, third-generation and fourth-generation chimeric antigen receptors.

[0331] In some embodiments of the present invention, the structure of the chimeric antigen receptor from N-terminus to C-terminus includes, in sequence, an extracellular antigen binding region, a hinge region, a transmembrane region, a co-stimulatory signaling domain, and an intracellular signaling domain.

[0332] In some embodiments of the present invention, the target cell is selected from at least one of cancer cells and autoimmune disease-related cells.

[0333] In some embodiments of the present invention, the autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Sjögren's syndrome, myasthenia gravis, celiac disease, type 1 diabetes, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis and scleroderma.

[0334] In some embodiments of the present invention, the target cells are cancer cells, including solid cancer cells and blood cancer cells.

[0335] In some embodiments of the present invention, the blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, high-grade B-cell lymphoma and multiple myeloma.

[0336] In some embodiments of the present invention, the cancer cells are blood cancer cells;

[0337] Preferably, the blood cancer is CD19 + Blood cancer;

[0338] More preferably, the CD19 + 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 and high-grade B-cell lymphoma.

[0339] In some embodiments of the present invention, the cancer cell is a blood cancer cell, and the blood cancer is selected from CD19+ Blood Cancers and CD33 + Blood cancer;

[0340] Preferably,

[0341] CD19 + a blood cancer 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;

[0342] CD33 + The 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).

[0343] In another aspect, the present invention further provides a TCP, which is any of the aforementioned TCPs provided by the present invention.

[0344] In another aspect, the present invention further provides a polynucleotide encoding any of the aforementioned TCPs provided by the present invention.

[0345] In some embodiments of the present invention, the polynucleotide is isolated.

[0346] In another aspect, the present invention also provides a method for transducing T cells, comprising contacting any one of the aforementioned viral vectors provided by the present invention with T cells.

[0347] In some embodiments of the present invention, the T cells are selected from at least one of activated T cells and non-activated T cells.

[0348] In some embodiments of the present invention, the contact occurs in vivo and / or in vitro in a subject; the subject is an individual who is administered T cells transduced by the method for transducing T cells and / or any of the aforementioned viral vectors provided by the present invention.

[0349] In another aspect, the present invention also provides an engineered T cell, which comprises the polynucleotide encoding any one of the aforementioned TCP molecules provided by the present invention and / or expresses any one of the aforementioned TCP molecules.

[0350] In some embodiments of the present invention, the engineered T cells are prepared by contacting T cells with any of the aforementioned viral vectors provided by the present invention.

[0351] In some embodiments of the present invention, the T cells are selected from at least one of activated T cells and non-activated T cells.

[0352] In some embodiments of the present invention, the contacting occurs in vivo and / or in vitro in a subject, and the subject is an individual who is administered the engineered T cells and / or any of the aforementioned viral vectors provided by the present invention.

[0353] In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implantation device administration.

[0354] In another aspect, the present invention also provides a composition comprising a pharmaceutically acceptable excipient or carrier and (a) any one of the aforementioned viral vectors provided by the present invention or (b) any one of the aforementioned engineered T cells.

[0355] In another aspect, the present invention also provides the use of any of the aforementioned TCPs, polynucleotides, viral vectors, engineered T cells or compositions provided by the present invention in the preparation of a drug for preventing and / or treating cancer.

[0356] In another aspect, the present invention also provides a method for treating cancer in a subject or killing cancer cells in a subject, comprising administering to the subject any one of the aforementioned viral vectors, engineered T cells or compositions provided by the present invention.

[0357] In some embodiments of the present invention, the cancer includes solid cancer and blood cancer.

[0358] In some embodiments of the present invention, the cancer is a blood cancer.

[0359] In some embodiments of the present invention, the blood cancer is selected from marginal zone lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, primary central nervous system lymphoma, primary mediastinal lymphoma B-cell lymphoma, small lymphocytic lymphoma, B-cell prolymphocytic leukemia, follicular lymphoma, Burkitt lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin lymphoma, high-grade B-cell lymphoma and multiple myeloma.

[0360] In some embodiments of the present invention, the blood cancer is CD19 + Blood cancer.

[0361] In some embodiments of the present invention, the CD19 +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 and high-grade B-cell lymphoma.

[0362] In some embodiments of the present invention, the blood cancer is selected from CD19 + Blood Cancers and CD33 + Blood cancer;

[0363] Preferably,

[0364] CD19 + a blood cancer 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;

[0365] CD33 + The 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).

[0366] In some embodiments of the present invention, the cancer cells express at least one antigen selected from CD19, CD20, CD33, MSLN, CD79B, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2 and GUCY2C.

[0367] In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implant device.

[0368] The beneficial effects of the present invention include:

[0369] The viral vector provided by the present invention comprises (a) a polynucleotide encoding any of the aforementioned TCPs, which can effectively reduce false transduction when the viral vector transduces target cells; (b) comprises a primary signaling molecule for T cell activation, such as an anti-CD3 antibody or an antigen-binding fragment thereof, and can further comprise a secondary signaling molecule for T cell activation, such as an anti-CD28 antibody or an antigen-binding fragment thereof; compared with other T cell targeting molecules, such as an anti-CD7 antibody or an antigen-binding fragment thereof, the viral vector can more effectively activate and stimulate T cells, thereby achieving higher expression efficiency and better killing efficiency of the TCP molecule in T cells;

[0370] More importantly, the inventors of the present invention discovered for the first time that when the primary signal molecule for T cell activation is an anti-CD3 antibody or an antigen-binding fragment thereof and cannot bind to the TSP contained in the TCP, for example, when the anti-CD3 antibody or its antigen-binding fragment is derived from the anti-CD3ε antibody UCHT1 (such as the UCHT1-scFv), the TSP is CD3γ rather than CD3ε, and the transduction efficiency of the viral vector is higher.

[0371] definition:

[0372] "T cells": T cells are one of the important white blood cells in the human immune system and play an important role in acquired immune responses. One of the main functions of T cells is immune-mediated cell death, which is mainly performed by two T cell subtypes: CD8 + T cells (Cytotoxic T Cell, cytotoxic T cells) and CD4 + T cells (Helper T Cell, helper T cells).

[0373] In some embodiments of the present invention, the T cells are CD4 + / CD8 - 、CD4 - / CD8 + 、CD4 + / CD8 + 、CD4 - / CD8 - T cells or a combination thereof. In some embodiments of the present invention, CD4 + T cells express TCP molecules and bind to target cells such as CD19 + Cancer cells then produce IL-2, IFN, TNF or a combination thereof. In some embodiments of the present invention, CD8 + T cells lyse antigen-specific target cells after expressing TCP molecules and binding to target cells.

[0374] "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.

[0375] "TCR / CD3 complex subunit or its functional fragment": TCR / CD3 complex subunit is selected from at least one of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3ζ, CD3δ and CD3ε; and its functional fragment is the minimum unit of the TCR / CD3 complex subunit, including the extracellular, transmembrane, intracellular, variable and constant domains, which are sufficient to perform their respective functions.

[0376] In some embodiments of the present invention, the TCP molecules provided by the present invention can be (a) incorporated into endogenous TCR / CD3 complexes, endogenous TCR / CD3 complex subunits or functional fragments thereof in T cells; and / or (b) functionally interact with endogenous TCR / CD3 complexes, endogenous TCR / CD3 complex subunits or functional fragments thereof, for example, forming a TCR / CD3 complex or a functional fragment thereof with an endogenous CD3 subunit or a functional fragment thereof, thereby being expressed outside the membrane and anchored on the cell membrane of the T cell.

[0377] Unlike CAR molecules, cells other than T cells do not contain TCR / CD3 complex subunits such as TCRα, TCRβ, CD3γ, CD3ε, CD3γ, CD3ζ and CD3δ. Therefore, even under the action of signal peptides, the TCP molecule will not or relatively rarely be expressed in cells other than T cells, such as the common packaging cells HEK-293T cells. Therefore, it will not be transferred to the envelope of the packaged lentiviral vector or retroviral vector during the budding process.

[0378] "CD19": CD19 is the most widely used target in CAR-T therapy and has been proven to be effective and safe in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma. CD19 is widely and specifically expressed throughout the developmental stages of B cells until terminal differentiation into plasma cells. Therefore, CD19 has perfect coverage for B-cell malignancies, which has led to a very high complete remission rate (CRR) in CAR-T-19 therapy (Wei, J., Han, X., Bo, J. et al. Target selection for CAR-T therapy. J Hematol Oncol 12, 62 (2019)).

[0379] "CD33": CD33 is a sialoadhesive protein composed of a membrane-proximal immunoglobulin variable region (IgV) and a membrane-proximal immunoglobulin constant region (IgC) domain. It has become a viable target due to its almost universal expression on acute myeloid leukemia (AML) cells. Notably, CD33 is also present on precursor / mature myeloid cells and hematopoietic stem cells (HSCs) but is not essential for the development and function of human myeloid cells. This provides the possibility of adopting strategies to eliminate all CD33-positive malignant and normal hematopoietic cell populations, followed by hematopoietic restoration by using engineered CD33-negative HSCs (Freeman R, Shahid S, Khan AG, et al. Developing a membrane-proximal CD33-targeting CAR T cell. J Immunother Cancer. 2024 May 20;12(5):e009013.).

[0380] "Flexible linkers": Flexible linkers are usually used when the connected domains need to move or interact to a certain extent (Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69.). Flexible linkers are usually composed of small, non-polar (such as Gly) or polar (such as Ser or Thr) amino acids (Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990; 211: 943–958.). These small amino acids provide flexibility while also allowing the movement of the connected functional domains. Commonly used flexible linker peptides are described in Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69, which is incorporated herein by reference in its entirety. The present invention does not particularly limit the flexible linker peptide that connects the TSP and the antigen-binding region, as long as it can provide a certain degree of flexibility between the antigen-binding region and the TSP, including but not limited to (G4S) n Connector peptide, connector peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23) or connector peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 63); wherein, n=1 to 4, optionally n=3, i.e. (G4S)3 connector peptide (connector peptide 2).

[0381] "Antibody" refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to specifically bind to an antigen. "Antibody" herein encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

[0382] 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.

[0383] In the context of antibodies, the term "variable region" or "variable domain" refers to the domain of the antibody heavy chain or light chain that is involved in binding the antibody to the antigen. The variable regions of the heavy and light chains (VH and VL regions, respectively) of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007)). A single VH region or VL region may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using VH or VL regions from antibodies that bind to that specific antigen to screen for libraries of complementary VL or VH regions, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0384] The terms "complementarity determining region" and "CDR", synonymous with "hypervariable region" or "HVR", are known in the art to refer to non-contiguous sequences of amino acids within an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0385] In some embodiments of the present invention, the CDR regions are identified according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, or the Contact numbering scheme.

[0386] In some embodiments of the present invention, the CDR regions are identified according to the Kabat numbering scheme.

[0387] The term "antibody" herein also includes single-chain variable region fragments ("scFv").

[0388] The term "antibody" herein also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by dromedary camels (Camelus Dromedarius), Bactrian camels (Camelus Bactrianus), llamas (Lama Glama), guanacos (Lama Guanicoe) and alpacas (Vicugna Pacos), as well as immunoglobulin new antigen receptors (Ig New Antigen Receptor, IgNAR) found in cartilaginous fish such as sharks.

[0389] The terms "VHH domain" and "single domain antibody" (sdAb) have the same meaning and are used interchangeably herein. They refer to the construction of a single domain antibody (sdAb) consisting solely of a single heavy chain variable region by cloning the variable region of a heavy chain antibody. This is the smallest fully functional antigen-binding fragment. 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 domain antibody consisting solely of a single heavy chain variable region.

[0390] "Antibody" herein also includes monoclonal antibodies or antigen-binding portions thereof. Monoclonal antibodies or antigen-binding portions thereof can 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).

[0391] 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).

[0392] "Antibody" herein also includes the heavy chain variable region (VH) or light chain variable region (VL) of the antibody.

[0393] Herein, "antigen-binding fragment" refers to a fragment that does not have the entire structure of an intact antibody and only contains a portion or a partial variant of the intact antibody, wherein the portion or partial variant has the ability to bind to an antigen.

[0394] Exemplary, herein, "antibody or antigen-binding fragment thereof" includes but is not limited to immunoglobulins (full-length antibodies), half antibodies, Fab, Fab', F(ab')2, Fv fragments, single-chain variable region fragments (scFv), disulfide bond-stabilized antibodies (dsFv), the heavy chain variable region (VH) or light chain variable region (VL) of antibodies, Fd fragments consisting of VH and CH1 domains, linear antibodies and single-domain antibodies (nanoantibodies); the heavy chain (VH) of the scFv is connected to the light chain (VL) by a connecting peptide.

[0395] In some embodiments of the present invention, there is no particular limitation on the order in which the scFv comprises the VH region or the VL region from the N-terminus to the C-terminus, such as VH-Linker-VL or VL-Linker-VH from the N-terminus to the C-terminus; the connecting peptide can be selected from a flexible connecting peptide.

[0396] "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.

[0397] "Receptor binding fragment" refers to a fragment that lacks the full structure of a complete ligand and contains only a portion or partial variant of the complete ligand, which possesses the ability to bind to the receptor. Exemplarily, "receptor binding fragment" herein includes, but is not limited to, the extracellular domain, functional fragment, epitope, binding region, and variable region of the ligand.

[0398] 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, which 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).

[0399] "Chimeric Antigen Receptor": Chimeric Antigen Receptor (CAR) refers to an artificial cell surface receptor that has been modified to be expressed on immune effector cells such as lymphocytes and specifically binds to antigens, which at least comprises (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 comprise a hinge region, and the intracellular structure may further comprise one or more costimulatory molecules to form a costimulatory signaling domain. The CAR molecule 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.

[0400] "Chimeric": The term "chimeric" refers to any nucleic acid molecule or protein that is non-endogenous and comprises a combination of sequences joined or linked together that are not naturally joined or linked together in nature. For example, a chimeric nucleic acid molecule can comprise nucleic acids encoding various domains from multiple different genes. As another example, a chimeric nucleic acid molecule can comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature.

[0401] "Antigen": The terms "antigen" and "Ag" refer to a molecule capable of inducing an immune response. The induced immune response may include the production of antibodies and / or the activation of specific immune competent cells. Macromolecules including proteins, glycoproteins and glycolipids can be used as antigens. Antigens can be derived from recombinant or genomic DNA. As contemplated herein, an antigen need not be (i) encoded solely by the full-length nucleotide sequence of a gene or (ii) fully encoded by a gene. Antigens can be generated or synthesized, or the antigen can be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

[0402] "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%.

[0403] "Nucleic acid" refers to any compound and / or substance including a polymer containing nucleotides, such as a polynucleotide. As used herein, "nucleic acid," "polynucleotide," and "gene" are used synonymously. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. As used herein, the term "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. "Nucleic acid" can be linear or circular. In addition, "nucleic acid" includes both a sense strand (coding strand) and an antisense strand (template strand), as well as single-stranded and double-stranded forms. Furthermore, the "nucleic acids" described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include nucleotide bases modified with derivatized sugars, phosphate backbone linkages, or chemically modified residues.

[0404] "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.

[0405] "Transgene": i.e., Transgene, also known as load gene (Payload gene). As used herein, the term "transgene" refers to a gene or polynucleotide encoding a protein of interest (e.g., any of the aforementioned TCPs, etc.), the expression of which is desired in host cells / target cells and has been transferred into the cells by genetic engineering techniques. Transgenes can encode therapeutically significant proteins as well as proteins that serve as reporters, tags, markers, suicide proteins, etc. Transgenes can be derived from natural sources, modifications of natural genes, or recombinant or synthetic molecules. In certain embodiments, transgenes are components of viral vectors.

[0406] "Expression cassette": As used herein, the term "expression cassette" refers to a unique component of a vector nucleic acid that comprises at least one transgene and regulatory sequences (e.g., promoter, 3'UTR) that control its expression in a host cell. A tandem expression cassette refers to a component of a vector nucleic acid that comprises at least two transgenes that are under the control of a set of identical regulatory sequences for tandem expression of the at least two transgenes. In certain embodiments, the tandem expression cassette comprises at least two transgenes under the control of the same promoter. In certain embodiments, the first transgene and the second transgene are separated by an internal ribosome entry site (IRES), a furin cleavage site, or a self-cleaving viral 2A peptide to allow co-expression of two proteins from a single mRNA.

[0407] 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.

[0408] "Encoding": refers to the inherent property of a specific polynucleotide sequence (such as DNA, cDNA and mRNA sequences) used as a template for synthesizing other polymers and macromolecules in biological processes, wherein the template has a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. Therefore, if the transcription and translation of the mRNA corresponding to the polynucleotide produces a protein in a cell or other biological system, the polynucleotide encodes the protein. Both the coding strand and the non-coding strand can be referred to as encoding proteins or other products of the polynucleotide. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.

[0409] "Self-cleaving peptide" or "self-cleaving peptide" or "2A peptide": refers to a self-cleaving peptide that is configured to generate two or more proteins from a single open reading frame, including FT2A peptide, F2A peptide, E2A peptide, T2A peptide and P2A peptide, etc. 2A peptides are 18 to 22 residues long viral oligopeptides that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. "2A peptide" can refer to peptides with different amino acid sequences. In the present disclosure, it should be understood that when a viral vector contains two or more 2A peptides, the 2A peptides may be the same or different from each other. Detailed methods for designing and using 2A peptides are provided by Szymczak-Workman et al. (2012) Cold Spring Harb. Protoc. 2012: 199-204.

[0410] "Exogenous" refers to any molecule that originates from outside an organism, including nucleic acids, proteins, peptides, or small molecule compounds. In contrast, the term "endogenous" refers to any molecule that originates from within an organism (i.e., produced naturally by the organism).

[0411] "Promoter": As used herein, the term "promoter" is defined as a DNA sequence that is recognized by the cellular synthetic machinery or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence. As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

[0412] 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.

[0413] 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.

[0414] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is of the tissue type corresponding to the promoter.

[0415] "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 genetic material during the virus's 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 they infect target cells by fusing the viral envelope with the cell membrane.

[0416] "Lentivirus": Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. This greater complexity allows the virus to regulate its life cycle, as it does during latent infection. Lentiviruses belong to a genus of retroviruses that can infect both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0417] "Lentiviral vector": A 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 the 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 results in biosafety for lentiviral vectors.

[0418] As used herein, the term "lentiviral vector" is intended to mean a lentiviral particle that includes a viral envelope, has at least one characteristic of a lentivirus, and is capable of invading target cells without the ability to replicate itself.

[0419] Commonly used pseudotyped lentiviral vectors include so-called third-generation lentiviral packaging systems. Third-generation lentiviral packaging systems typically consist of four plasmids: a transfer plasmid (encoding a gene of interest, "GOI"), such as a transgene; a GagPol plasmid; a Rev plasmid; and an envelope plasmid (encoding a viral glycoprotein gene, such as VSV-G or its variants or Cocal-G or its variants).

[0420] The "transfer plasmid" contains the lentiviral vector backbone genome and 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 process of the viral genome. Through these sequences, the lentivirus can integrate the transgene into the genome of the host cell. For safety reasons, the transfer plasmid is usually designed so that the resulting viral vector cannot replicate itself, for example, the transfer plasmid lacks the genetic elements necessary to produce an infectious lentiviral vector in the host cell. In addition, the transfer plasmid can be designed to lack the 3'LTR, thereby making the virus "self-inactivating". Compared with the traditional second-generation pseudotype lentiviral vector packaging system (usually a single packaging plasmid containing nucleic acids encoding Gag, Pol, Rev and Tat and a separate envelope plasmid), the TAT gene is eliminated from the third-generation pseudotype lentiviral vector packaging system by adding a chimeric 5'LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) to the transfer plasmid. The transfer plasmid usually contains a Ψ sequence (Psi sequence, also known as Ψ packaging signal) located downstream of the 5'LTR. The Ψ sequence is responsible for packaging the transgenic RNA into the viral vector. The Ψ sequence ensures that only RNA containing the transgene is packaged into the viral vector. The transfer plasmid may also optionally contain an internal ribosome entry site (IRENS). In some embodiments, the transfection site (IRES) can be used to allow simultaneous translation of two or more open reading frames (ORFs) on a single mRNA, thereby achieving multi-gene expression. Some transfer plasmids, such as the lentiviral master plasmid / transfer plasmid used in some embodiments of the present invention, may also contain a selectable marker gene, such as an antibiotic resistance gene (e.g., PuroR, encoding puromycin resistance) or a fluorescent protein gene (e.g., GFP), for screening or tracking transduced cells.

[0421] For details on transfer plasmids in lentiviral vector packaging systems, see Dull, et al., J. Virol. 72: 8463-71 (1998); Miyoshi, et al., J. Virol. 72: 8150-57 (1998).

[0422] 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 that contains the lentiviral structural proteins, which include the matrix, capsid, and nucleocapsid; the Pol gene encodes the Pol polyprotein precursor that provides the lentiviral enzyme functions necessary for replication, which 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 promotes the transport of incompletely spliced ​​viral RNA from the cell nucleus to the cytoplasm by interacting with the host cell's nuclear export machinery. These unspliced ​​RNAs can be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral vectors.

[0423] Exemplarily, the packaging plasmid includes but is not limited to pMD2.G, pRSV-rev, pMDLG-pRRE and pRRL-GOI.

[0424] 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.

[0425] In contrast to pseudotyped lentiviral packaging systems, pseudotyped retroviral packaging systems typically do not include a Rev plasmid. This is because genomic RNA from retroviruses such as Moloney Murine Leukemia Virus (MMLV) is naturally transported from the nucleus to the cytoplasm for translation and assembly, thus eliminating the need for specialized nuclear export mechanisms such as the Rev protein. Pseudotyped retroviral packaging systems typically consist of a transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transgene sequence contained in the transfer plasmid is flanked by long terminal repeats (LTRs), which facilitate integration of the transfer plasmid sequence into the host genome. Typically, during viral transduction, sequences between and including the LTRs are integrated into the host genome. The backbone genome of MMLV or murine stem cell virus (MSCV), including its respective LTRs, is typically used to construct the transfer plasmid in pseudotyped retroviral packaging systems. The GagPol packaging plasmid contains the Gag gene and the Pol gene; the envelope plasmid generally contains a polynucleotide encoding a viral glycoprotein, such as VSV-G or Cocal-G. In some embodiments of the present invention, the envelope plasmid may further contain a nucleic acid encoding the T cell activation primary signaling molecule and / or the T cell activation secondary signaling molecule.

[0426] In some embodiments, the production cells are transfected with a defined ratio of transfer plasmids, GagPol plasmids, envelope plasmids, and Rev plasmids. In some embodiments, the ratio of each plasmid is determined by mass, and there is no particular limitation as long as it can package a non-integrated lentiviral vector with biological activity. In some embodiments, the mass of each of the transfer plasmid and the GagPol plasmid is higher than the mass of each of the envelope plasmid and the Rev plasmid. In some embodiments, the defined ratio of the transfer plasmid, GagPol plasmid, envelope plasmid, and Rev plasmid is about 1:1:1:1 to about 9:4:2:2; in some embodiments of the present invention, the envelope plasmid may contain a nucleic acid encoding the T cell activation primary signaling molecule and / or the T cell activation secondary signaling molecule.

[0427] In some embodiments, the envelope plasmid comprises a tandem expression cassette encoding any one of the aforementioned VSV-G or its variants or Cocal-G or its variants and the T cell activation primary signal molecule and / or T cell activation secondary signal molecule as disclosed herein. In a specific embodiment, the tandem expression cassette contained in the envelope plasmid comprises a polynucleotide encoding a first signal peptide, a polynucleotide encoding the T cell activation primary signal molecule and / or the T cell activation secondary signal molecule, a polynucleotide encoding an internal ribosome entry site (IRES), a furin cleavage site or one of the viral 2A peptides, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding VSV-G or its variants or Cocal-G or its variants. In certain embodiments, the polynucleotide encoding VSV-G or its variants or Cocal-G or its variants is located at the 5' end of the polynucleotide encoding the T cell activation primary signal molecule and / or the T cell activation secondary signal molecule. In other embodiments, the polynucleotide encoding VSV-G or its variants or Cocal-G or its variants is located at the 3' end of the polynucleotide encoding the T cell activation primary signal molecule and / or the T cell activation secondary signal molecule. The polynucleotide encoding the T cell activation primary signaling molecule and / or the T cell activation secondary signaling molecule and the polynucleotide encoding VSV-G or its variant or Cocal-G or its variant are separated in a tandem cassette by a polynucleotide encoding an IRES, a furin cleavage site or a viral 2A peptide, which allows the co-expression of the two proteins from a single mRNA. In certain embodiments, the viral 2A peptide is porcine Teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A) or variants thereof.

[0428] The use of lentiviral / retroviral vector packaging systems relies on a "packaging cell line." Generally speaking, a packaging cell line is a cell line that, when a transfer plasmid or one or more packaging plasmids are introduced into the cells, 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 Harbor Laboratory Press, 1997, p. 447, which is incorporated herein by reference in its entirety.

[0429] Exemplarily, various plasmids can be introduced into the packaging cell line using transfection methods including chemical-mediated transfection methods, physical-mediated transfection methods or biological-mediated transfection methods. For example, chemical-mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran or PEI (Polyethylenimine, polyethyleneimine transfection reagent), and physical-mediated transfection methods include transfection methods such as electroporation.

[0430] 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.

[0431] Exemplarily, the packaging cell / host cell is selected from CHO cells, BHK cells, MDCK cells, C3H-10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells and 211 cells;

[0432] Preferably, the packaging cell / host cell is a HEK-293T cell.

[0433] "Retrovirus" and "Retroviral Vector": Retrovirus and Retroviral Vector. "Retrovirus" refers to an RNA virus with a single-stranded positive-sense RNA molecule. Retroviruses contain reverse transcriptase and integrase. After entering the target cell, the retrovirus uses its reverse transcriptase to transcribe its RNA molecule into a DNA molecule. Subsequently, the DNA molecule is integrated into the host cell genome using integrase. After integration into the host cell genome, the sequence from the retrovirus is called a provirus (e.g., a proviral sequence or a proviral sequence). Retroviral vectors typically refer to pseudotyped retroviral vectors derived from retroviruses, illustratively from γ-retroviruses. Unlike lentiviral vectors that can transduce dividing and non-dividing cells, retroviral vectors can only transduce dividing cells, and the exogenous transgenes they can carry are generally relatively small. For a comparison and discussion of lentiviral vectors and retroviral vectors, see Stripecke, R., Kasahara, N. (2007). Lentiviral and Retroviral Vector Systems. In: Hunt, KK, Vorburger, SA, Swisher, SG (eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

[0434] 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.

[0435] "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.

[0436] "Variant": A variant refers to a mutant having at least about 50% identity to the amino acid sequence of a non-mutant (wild type), and "at least about 50% identity" refers to about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the amino acid sequence of the non-mutant (wild type); or, a variant refers to a variant that is identical to the nucleic acid sequence encoding the non-mutant (wild type). "At least 50% identity" means that the nucleic acid sequence encoding the variant is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the nucleic acid sequence encoding the non-mutant (wild type).

[0437] In some embodiments of the present invention, variants include mutants comprising conservative substitutions relative to non-mutants. "Conservative substitutions" are considered in the art to replace an amino acid with another amino acid having similar properties. For example, conservative substitutions are well known in the art (see, for example, WO97 / 09433, page 10, published on March 13, 1997; Lehninger, Biochemistry, 2nd edition; Worth Publishers, Inc. NY: NY (1975), pages 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), page 8).

[0438] "Pharmaceutically acceptable excipient or carrier": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, preservatives, and / or adjuvants. Excipients are preferably nontoxic or substantially nontoxic to the recipient at the dosages and concentrations employed. Such excipients include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, pharmaceutical compositions may contain substances for improving, maintaining, or preserving, for example, the pH, osmotic properties, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition. The optimal pharmaceutical composition can be determined based on the intended route of administration, mode of delivery, and desired dosage.

[0439] Pharmaceutical compositions for in vivo administration are typically provided as sterile formulations. Sterilization is achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization, reconstitution, or dilution. The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, they can be prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposomal carriers, bioerodible microparticles or porous beads, and depot injection) are also known to those skilled in the art.

[0440] Once the pharmaceutical composition is formulated, it is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form (e.g., lyophilized) that is redissolved before administration. The present invention also provides a test kit for producing a single-dose administration unit. The test kit of the present invention can each contain a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, a test kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilizing syringe) is provided.

[0441] "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.

[0442] A "disease" is a state of health in a subject in which the subject is unable to maintain homeostasis and in which the subject's health continues to deteriorate if the disease does not improve. In contrast, a "disorder" or "adverse condition" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's health is less favorable than it would be in the absence of the disorder or adverse condition. Without treatment, a disorder or adverse condition does not necessarily result in a further deterioration in the subject's health.

[0443] "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.

[0444] "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.

[0445] As used herein, "treatment" includes any beneficial or desired effect associated with treatment."Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or its associated symptoms.

[0446] 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.

[0447] 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.

[0448] "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.

[0449] “And / or”: should be understood to mean one or two alternatives.

[0450] "About" / "approximately": As used herein, the term "about" refers to the usual error range for the corresponding value as readily known to those skilled in the art, including, by way of example, but not limited to, reference to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. Reference herein to "about" a value or parameter includes (and describes) embodiments for the value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0451] "Comprising": As used herein, unless the context requires otherwise, the word "comprising" will be understood to mean the inclusion of the specified steps, elements, or groups of steps or elements, but not the exclusion of any other steps, elements, or groups of steps or elements. In some embodiments of the present invention, the terms "including," "having," "containing," and "comprising" are used synonymously.

[0452] "Embodiments": Reference throughout this specification to "some embodiments," "some embodiments," "embodiments," "specific embodiments," "related embodiments," "an embodiment," "another embodiment," or "other embodiments" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, the various appearances of the foregoing phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0453] "Prevention": As used herein, "prevention" and similar words, such as "preventing," refer to methods used to prevent, inhibit, or reduce the likelihood of the occurrence or recurrence of a condition. As used herein, "prevention" and similar words also include lessening the intensity, effects, symptoms, and / or burden of a disease or condition prior to onset or recurrence.

[0454] "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 to transient transfection and transient expression (Transient Expression).

[0455] "Specific binding": As used herein, the term "specific binding" refers to the binding that occurs between paired molecular species (e.g., a receptor and a ligand, an antibody and an antigen). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, the specific binding between one or more species is direct. In some embodiments of the invention, the affinity of the specific binding is about 2 times greater than background binding (non-specific binding), about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.

[0456] "Sequence identity": Generally speaking, "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 with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity." Whether it is a nucleic acid or amino acid sequence, 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. For example, the advanced BLAST computer program available from the National Institutes of Health can also be used to compare sequence information to determine the percent identity. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). Briefly, the BLAST program defines identity as the number of aligned symbols (usually nucleotides or amino acids) that are identical divided by the total number of shorter symbols in the two sequences. The program can be used to determine percent identity over the entire length of the compared proteins.

[0457] "Signal peptide": Signal peptide, sometimes also called signal sequence, targeting signal, localization signal, localization sequence, transit peptide or leader peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), present at the N-terminus of most newly synthesized proteins that enter the secretory pathway (occasionally non-classically present at the C-terminus or internally) (Owji, et al., A comprehensive review of signal peptides: Structure, roles, and applications, European Journal of Cell Biology. 97(6): 422-441. (2018)) (Blobel G, Dobberstein B, et al., Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma, The Journal of Cell Biology, 67(3):835-51.(1975)).

[0458] Signal peptides are short peptides present at the N-terminus of newly synthesized proteins that are specific for the plasma membrane or secretory pathway. Signal sequences typically contain a short stretch of hydrophilic, positively charged amino acids at the N-terminus, a central hydrophobic domain of 5-15 residues, and a C-terminal region with a signal sequence cleavage site. In eukaryotes, signal sequences cause newly synthesized proteins to translocate to the endoplasmic reticulum, where the protein is cleaved by a signal peptidase to produce the mature protein, which then enters its appropriate destination. The diversity of signal sequence length and amino acid composition makes it difficult to accurately predict the cleavage site. For the polypeptide sequences disclosed herein, when referring to a signal sequence, polypeptide sequences in which no signal sequence or a partial signal sequence is present are also contemplated.

[0459] The effect of signal peptide is to promote cell transfer protein, usually transferred to the cell membrane. In prokaryotes, the newly synthesized protein is guided to the SecYEG protein conduction channel present in the plasma membrane by signal peptide. There is a homologous system in eukaryotes, in which the newly synthesized protein is guided to the Sec6L channel by signal peptide, 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 commonly referred to as transporters, and the transport by the channel is called translocation. When secreted protein passes through the channel, the transmembrane region may diffuse through the side gate in the translocon to be distributed in the surrounding membrane.

[0460] "MOI": Multiplicity of Infection (MOI) refers to the number of viral particles added to each cell during infection. For example, when one million viral particles are added to one million cells, MOI = 1.

[0461] "Operably": A polynucleotide is "operably linked" when it is in a functional relationship with another polynucleotide. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned so as to promote translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the polynucleotides being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0462] "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.

[0463] Methods for introducing vectors such as viral vectors or isolated polynucleotides into mammalian cells are known in the art. The described vectors can be transferred to immune effector cells by physical, chemical or biological methods.

[0464] Physical methods for introducing vectors or isolated polynucleotides into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, EF and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments of the present invention, vectors are introduced into cells by electroporation. In some embodiments of the present invention, vectors are introduced into cells by PEI (Polyethylenimine, polyethyleneimine transfection reagent) transfection reagent.

[0465] 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.

[0466] Chemical methods for introducing vectors or isolated polynucleotides into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome.

[0467] All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety, just as if each individual publication, document, or patent were not specifically and individually indicated as being incorporated by reference in its entirety. In the event of a conflict, the present application (including any definitions herein) will control. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be taken as an admission or any form of suggestion or that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0468] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. BRIEF DESCRIPTION OF THE DRAWINGS

[0469] 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;

[0470] 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;

[0471] Figure 3 shows the expression of CD3 in PBMCs of the CD19-TCP-E / G molecule in each group after the control group lentiviral vector m-7-E, the targeted lentiviral vectors m-TCP-E and m-TCP-G were transduced into non-activated human PBMCs in Example 2. + Flow cytometry results of membrane expression efficiency in T cells;

[0472] Figure 4 shows the CD3 expression in each group of PBMCs after the targeted lentiviral vector m-TCP-G activated and stimulated the non-activated PBMCs of Donor 1 and Donor 2 in Example 3. + T cell proliferation curve;

[0473] 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;

[0474] 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;

[0475] Figure 7: In Example 6, the control group lentiviral vector m-7-G, the targeted lentiviral vector m-3-G and m-TCP-G were used to transduce non-activated human PBMCs in each group, wherein CD3 + Flow cytometry results of the efficiency of T cells expressing the CD19-TCP-G molecule;

[0476] Figure 8: The targeted lentiviral vectors m-TCP-G and m-86-G were used to transduce non-activated human PBMCs in Example 7, where CD3 + Flow cytometry results of the efficiency of T cells expressing the CD19-TCP-G molecule;

[0477] Figure 9: CD3 expression in non-activated human PBMCs after transduction of the targeted lentiviral vector m-a33-G in Example 8 + Figure 2 is a flow cytometry result showing the efficiency of T cells expressing the CD33-TCP-G molecule.

[0478] Figure 10: CD33-TCP-T cells kill CD33 in vitro in Example 8 + Figure 3 shows the flow cytometry results of the killing efficiency of target MOLM-13 cells. DETAILED DESCRIPTION

[0479] The following is a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments, so that the purpose, features and effects of the present invention are fully understood. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

[0480] In the following examples, the experimental methods without specific conditions are based on conventional methods and conditions known in the art, or are selected according to the product specifications. Reagents and raw materials not specified in the present invention are all commercially available.

[0481] Example 1: Construction of a lentiviral vector m-TCP-E / G targeting non-activated T cells

[0482] 1. Design of membrane-expressed anti-CD3 antibody × anti-CD28 antibody structure

[0483] In this embodiment, the polynucleotides encoding membrane-expressed anti-CD3 antibody × anti-CD28 antibody (membrane-expressed anti-CD3 × anti-CD28 dual antibody) are as follows from the 5' end to the 3' end: a polynucleotide encoding a human CD8α signal peptide, a polynucleotide encoding the UCHT1-scFv, a polynucleotide encoding a human CD8α hinge region, a polynucleotide encoding a human CD8α transmembrane region, a polynucleotide encoding an FT2A peptide, a polynucleotide encoding a human CD8α signal peptide, a polynucleotide encoding the 15E8-scFv, a polynucleotide encoding a human CD8α hinge region, and a polynucleotide encoding a human CD8α transmembrane region;

[0484] (1) The amino acid sequence of the human CD8α signal peptide is shown in SEQ ID NO: 12;

[0485] (2) The amino acid sequence of the UCHT1-scFv is shown in SEQ ID NO: 13;

[0486] (3) the amino acid sequence of the human CD8α hinge region is shown in SEQ ID NO: 14;

[0487] (4) The amino acid sequence of the human CD8α transmembrane region is shown in SEQ ID NO: 15;

[0488] (5) The amino acid sequence of the FT2A peptide is shown in SEQ ID NO: 16;

[0489] (6) The amino acid sequence of the 15E8-scFv is shown in SEQ ID NO: 17.

[0490] Under the action of the self-cleaving peptide FT2A peptide, the membrane-expressed anti-CD3 antibody, UCHT1-scFv (membrane-expressed UCHT1-scFv) and the membrane-expressed anti-CD28 antibody, 15E8-scFv (membrane-expressed 15E8-scFv) are separately expressed on the cell membrane of the packaging cell, and as the lentiviral vector buds out, they are transferred to the envelope of the lentiviral vector, thereby enabling the lentiviral vector to have the ability to target and activate non-activated T cells.

[0491] 2. Construction of T cell receptor chimeric protein

[0492] In this example, a TCP molecule targeting CD19 (CD19-TCP) was constructed; the polynucleotides encoding the CD19-TCP molecule were, from the 5' end to the 3' end, the following: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding the extracellular antigen binding region targeting human CD19, a polynucleotide encoding the connecting peptide 1, and a polynucleotide encoding human CD3ε (CD19-TCP-E) or human CD3γ (CD19-TCP-G);

[0493] (1) The extracellular antigen binding region is the FMC63-scFv that can specifically bind to human CD19; the amino acid sequence of the VH region of the FMC63-scFv is shown in SEQ ID NO: 18, the amino acid sequence of the VL region of the FMC63-scFv is shown in SEQ ID NO: 19; the amino acid sequence of the FMC63-scFv is shown in SEQ ID NO: 64; the amino acid sequences of the HCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 65-67, respectively, and the amino acid sequences of the LCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 68-70, respectively;

[0494] (2) The amino acid sequence of human CD3ε is shown in SEQ ID NO: 21, wherein the CD3ε does not contain its signal peptide and only contains its extracellular domain, transmembrane region and intracellular domain;

[0495] (3) The amino acid sequence of human CD3γ is shown in SEQ ID NO: 22, wherein the CD3γ does not contain its signal peptide, but only contains its extracellular domain, transmembrane region, and intracellular domain;

[0496] (4) The amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO: 23;

[0497] The C-terminus of the extracellular antigen-binding region is operably connected to the N-terminus of the human CD3ε or human CD3γ through the connecting peptide 1;

[0498] The amino acid sequence of CD19-TCP-E is shown in SEQ ID NO: 100;

[0499] The amino acid sequence of the CD19-TCP-G is shown in SEQ ID NO: 101.

[0500] Human CD3ε, Uniprot ID: P07766.

[0501] Human CD3ε without its signal peptide:

[0502] Human CD3γ, Uniprot ID: P09693.

[0503] Human CD3γ without its signal peptide:

[0504] 3. Packaging of Targeted Lentiviral Vector m-TCP-E / G

[0505] A. Prepare the following four plasmids to package the targeted lentiviral vector m-TCP-E / G:

[0506] 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;

[0507] 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;

[0508] The targeted lentiviral vector m-TCP-G comprises a polynucleotide encoding the CD19-TCP-G molecule;

[0509] The targeted lentiviral vector m-TCP-E comprises a polynucleotide encoding the CD19-TCP-E molecule;

[0510] 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.

[0511] Mutant VSV-G (K47 deletion, T214N, and T352A)

[0512] B. Packaging of the targeted lentiviral vector m-TCP-E / G:

[0513] (a) mixing the four plasmids, transfecting the four plasmids into the packaging cell line HEK-293T cell line using a PEI reagent, and packaging the targeted lentiviral vector m-TCP-G or m-TCP-E;

[0514] The specific steps are:

[0515] Prepare the HEK-293T cell culture system: filter 56 mL of FBS into 500 mL of DMEM / high glucose (10% FBS) and add 4 mL of P / S (double antibody, penicillin × streptomycin), shake well, and place in a CO2 incubator to preheat for transfection and neutralization.

[0516] On Day 0, 4.5×10 HEK-293T cells were seeded in a 10 cm culture dish. 6 About 48 hours after inoculation, when the cell confluence is 80-90%, the four plasmids are transfected into packaging cells HEK-293T cells using PEI reagent, including:

[0517] 9 μg CD19-TCP-E / G main plasmid, 4 μg pMDLg / pRRE packaging plasmid, 2 μg pRSV-REV packaging plasmid and the 2 μg envelope plasmid 1 were added to 1 mL of Opti-MEM medium, shaken and added with 64 μL of PEI reagent, pipetted evenly and allowed to stand for 10 minutes, and then added to the culture medium of HEK-293T cells. The culture medium was renewed after 6 hours, and the culture supernatant was collected 48 hours after transfection, filtered using a 0.45 μm filter membrane, centrifuged at 50,000 g for 2.5 hours, and the supernatant was discarded; the lentiviral vector m-TCP-E or m-TCP-G was resuspended in 200 μL of 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.

[0518] 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.

[0519] Opti-MEM alpha Reduced Serum Medium: Brand: GIBCO, Catalog Number: #SP0272;

[0520] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog Number: #C12430500BT; FBS: Brand: EXCELL, Catalog Number: #FSP500;

[0521] F12 culture medium: Brand: GIBCO, catalog number: #C11330500BT;

[0522] Syringe filter: Brand: SORFA, item number: #622120.

[0523] 4. Packaging of the control group lentiviral vector m-CAR

[0524] Referring to the above-mentioned method for packaging the targeting lentiviral vector m-TCP-E / G, the control group lentiviral vector m-CAR was packaged.

[0525] Prepare the following four plasmids: the envelope plasmid 1, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, and a main plasmid carrying a polynucleotide encoding a CD19-CAR molecule;

[0526] The structure of the CD19-CAR molecule from N-terminus to C-terminus is: an extracellular antigen binding region, the human CD8α hinge region, the human CD8α transmembrane region, a human 4-1BB co-stimulatory signaling domain, and a human CD3ζ intracellular signaling domain; the extracellular antigen binding region is the FMC63-scFv;

[0527] The polynucleotide encoding the CD19-CAR molecule is operably linked to the polynucleotide encoding the human CD8α signal peptide, and the human CD8α signal peptide is located at the N-terminus of the CD19-CAR molecule;

[0528] (1) The amino acid sequence of the 4-1BB costimulatory domain is shown in SEQ ID NO: 49;

[0529] (2) The amino acid sequence of the intracellular signaling domain of CD3ζ is shown in SEQ ID NO:50.

[0530] Referring to the above-mentioned method for packaging the targeted lentiviral vector m-TCP-E / G, the control group lentiviral vector m-CAR was packaged and frozen at -80°C; at the same time, the packaging cell HEK-293 cells were collected, and the expression of the CD19-CAR molecule in the HEK-293T cells was detected by flow cytometry. The results are shown in Figure 1.

[0531] As shown in Figure 1, during the packaging of the targeted lentiviral vectors m-TCP-E, m-TCP-G and the control lentiviral vector m-CAR, the CD19-CAR molecule was expressed in large quantities outside the membrane in the packaging cell HEK-293T cells (positive rate of about 74.76%), while the CD19-TCP-E molecule (positive rate of about 4.21%) and CD19-TCP-G molecule (positive rate of about 1.12%) were rarely expressed outside the membrane in the packaging cell HEK-293T cells; therefore, based on the mechanism of budding of the lentiviral vector in the packaging cells, it can be reasonably inferred that the content of the CD19-TCP-E / G molecule in the viral envelope of the lentiviral vector will also be significantly reduced relative to the CD19-CAR molecule, thereby effectively reducing false transduction when the targeted lentiviral vector m-TCP-E / G is used to transduce T cells; and the ability of the targeted lentiviral vector m-TCP-E / G to bind to cancer cell surface antigens and / or transduce cancer cells is significantly reduced relative to the control lentiviral vector m-CAR.

[0532] 5. Detection of the transduction efficiency of Nalm-6 cells transduced with the targeted lentiviral vector m-TCP-E / G and the control lentiviral vector m-CAR

[0533] On Day 0, at an MOI of 5, the targeted lentiviral vectors m-TCP-E, m-TCP-G and the control lentiviral vector m-CAR were added to the Nalm-6 cell culture system (1640 medium + 10% FBS), and 2×10 5 CD19 + Nalm-6 cells (human B lymphoid leukemia cells); Day 2, flow cytometry was used to detect the expression of CD19 in Nalm-6 cells in each group. The results are shown in Figure 2.

[0534] As shown in Figure 2, compared with the positive rate of 97.03% in the blank control group, the expression level of CD19 in the Nalm-6 cell culture system to which the control group lentiviral vector m-CAR was added was significantly reduced (positive rate 34.69%), which proves that the control group lentiviral vector m-CAR can effectively bind to the surface antigen CD19 of Nalm-6 cells through the FMC63-scFv contained in its viral envelope, thereby reducing the expression level of CD19 on the surface of the Nalm-6 cells; and in the Nalm-6 cell culture system to which the targeting lentiviral vector m-TCP-E or m-TCP-G was added, the positive rate of CD19 was maintained at 97.07% or 97.61%, respectively, which proves that the viral envelope of the targeting lentiviral vector m-TCP-E / G does not contain or almost does not contain the FMC63-scFv, making it difficult to specifically bind to the Nalm-6 cell surface antigen CD19, and can effectively maintain the expression abundance of the surface antigen CD19 of Nalm-6 cells. This proves that the TCP molecule provided by the present invention is specifically expressed only in T cells, and is not expressed or hardly expressed in cells other than T cells, such as packaging cells HEK-293T cells.

[0535] Flow cytometry antibody for detecting CD19: CD19-FITC antibody (brand: BD, catalog number: #555412).

[0536] 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

[0537] 1. Packaging control group lentiviral vector m-7-E

[0538] Prepare the following four plasmids to package the control lentiviral vector m-7-E:

[0539] An envelope plasmid (envelope plasmid 2) carrying a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding a membrane-expressed anti-CD7 antibody, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and the CD19-TCP-E main plasmid; the envelope plasmid 2 is synthesized by conventional molecular cloning methods;

[0540] 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;

[0541] The structure of the polynucleotide encoding the membrane-expressed anti-CD7 antibody from the 5' end to the 3' end is: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding a scFv that specifically binds to human CD7 (scFv derived from TH-69, TH69-scFv), a polynucleotide encoding the human CD8α hinge region, and a polynucleotide encoding the human CD8α transmembrane region;

[0542] 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;

[0543] (1) The amino acid sequence of the VH region of the TH69-scFv is shown in SEQ ID NO: 25; the amino acid sequence of the VL region of the TH69-scFv is shown in SEQ ID NO: 26; the amino acid sequence of the TH69-scFv is shown in SEQ ID NO: 71, the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 72-74, respectively, and the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 75-77, respectively;

[0544] (2) the amino acid sequence of the (G4S)3 / connector peptide 2 is shown in SEQ ID NO: 20;

[0545] Referring to the packaging method of the targeted lentiviral vector m-TCP-E / G in Example 1, the control lentiviral vector m-7-E and the targeted lentiviral vectors m-TCP-E and m-TCP-G were packaged in the same batch.

[0546] 2. Transduction of non-activated T cells with targeted lentiviral vectors m-TCP-E / G and m-7-E

[0547] Day 0, take 3 groups of 1×10 6Healthy human non-activated PBMCs were resuspended in 200 μL of PBMCs culture medium, wherein the PBMCs culture medium included XVT medium, IL-7 with a final concentration of 20 ng / mL, and IL-15 with a final concentration of 20 ng / mL; the targeted lentiviral vectors m-TCP-G and m-TCP-E, and the control group lentiviral vector m-7-E were added to the three groups of human non-activated PBMCs culture medium at an MOI of 5, mixed well, and cultured in a 5% CO2, 37°C incubator;

[0548] On Day 5, flow cytometry was used to detect the expression of the CD19-TCP-E / G molecule in the three groups of PBMCs. The results are shown in FIG3 .

[0549] As shown in Figure 3, in the culture medium of the three groups of PBMCs, the control group lentiviral vector m-7-E, the targeted lentiviral vectors m-TCP-E and m-TCP-G respectively transduced CD3 + The expression efficiency of the CD19-TCP-E / G gene delivered by T cells was approximately 5.25%, 12.65%, and 34.81%, respectively;

[0550] First, compared with the control group lentiviral vector m-7-E, the targeted lentiviral vectors m-TCP-G and m-TCP-E stably integrated the polynucleotide encoding the CD19-TCP-E / G molecule into the T cell genome, and the efficiency of the CD19-TCP-E / G molecule in T cell membrane expression was significantly improved; this may be because, compared with the anti-CD7 antibody that cannot effectively activate and stimulate non-activated T cells, the T cell activation primary signal molecule anti-CD3 antibody and the secondary signal molecule anti-CD28 antibody can effectively activate and stimulate non-activated T cells in human non-activated PBMCs, and the efficiency of activated T cells in assembling and expressing TCR / CD3 complexes and the CD19-TCP-E / G molecules (which may be incorporated into endogenous TCR / CD3 complexes and / or functionally interact with endogenous TCR / CD3 complexes) is better than that of significantly non-activated T cells.

[0551] Secondly, as shown in Figure 3, compared with the targeted lentiviral vector m-TCP-E, after the targeted lentiviral vector m-TCP-G transduced human non-activated T cells, the efficiency of the CD19-TCP-G molecule (positive rate of approximately 34.81%) membrane expression was significantly higher than that of the CD19-TCP-E molecule (positive rate of approximately 12.65%).

[0552] This may be because the membrane-expressed anti-CD3 antibody used in this example, and the UCHT1-scFv is an anti-CD3ε antibody; during the packaging process of the targeted lentiviral vector m-TCP-E, the UCHT1-scFv can specifically bind to the CD3ε contained in the CD19-TCP-E molecule in the packaging cell HEK-293T cells, thereby reducing the content of UCHT1-scFv that can be expressed on the cell membrane of the HEK-293T cells and transferred to the envelope of the targeted lentiviral vector m-TCP-E with budding, thereby reducing the ability of the targeted lentiviral vector m-TCP-E to target and activate and stimulate non-activated T cells, ultimately leading to a decrease in transduction efficiency and / or a decrease in the efficiency of membrane expression of the CD19-TCP-E molecule in T cells with relatively low activation levels;

[0553] During the packaging of the targeted lentiviral vector m-TCP-G, because the TCR / CD3 complex subunit contained in the CD19-TCP-G molecule is CD3γ, the anti-CD3ε antibody UCHT1-scFv cannot specifically bind to CD3γ, and therefore will not lead to a reduction in the content of UCHT1-scFv available for the specific T cell surface antigen CD3ε in the envelope of the targeted lentiviral vector m-TCP-G, and thus will not affect the ability of the targeted lentiviral vector m-TCP-G to target and activate and stimulate non-activated T cells, and ultimately will not lead to a decrease in transduction efficiency and / or a decrease in the efficiency of the CD19-TCP-G molecule to express outside the membrane in T cells with a relatively high degree of activation.

[0554] Therefore, when using an anti-CD3 antibody or an antigen-binding fragment thereof as a primary signal molecule for T cell activation and simultaneously using the TCP molecule provided by the present invention, in order to maximize the transduction efficiency of the constructed targeted lentiviral vector, the anti-CD3 antibody or antigen-binding fragment thereof used should not specifically bind to the TSP polypeptide contained in the TCP molecule.

[0555] XVT medium: Trade name: PRIME-XV T cell CDM, brand: IRVINE (FUJIFILM), catalog number: #91154;

[0556] IL-7: Trade Name: IL-7 Protein, Human, Recombinant, Brand: Sino Biological, Catalog Number: #11821-HNAE;

[0557] IL-15: Trade Name: IL-15 Protein, Human, Recombinant (His Tag), Brand: Sino Biological, Catalog Number: #10360-H07E;

[0558] Flow cytometry antibody for detecting CD3: Trade name: FITC Mouse Anti-Human CD3; Brand: BIOLEGEND, Catalog Number: #555339.

[0559] Example 3: Targeted lentiviral vector m-TCP-G can effectively activate and stimulate non-activated T cells

[0560] On Day 0, two groups of 2×10 5 Human non-activated PBMCs were resuspended in 200 μL of the human non-activated PBMCs culture medium;

[0561] On Day 0, at an MOI of 5, the targeted lentiviral vector m-TCP-G was added to the culture medium of one of the non-activated PBMCs of Donor 1 and Donor 2, mixed, and the CD3 + The results of T cell proliferation are shown in FIG4 .

[0562] As shown in FIG4 , 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.

[0563] Example 4: CD19-TCP-T cells kill target cells in vitro

[0564] Day 0, take two groups of 5×10 6 Non-activated PBMCs from a healthy individual, Donor 1, were resuspended in 200 μL of the PBMCs culture medium. One group served as the control group, and the other group was added with the targeted lentiviral vector m-TCP-G at an MOI of 5 to prepare CD19-TCP-T cells (experimental group).

[0565] On Day 1, 1 mL of DPBS was added to each of the control and experimental groups for washing once;

[0566] On Day 5, 3×10 cells were collected from the control group and the experimental group respectively. 5 According to the effector-target ratio E:T=3:1, 1×10 5 target cells Nalm-6 cells, mixed;

[0567] On Day 7, flow cytometry was used to detect the killing of target Nalm-6 cells in the control group and the experimental group, respectively. The results are shown in Figure 5.

[0568] As shown in FIG5 , the CD19-TCP-T cells prepared after the targeted lentiviral vector m-TCP-G transduced T cells can efficiently kill the target cells Nalm-6 cells.

[0569] Example 5: Targeted lentiviral vector m-TCP-G kills cancer cells in vivo

[0570] 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;

[0571] On Day 0, 5×10 5 Nalm-6 cells carrying luciferase were injected into the peritoneal cavity of each group of mice with 200 μL of 15 mg / mL luciferin sodium salt (brand: Yisheng Bio, catalog number: #40901ES10), and in vivo imaging was performed on the three groups of mice respectively;

[0572] Four hours later, 1×10 7 Individual non-activated PBMCs were injected into the experimental group mice alone. 6 The targeted lentiviral vector m-TCP-G viral supernatant of TU;

[0573] On Day 5 and Day 7, live imaging was performed on the three groups of mice, and the results are shown in Figure 6.

[0574] As shown in Figure 6, the targeted lentiviral vector m-TCP-G can transduce human PBMCs cells to prepare CD19-TCP-T cells in the experimental group mice, and effectively kill Nalm-6 cells.

[0575] 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

[0576] 1. Packaging of Lentiviral Vector m-3-G

[0577] Referring to the method for packaging the targeted lentiviral vector m-TCP-E / G in Example 1, the control lentiviral vector m-7-G, targeted lentiviral vectors m-3-G and m-TCP-G were packaged in the same batch;

[0578] Among them, the targeted lentiviral vector m-3-G: (a) the envelope contains a T cell targeting molecule that is a primary signal molecule for T cell activation, the membrane expresses UCHT1-scFv, and does not contain secondary signal molecules for T cell activation and other T cell targeting molecules; (b) contains a polynucleotide encoding the CD19-TCP-G; specifically, the envelope plasmid 1 is replaced with an envelope plasmid (envelope plasmid 3) containing a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding the membrane-expressed UCHT1-scFv.

[0579] On Day 0, at an MOI of 5, 1×10 6 Non-activated PBMCs from a healthy individual, Donor 1;

[0580] Day 5, flow cytometry was used to detect the CD3 + In T cells, the antigen binding region contained in the CD19-TCP-G molecule and the expression of the FMC63-scFv are shown in FIG7 .

[0581] As shown in Figure 7, first, compared with the control group lentiviral vector m-7-G, the targeted lentiviral vectors m-3-G and m-TCP-G transduced human non-activated PBMCs, in which CD3 + T cells express the CD19-TCP-G molecule more efficiently; this may be because membrane-expressed anti-CD7 antibodies cannot effectively activate and stimulate non-activated T cells. For TCP molecules whose expression is limited in T cells and whose expression efficiency may be more dependent on the degree of T cell activation, the transduction efficiency of lentiviral vectors whose viral envelope contains at least primary signaling molecules for T cell activation is relatively higher.

[0582] Secondly, compared with the targeted lentiviral vector m-3-G, the targeted lentiviral vector m-TCP-G transduced human non-activated PBMCs, in which CD3 + T cells expressed the CD19-TCP-G molecule significantly more efficiently;

[0583] This may be because the envelope of the targeted lentiviral vector m-3-G only contains the antigen-specific first signal that simulates the T cell activation signal, that is, the membrane-expressed anti-CD3 antibody of the T cell activation primary signal molecule, but lacks the T cell activation secondary signal molecules such as anti-CD28 antibodies that simulate the second signal, T cell activation co-stimulatory / secondary signal; therefore, compared with the targeted lentiviral vector m-TCP-G whose envelope contains both T cell activation primary and secondary signal molecules, the ability of the targeted lentiviral vector m-3-G to activate and stimulate non-activated T cells is relatively low, resulting in a low efficiency of membrane expression of the TCP molecule in relatively underactivated T cells.

[0584] Example 7: Packaging of a targeted lentiviral vector m-86-G containing an anti-CD3 antibody and CD86

[0585] 1. Design of membrane-expressed anti-CD3 antibody × CD86 construct

[0586] In this embodiment, the polynucleotides encoding membrane-expressed anti-CD3 antibody × CD86 (membrane-expressed anti-CD3 antibody × CD86) are, from 5' to 3' end, the following: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding the UCHT1-scFv, a polynucleotide encoding the human CD8α hinge region, a polynucleotide encoding the human CD8α transmembrane region, a polynucleotide encoding the FT2A peptide, a polynucleotide encoding the human CD86 signal peptide, a polynucleotide encoding the human CD86 extracellular domain, and a polynucleotide encoding the human CD86 transmembrane region;

[0587] Human CD86, Uniprot ID: P42081;

[0588] (1) The amino acid sequence of the human CD86 signal peptide is shown in SEQ ID NO: 30;

[0589] (2) The amino acid sequence of the human CD86 extracellular domain is shown in SEQ ID NO: 31;

[0590] (3) The amino acid sequence of the human CD86 transmembrane region is shown in SEQ ID NO: 32.

[0591] The targeted lentiviral vectors m-TCP-G and m-86-G were packaged in the same batch:

[0592] Referring to the method for packaging the targeted lentiviral vector m-TCP-E / G in Example 1, the packaging envelope comprises the targeted lentiviral vector m-86-G expressing the membrane-expressing anti-CD3 antibody × CD86; specifically, the envelope plasmid 1 is replaced with an envelope plasmid (envelope plasmid 4) carrying a polynucleotide encoding the mutant VSV-G and a polynucleotide encoding the membrane-expressing anti-CD3 antibody × CD86.

[0593] 2. Transduction of human non-activated PBMCs with the targeted lentiviral vector m-86-G

[0594] Day 0, referring to the method of transducing non-activated human PBMCs with the targeted lentiviral vector m-TCP-E / G in Example 1, at an MOI of 5, 1×10 6 Non-activated PBMCs from a healthy individual, Donor 1;

[0595] Day 5, flow cytometry was used to detect the CD3 + The expression of the CD19-TCP-G molecule in T cells is shown in FIG8 .

[0596] As shown in Figure 8, the targeted lentiviral vectors m-TCP-G and m-86-G were used to transduce human non-activated PBMCs, in which CD3 + The efficiency of CD19-TCP-G expression in T cells was approximately 12.66% and 16.15%, respectively; the targeted lentiviral vectors m-TCP-G and m-86-G could effectively transduce CD3 + T cells expressing the CD19-TCP-G.

[0597] Example 8: Construction of a targeted lentiviral vector containing a polynucleotide encoding a TCP molecule targeting CD33

[0598] 1. Construction of TCP containing CD33 targeting

[0599] Referring to the CD19-TCP-G constructed in Example 1, a TCP molecule targeting human CD33 (Uniprot ID: P20138) (CD33-TCP-G) was constructed; specifically, the FMC63-scFv was replaced with an antigen binding region targeting human CD33;

[0600] The polynucleotides encoding the CD33-TCP-G molecule are as follows from the 5' end to the 3' end: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding the extracellular antigen binding region targeting human CD33, a polynucleotide encoding the connecting peptide 1, and a polynucleotide encoding the human CD3γ (CD33-TCP-G);

[0601] 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 NOs:79-81, respectively; and the amino acid sequences of the LCDR1-3 regions of the Gemtuzumab-scFv are shown in SEQ ID NOs:82-84, respectively.

[0602] 2. Packaging of the Targeted Lentiviral Vector m-a33-G

[0603] Referring to the method for packaging the targeted lentiviral vector m-TCP-E / G in Example 1, m-a33-G was packaged.

[0604] 3. Use the targeted lentiviral vector m-a33-G to transduce non-activated T cells to prepare TCP-T cells

[0605] Referring to the method for transducing human non-activated PBMCs with the targeted lentiviral vector m-TCP-E / G in Example 2, on Day 0, non-activated PBMCs of Donor 1 (a healthy individual) were transduced with the targeted lentiviral vector m-a33-G to prepare CD33-TCP-T cells;

[0606] Day 5, flow cytometry was used to detect the expression of CD33-TCP-G molecules in CD3 + The results of the membrane expression efficiency in T cells are shown in FIG9 .

[0607] As shown in Figure 9, the targeting vector m-a33-G can effectively transduce CD3 + T cells, the membrane expression efficiency of the CD33-TCP-G molecule was about 27.84%.

[0608] 4. CD33-TCP-T cells kill target cells in vitro

[0609] Referring to the method of killing target cells Nalm-6 cells by CD19-TCP-T cells in Example 4, the CD33-TCP-T cells were used to kill CD33 in vitro. + Target cells MOLM-13 cells (human acute myeloid leukemia cells), Day 7, flow cytometry was used to detect the killing efficiency, and the results are shown in Figure 10;

[0610] As shown in Figure 10, the CD33-TCP-T cells can effectively kill CD33 + MOLM-13 cells.

[0611] Antibodies used in flow cytometry:

[0612] Detection of CD33: FITC-CD33; Brand: BD, Catalog No.: #561818;

[0613] Detection CD3: Brand: ACRO, Product Number: #CD3-HP2E3-200tests.

Claims

1. A viral vector, characterized in that (a) the viral vector comprises a polynucleotide encoding a T cell receptor chimeric protein (TCP); and (b) the surface of the viral vector contains T cell activation signal molecules; Wherein, the TCP includes: (i) a TCR / CD3 complex subunit related peptide (TSP), wherein the TSP comprises at least one of (1) a TCR / CD3 complex subunit, (2) a functional fragment of a TCR / CD3 complex subunit, (3) a TCR / CD3 complex subunit variant, and (4) a variant of a functional fragment of a TCR / CD3 complex subunit; and (ii) Antigen binding region.

2. The viral vector according to claim 1, characterized in that The viral vector is a lentiviral vector (LVV) or a retroviral vector (RVV).

3. The viral vector according to claim 1 or 2, characterized in that The TCR / CD3 complex subunit is selected from at least one of TCRα (TCRA), TCRβ (TCRB), TCRγ (TCRG), TCRδ (TCRD), CD3γ (CD3G), CD3δ (CD3D), CD3ζ (CD3Z) and CD3ε (CD3E).

4. The viral vector according to any one of claims 1 to 3, characterized in that The TCR / CD3 complex subunit functional fragment comprises at least one of the extracellular region, transmembrane region, intracellular region, variable region and constant region of the TCR / CD3 complex subunit.

5. The viral vector according to claim 4, characterized in that The TSP comprises CD3γ or a functional fragment thereof; Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3γ; More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3γ and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3ζ and CD3δ; Still more preferably, the TSP comprises the transmembrane region and intracellular region of CD3γ and the extracellular region of (a) CD3δ, (b) CD3ζ or (c) CD3ε; Most preferably, the C-terminus of the extracellular region of CD3δ, the extracellular region of CD3ζ or the extracellular region of CD3ε is located towards the N-terminus of the transmembrane region of CD3γ, and the C-terminus of the transmembrane region of CD3γ is located towards the N-terminus of the intracellular region of CD3γ.

6. The viral vector according to claim 4, characterized in that The TSP comprises CD3ε or a functional fragment thereof. Preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ε; More preferably, the TSP comprises the transmembrane region and the intracellular region of CD3ε and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3ζ and CD3δ; More preferably, the TSP comprises the transmembrane region and intracellular region of CD3ε; and (a) the extracellular region of CD3γ, (b) the extracellular region of CD3ζ, or (c) the extracellular region of CD3δ; Most preferably, the C-terminus of the extracellular region of CD3γ, the extracellular region of CD3ζ or the extracellular region of CD3δ is located in the N-terminal direction of the transmembrane region of CD3ε, and the C-terminus of the transmembrane region of CD3ε is located in the N-terminal direction of the intracellular region of CD3ε.

7. The viral vector according to claim 4, characterized in that The TSP comprises TCRα or a functional fragment thereof and TCRβ or a functional fragment thereof; Preferably, the TSP comprises the constant region of TCRα and the constant region of TCRβ.

8. The viral vector according to claim 7, characterized in that The constant region of the TCRα is mutated, and the mutation includes a cysteine substitution in the constant region of the TCRα; Preferably, the TCRα constant region is derived from a human or mouse TCRα constant region, the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 39, and the mouse TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 40; The mutation includes replacing the 47th amino acid Threonine T of the human TCRα constant region with Cysteine C (human TCRα constant region variant 1) or replacing the 47th amino acid Threonine T of the mouse TCRα constant region with Cysteine C (mouse TCRα constant region variant 1); The human TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:41, and the mouse TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:

42.

9. The viral vector according to claim 7 or 8, characterized in that The TCRβ constant region is mutated, and the mutation includes a cysteine substitution in the TCRβ constant region; Preferably, the TCRβ constant region is derived from a human TCRβ constant region, and the human TCRβ constant region comprises the amino acid sequence shown in SEQ ID NO: 43 (hTRBC1) or SEQ ID NO: 44 (hTRBC2), and the mutation includes replacing the 56th amino acid serine S of the human TCRβ constant region with cysteine C (human TCRβ constant region variant 1), and the human TCRβ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO:

46.

10. The viral vector according to any one of claims 7 to 9, characterized in that The TCRα constant region is mutated, wherein the mutation comprises replacing at least one uncharged amino acid in the TCRα constant region with a hydrophobic amino acid; Preferably, the TCRα constant region is derived from a human TCRα constant region, the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 39, and the mutation comprises replacement of at least one of amino acids 115, 118, and 119 of the human TCRα constant region with a hydrophobic amino acid; More preferably, the mutations include at least one of the following mutations in the human TCRα constant region: substitution of amino acid serine S at position 115 with leucine L (S115T), substitution of amino acid glycine G at position 118 with valine V (G118V), and substitution of amino acid phenylalanine F at position 119 with leucine L (F119L); Further preferably, the mutation includes replacing the 115th amino acid serine S of the human TCRα constant region with leucine L, replacing the 118th amino acid glycine G with valine V, and replacing the 119th amino acid phenylalanine F with leucine L (human TCRα constant region variant 2), and the human TCRα constant region variant 2 comprises the amino acid sequence shown in SEQ ID NO:

47.

11. The viral vector according to any one of claims 7 to 10, characterized in that The TCRα constant region is derived from a human TCRα constant region, comprising the amino acid sequence set forth in SEQ ID NO:39; the human TCRα constant region is mutated, wherein the mutations comprise substitution of amino acid threonine T at position 47 of the human TCRα constant region with cysteine C, substitution of amino acid serine S at position 115 with leucine L, substitution of amino acid glycine G at position 118 with valine V, and substitution of amino acid phenylalanine F at position 119 with leucine L (human TCRα constant region variant 3), comprising the amino acid sequence set forth in SEQ ID NO:48; Preferably, the TCRα constant region and the TCRβ constant region are derived from a human TCRα constant region and a human TCRβ constant region; the human TCRα constant region comprises the amino acid sequence as shown in SEQ ID NO: 39, and the human TCRβ constant region comprises the amino acid sequence as shown in SEQ ID NO: 43 or SEQ ID NO: 44; the human TCRα constant region and the human TCRβ constant region are mutated, and the mutations include substitution of amino acid 47 of the human TCRα constant region with cysteine, substitution of amino acid serine S at position 115 with leucine L, substitution of amino acid glycine G at position 118 with valine V, and substitution of amino acid phenylalanine F at position 119 with leucine L (human TCRα constant region variant 3), and substitution of amino acid serine S at position 56 of the human TCRβ constant region with cysteine C (human TCRβ constant region variant 1); the human TCRα constant region variant 3 comprises the amino acid sequence as shown in SEQ ID NO: 48, and the human TCRβ constant region variant 1 comprises the amino acid sequence as shown in SEQ ID NO: 45 or SEQ ID The amino acid sequence shown in NO:

46.

12. The viral vector according to any one of claims 1 to 11, characterized in that The TSP may further comprise the hinge region of the TCR / CD3 complex subunit.

13. The viral vector according to any one of claims 1 to 12, characterized in that The antigen binding region is operably linked to the N-terminus of the TSP via a linker peptide.

14. The viral vector according to claim 13, characterized in that The connecting peptide includes a flexible connecting peptide.

15. The viral vector according to claim 14, characterized in that The flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence of the connecting peptide 1 are as shown in SEQ ID NO: 23; wherein n=1 to 4; Preferably, the flexible connecting peptide is connecting peptide 1 or (G4S)3 connecting peptide (connecting peptide 2).

16. The viral vector according to any one of claims 1 to 15, characterized in that The antigen binding region binds to a disease-associated antigen.

17. The viral vector according to claim 16, characterized in that The disease is selected from cancer and autoimmune disease; the cancer includes hematological cancer and solid cancer.

18. The viral vector according to claim 17, characterized in that 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, CD40 L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD 248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, Erb B4, MUC16, MAGE-3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, uPAR, GCC (guanylate cyclase C), EPCAM, Nectin4, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.

2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut At least one of hsp70-2, CD79A, CD79B, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, and NKG2D; Preferably, the disease-associated antigen is selected from at least one of CD19, CD20, CD33, MSLN, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2 and GUCY2C.

19. The viral vector according to any one of claims 1 to 18, characterized in that The antigen binding region comprises an antibody or an antigen binding fragment thereof and / or a ligand or a receptor binding fragment thereof, wherein 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 bond-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 VHH).

20. The viral vector according to any one of claims 1 to 19, characterized in that The TCP does not contain a signal peptide.

21. The viral vector according to any one of claims 1 to 19, characterized in that The TCP comprises a signal peptide.

22. The viral vector according to claim 21, characterized in that The signal peptide is selected from the following signal peptides: CD8α signal peptide, CD28 signal peptide, IgG signal peptide, HLA-A signal peptide, CD3γ signal peptide, CD3δ signal peptide, CD3ζ signal peptide and CD3ε signal peptide.

23. The viral vector according to claim 22, characterized in that The signal peptide is CD8α signal peptide.

24. The viral vector according to any one of claims 1 to 23, characterized in that The TCP also comprises a costimulatory signaling domain; Preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of at least one of the following proteins: CD28, 4-1BB, CD27, CD2, CD3, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαR1γ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88; preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of 4-1BB and / or CD28; More preferably, the costimulatory signaling domain is derived from the costimulatory signaling domains of 4-1BB and CD28.

25. The viral vector according to any one of claims 1 to 24, characterized in that When expressed in T cells, the TCP (a) is incorporated into the endogenous TCR / CD3 complex or TCR / CD3 complex subunit or a functional fragment thereof; or (b) functionally interacts with the endogenous TCR / CD3 complex or an endogenous TCR / CD3 complex subunit or a functional fragment thereof.

26. The viral vector according to any one of claims 1 to 25, characterized in that The TCP is not expressed on the membrane in cells other than T cells, or the efficiency of the TCP's expression on the membrane in cells other than T cells is lower than the efficiency of the TCP's expression on the membrane in T cells.

27. The viral vector according to any one of claims 1 to 26, characterized in that The T cell activation signal molecule includes a T cell activation primary signal molecule.

28. The viral vector according to claim 27, characterized in that The T cell activation primary signal molecule binds to at least one of a TCR / CD3 complex subunit and a TCR / CD3 complex subunit functional fragment; the TCR / CD3 complex subunit is selected from at least one of CD3ε, CD3γ, CD3δ, CD3ζ, TCRγ, TCRδ, TCRα and TCRβ.

29. The viral vector according to claim 28, characterized in that The TCR / CD3 complex subunit is a human TCR / CD3 complex subunit.

30. The viral vector according to claim 28 or 29, characterized in that The T cell activation primary signal molecule includes an anti-CD3 antibody or an antigen-binding fragment thereof.

31. The viral vector according to claim 30, characterized in that The anti-CD3 antibody is selected from at least one of OKT3, UCHT1, YTH12.5 and TR66.

32. The viral vector according to claim 30, characterized in that The anti-CD3 antibody is SP34.

33. The viral vector according to any one of claims 30 to 32, characterized in that The anti-CD3 antibody or antigen-binding fragment thereof cannot bind to the TSP.

34. The viral vector according to any one of claims 30 to 33, characterized in that The anti-CD3 antibody is an anti-CD3ε antibody, and the TSP is not CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

35. The viral vector according to claim 34, characterized in that The anti-CD3ε antibody or antigen-binding fragment thereof is UCHT1 or an antigen-binding fragment thereof; Preferably, the antigen-binding fragment of UCHT1 is a scFv (UCHT1-scFv); More preferably, the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NOs: 81-83, respectively, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown as SEQ ID NOs: 84-86, respectively.

36. The viral vector according to claim 34, characterized in that The anti-CD3ε antibody or antigen-binding fragment thereof is OKT3 or an antigen-binding fragment thereof; Preferably, the antigen-binding fragment of OKT3 is scFv (OKT3-scFv); More preferably, the amino acid sequences of the HCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NOs: 104-106, respectively, and the amino acid sequences of the LCDR1-3 regions of the OKT3-scFv are shown as SEQ ID NOs: 107-109, respectively.

37. The viral vector according to claim 34, characterized in that The anti-CD3ε antibody or antigen-binding fragment thereof is SP34 or an antigen-binding fragment thereof; Preferably, the antigen-binding fragment of SP34 is scFv (SP34-scFv); More preferably, the amino acid sequences of the HCDR1-3 regions of the SP34-scFv are shown as SEQ ID NOs: 113-115, respectively, and the amino acid sequences of the LCDR1-3 regions of the SP34-scFv are shown as SEQ ID NOs: 116-118, respectively.

38. The viral vector according to any one of claims 34 to 37, characterized in that The TSP is: (a) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; (b) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or (c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

39. The viral vector according to any one of claims 30 to 33, characterized in that The anti-CD3 antibody is an anti-CD3γ antibody, and the TSP is not CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

40. The viral vector according to claim 39, characterized in that The TSP is: (a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; (b) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or (c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

41. The viral vector according to any one of claims 30 to 33, characterized in that The anti-CD3 antibody is an anti-CD3δ antibody, and the TSP is not CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

42. The viral vector according to claim 41, characterized in that The anti-CD3δ antibody or antigen-binding fragment thereof is TR66 or an antigen-binding fragment thereof; Preferably, the antigen-binding fragment of TR66 is scFv (TR66-scFv).

43. The viral vector according to claim 41 or 42, characterized in that The TSP is: (a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; (b) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or (c) CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

44. The viral vector according to any one of claims 30 to 33, characterized in that The anti-CD3 antibody is an anti-CD3ζ antibody, and the TSP is not CD3ζ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

45. The viral vector according to claim 44, characterized in that The anti-CD3ζ antibody or antigen-binding fragment thereof is YTH12.5 or an antigen-binding fragment thereof; Preferably, the antigen-binding fragment of YTH12.5 is scFv (YTH12.5-scFv).

46. The viral vector according to claim 44 or 45, characterized in that The TSP is: (a) CD3ε, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; (b) CD3γ, or a functional fragment thereof, or a variant thereof, or a functional fragment thereof; or (c) CD3δ, or a functional fragment thereof, or a variant thereof, or a functional fragment of a variant thereof.

47. The viral vector according to any one of claims 1 to 46, characterized in that The T cell activation signaling molecules also include T cell activation secondary signaling molecules.

48. The viral vector according to claim 47, characterized in that The T cell activation secondary signaling molecule binds to CD28.

49. The viral vector according to claim 48, characterized in that The CD28 is human CD28.

50. The viral vector according to claim 48 or 49, characterized in that The T cell activation secondary signal molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof.

51. The viral vector according to claim 50, characterized in that The CD28 ligand or its receptor binding fragment includes CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.

52. The viral vector according to claim 51, characterized in that The T cell activation secondary signal molecule includes an anti-CD28 antibody or an antigen-binding fragment thereof; Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is a scFv derived from 15E8 (15E8-scFv), the amino acid sequence of which 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 NOs: 93-95, respectively, and the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 96-98, respectively.

53. The viral vector according to any one of claims 48 to 52, characterized in that The T cell activation secondary signal molecule further includes at least one ligand or receptor binding fragment thereof selected from ICOS ligand (ICOSL) or its receptor binding fragment, 4-1BB ligand (4-1BBL) or its receptor binding fragment and OX40 ligand (OX40L) or its receptor binding fragment.

54. The viral vector according to any one of claims 1 to 53, characterized in that The T cell activation signal molecule is directly or indirectly linked to a transmembrane polypeptide and displayed on the surface of the viral vector.

55. The viral vector according to claim 54, characterized in that The transmembrane polypeptide is selected from the transmembrane region of the following proteins: CD2, CD3, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD 37. CD40, CD45, CD64, CD80, CD86, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; Preferably, the transmembrane polypeptide is the CD8α transmembrane region.

56. The viral vector according to claim 54 or 55, characterized in that The T cell activation signal molecule is indirectly connected to the transmembrane polypeptide through a linker domain and is displayed on the surface of the viral vector; Preferably, the linker domain is selected from: (a) an immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from a wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge region; (b) a hinge region selected from the wild-type or modified hinge region of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (c) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; (d) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and (e) flexible linker peptide; More preferably, the connecting domain is the CD8α hinge region.

57. The viral vector according to any one of claims 1 to 56, characterized in that The surface of the viral vector contains glycoproteins, which are selected from the envelope glycoproteins of vesicular stomatitis virus strains and their variants, the envelope glycoproteins of baboon endogenous retrovirus BaEV and their variants, the envelope glycoproteins RD114 and their variants of feline endogenous retrovirus, and the envelope glycoproteins GALV and their variants of gibbon ape leukemia virus.

58. [Corrected 18.02.2025 according to Rule 26] The viral vector according to claim 57, characterized in that The transmembrane polypeptide is the glycoprotein, and the glycoprotein is directly or indirectly linked to the T cell activation signal molecule; Preferably, the glycoprotein is indirectly linked to the T cell activation signal molecule via a polypeptide linker.

59. The viral vector according to claim 58, characterized in that The T cell activation primary signaling molecule is directly or indirectly linked to the T cell activation secondary signaling molecule; Preferably, the T cell activation primary signal molecule is indirectly linked to the T cell activation secondary signal molecule via a polypeptide linker.

60. The viral vector according to claim 58 or 59, characterized in that The polypeptide linker is a flexible connecting peptide; Preferably, the flexible connecting peptide is selected from (G4S) n Connector peptide, Connector peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23) and Connector peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 63); wherein, n=1 to 4.

61. The viral vector according to any one of claims 58 to 60, characterized in that (a) The glycoprotein is passed through (G4S) n A connecting peptide indirectly connected to the anti-CD3 antibody or antigen-binding fragment thereof, wherein the anti-CD3 antibody or antigen-binding fragment thereof is connected to the anti-CD3 antibody or antigen-binding fragment thereof via (G4S) n A connecting peptide indirectly linked to (i) the anti-CD28 antibody or antigen-binding fragment thereof, (ii) the extracellular domain of CD80 (iii) or the extracellular domain of CD86; and / or (b) The glycoprotein is passed through (G4S) n A connecting peptide indirectly connected to (i) the anti-CD28 antibody or its antigen-binding fragment, (ii) the CD80 extracellular domain (iii) or the CD86 extracellular domain; the anti-CD28 antibody or its antigen-binding fragment, the CD80 extracellular domain or the CD86 extracellular domain is connected to the anti-CD28 antibody or its antigen-binding fragment, the CD80 extracellular domain or the CD86 extracellular domain via (G4S) n a linker peptide, indirectly linked to the anti-CD3 antibody or antigen-binding fragment thereof; Preferably, n=3; Preferably, the anti-CD3 antibody or antigen-binding fragment thereof is the UCHT1-scFv; Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is the 15E8-scFv.

62. The viral vector according to any one of claims 57 to 61, characterized in that The glycoprotein is selected from the envelope glycoprotein of a vesicular stomatitis virus strain and variants thereof.

63. The viral vector according to claim 62, characterized in that The envelope glycoproteins and variants thereof of the vesicular stomatitis virus strains include the following envelope glycoproteins and variants thereof: envelope glycoproteins and variants of the vesicular stomatitis virus Indiana strain, envelope glycoproteins and variants thereof of the vesicular stomatitis virus Cocal strain, envelope glycoproteins and variants thereof of the vesicular stomatitis virus Maraba strain, envelope glycoproteins and variants thereof of the vesicular stomatitis virus Morreton strain, envelope glycoproteins and variants thereof of the vesicular stomatitis virus Alagoas strain, envelope glycoproteins and variants thereof of the vesicular stomatitis virus New The envelope glycoprotein of Jersey strain and its variants, the envelope glycoprotein of Carajas strain and its variants, the envelope glycoprotein of Chandipura strain and its variants, the envelope glycoprotein of Eptesicus strain and its variants, the envelope glycoprotein of Isfahan strain and its variants, the envelope glycoprotein of Jurona strain and its variants, the envelope glycoprotein of Malpais strain and its variants, the envelope glycoprotein of Perinet strain and its variants, the envelope glycoprotein of Piry strain and its variants, the envelope glycoprotein of Radi strain and its variants, the envelope glycoprotein of Rhinolopus strain and its variants, and the envelope glycoprotein of Yug Bogdanovac strain and its variants.

64. The viral vector according to claim 63, characterized in that The glycoprotein is an 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 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 with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

65. The viral vector according to any one of claims 57 to 64, characterized in that The glycoprotein undergoes a first mutation, which reduces or loses the ability of the glycoprotein to bind to a glycoprotein receptor compared to before the first mutation occurs.

66. The viral vector according to claim 65, characterized in that The glycoprotein is selected from the envelope glycoprotein of a vesicular stomatitis virus strain and variants thereof.

67. The viral vector according to claim 66, characterized in that The glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the glycoprotein receptor is the low-density lipoprotein receptor LDL-R; the extracellular domain of the glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or 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 with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

68. The viral vector according to claim 67, characterized in that The first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 209, substitution or deletion of amino acid at position 347 in SEQ ID NO: 1 or SEQ ID NO:

2. 354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; and (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, substitution or deletion of amino acid 8, substitution or deletion of amino acid 9, substitution or deletion of amino acid 10, substitution or deletion of amino acid 47, substitution or deletion of amino acid 50, substitution or deletion of amino acid 51, substitution or deletion of amino acid 183, substitution or deletion of amino acid 179, substitution or deletion of amino acid 180, substitution or deletion of amino acid 182, substitution or deletion of amino acid 184, substitution or deletion of amino acid 209, substitution or deletion of amino acid 347, substitution or deletion of amino acid 358, substitution or deletion of amino acid 360, substitution or deletion of amino acid 361, substitution or deletion of amino acid 362, substitution or deletion of amino acid 364, substitution or deletion of amino acid 365, substitution or deletion of amino acid 366, substitution or deletion of amino acid 367, substitution or deletion of amino acid 368, substitution or deletion of amino acid 369, substitution or deletion of amino acid 370, substitution or deletion of amino acid 371, substitution or deletion of amino acid 372, substitution or deletion of amino acid 373, substitution or deletion of amino acid 374, substitution or deletion of amino acid 375, substitution or deletion of amino acid 376, substitution or deletion of amino acid 377, substitution or deletion of amino acid 378, substitution or deletion of amino acid 379, substitution or deletion of amino acid 371, substitution or deletion of amino acid 377 The present invention also includes substitution or deletion of the amino acid at position 350, substitution or deletion of the amino acid at position 352, substitution or deletion of the amino acid at position 353, substitution of the amino acid at position 354, deletion of amino acids at positions 1-18, deletion of amino acids at positions 19-36, deletion of amino acids at positions 37-51, deletion of amino acids at positions 314-384, deletion of amino acids at positions 321-374, deletion of amino acids at positions 331-364, deletion of amino acids at positions 344-354, and deletion of amino acids at positions 345-353.

69. The viral vector according to claim 68, characterized in that The first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, substitution of R354 in SEQ ID NO: 1 or SEQ ID NO: 2; and (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, deletion of amino acids 331-364, deletion of amino acids 344-354, substitution of K47, deletion of K47, substitution of R354 at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) deletion of amino acids 331 to 364, 344 to 354, K47Q, R354Q, or K47 located in SEQ ID NO: 1 or SEQ ID NO: 2; and (b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, amino acid deletions at positions 331 to 364, amino acid deletions at positions 344 to 354, K47Q, R354Q, and K47 deletion are located corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

70. The viral vector according to claim 69, characterized in that The first mutation includes a mutation in which the amino acid sequence comprises the following amino acids: (a) the amino acid lysine at position 47 of SEQ ID NO: 1 or SEQ ID NO: 2 is deleted; or (b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, the lysine residue at amino acid position 47 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 is missing.

71. The viral vector according to claim 67 or 68, characterized in that The first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution of K47, deletion of K47, substitution of I182, substitution of R354, or substitution of Y209 in SEQ ID NO: 1; (b) after optimal global alignment with SEQ ID NO: 1, substitution at K47, deletion of K47, substitution at I182, substitution at R354, and substitution at Y209 equivalent to SEQ ID NO: 1; (c) substitution of K47, deletion of K47, substitution of V182, substitution of R354, substitution of Y209 at SEQ ID NO: 2; and (d) after optimal global alignment with SEQ ID NO: 2, a substitution at K47, a deletion of K47, a substitution at V182, a substitution at R354, and a substitution at Y209 corresponding to SEQ ID NO: 2; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q located in SEQ ID NO: 1; (b) after optimal global alignment with SEQ ID NO: 1, located at K47Q or K47A, K47 deletion, I182E or I182D, R354Q or R354A, Y209Q equivalent to SEQ ID NO: 1; (c) K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, Y209Q at SEQ ID NO: 2; and (d) After optimal global alignment with SEQ ID NO: 2, it is located at K47Q or K47A, K47 deletion, V182E or V182D, R354Q or R354A, and Y209Q corresponding to SEQ ID NO:

2.

72. The viral vector according to claim 66, characterized in that The extracellular domain of the glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 33 or SEQ ID NO:

34.

73. The viral vector according to any one of claims 57 to 72, characterized in that The glycoprotein undergoes a second mutation, which enhances the ability of the glycoprotein to antagonize inactivation by complement relative to that before the second mutation occurs, or makes the glycoprotein not inactivated by complement.

74. The viral vector according to claim 73, characterized in that The glycoprotein is an 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 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 with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

75. The viral vector according to claim 74, characterized in that The second mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 146 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the amino acid mutation is selected from at least one of deletion, insertion and substitution of amino acids; More preferably, the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the amino acid position corresponding to 146 of SEQ ID NO: 1 or SEQ ID NO:

2.

76. The viral vector according to claim 75, characterized in that The second mutation includes an amino acid sequence as shown in SEQ ID NO: 1 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 1, comprising at least one of the following position mutations: (a) substitution of T214, substitution of T352, substitution of K50, or substitution of S146 in SEQ ID NO: 1; and (b) after optimal global alignment with SEQ ID NO: 1, substitutions at T214, T352, K50, and S146 corresponding to SEQ ID NO: 1; Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence: (a) T214N, T352A, K50T, S146T at SEQ ID NO: 1; and (b) After optimal global alignment with SEQ ID NO: 1, T214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO:

1.

77. The viral vector according to claim 76, characterized in that The second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) substitution of (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1; and (b) after optimal global alignment with SEQ ID NO: 1, substitutions at positions corresponding to (1) T214 and T352; or (2) T214, T352, K50, and S146 of SEQ ID NO: 1; Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) (1) T214N and T352A; or (2) T214N, T352A, K50T, and S146T at SEQ ID NO: 1; and (b) After optimal global alignment with SEQ ID NO: 1, located at (1) T214N and T352A; or (2) T214N, T352A, K50T, and S146T equivalent to SEQ ID NO:

1.

78. The viral vector according to claim 75, characterized in that The second mutation includes an amino acid sequence as shown in SEQ ID NO: 2 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity to the amino acid sequence as shown in SEQ ID NO: 2, comprising at least one of the following position mutations: (a) substitution of K214, substitution of T352, substitution of K50, substitution of S146 in SEQ ID NO: 2; and (b) after optimal global alignment with SEQ ID NO: 2, substitutions at positions corresponding to K214, T352, K50, and S146 of SEQ ID NO: 2; Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence: (a) K214N, T352A, K50T, S146T at SEQ ID NO: 2; and (b) After optimal global alignment with SEQ ID NO: 2, K214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO:

2.

79. The viral vector according to claim 78, characterized in that The second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) substitution of (1) K214 and T352; or (2) K214, T352, K50, and S146 of SEQ ID NO: 2; and (b) after optimal global alignment with SEQ ID NO: 2, at positions corresponding to (1) substitutions of K214 and T352; or (2) substitutions of K214, T352, K50, and S146 of SEQ ID NO: 2; Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) (1) K214N and T352A; or (2) K214N, T352A, K50T, and S146T at SEQ ID NO: 2; and (b) After optimal global alignment with SEQ ID NO: 2, located at (1) K214N and T352A; or (2) K214N, T352A, K50T, and S146T equivalent to SEQ ID NO:

2.

80. The viral vector according to claim 73, characterized in that The extracellular domain of the glycoprotein comprises the amino acid sequence 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.

81. A method for transducing T cells, characterized in that The method comprises contacting the viral vector of any one of claims 1 to 80 with T cells.

82. The method according to claim 81, characterized in that The T cells are selected from at least one of activated T cells and non-activated T cells.

83. The method according to claim 81 or 82, characterized in that The contacting occurs inside the body and / or outside the body of the subject; The subject is an individual who has been administered T cells transduced by the method for transducing T cells and / or the viral vector according to any one of claims 1 to 80.

84. An engineered T cell, characterized in that The engineered T cells are prepared by contacting T cells with the viral vector described in any one of claims 1-80.

85. The engineered T cell according to claim 84, wherein The T cells are selected from at least one of activated T cells and non-activated T cells.

86. The engineered T cell according to claim 84 or 85, wherein The contacting occurs inside the body and / or outside the body of the subject; The subject is an individual administered the engineered T cells and / or the viral vector of any one of claims 1-80.

87. The engineered T cell according to claim 86, characterized in that 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 implantation.

88. A composition, characterized in that The composition comprises a pharmaceutically acceptable excipient or carrier and (a) the viral vector of any one of claims 1-80 or (b) the engineered T cell of any one of claims 84-87.

89. Use of the viral vector of any one of claims 1-80, the engineered T cell of any one of claims 84-87, or the composition of claim 88 in the preparation of a medicament for preventing and / or treating cancer.

90. The use according to claim 89, characterized in that The disease is selected from at least one of cancer and autoimmune disease; the cancer includes solid cancer and blood cancer.

91. The use according to claim 90, characterized in that The cancer is a blood cancer.

92. The use according to claim 91, characterized in that The 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's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin's lymphoma, high-grade B-cell lymphoma, and multiple myeloma.

93. The use according to claim 91, characterized in that The blood cancer is selected from CD19 + Blood Cancers and CD33 + Blood cancer; Preferably, CD19 + a blood cancer 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; CD33 + The 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).

94. A method for treating cancer in a subject or killing cancer cells in a subject, characterized in that: The method comprises administering to the subject the viral vector of any one of claims 1-80, the engineered T cell of any one of claims 84-87, or the composition of claim 88.

95. The method according to claim 94, characterized in that 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.

96. The method according to claim 94 or 95, characterized in that Such cancers include solid cancers and blood cancers.

97. The method according to claim 96, characterized in that The cancer is a blood cancer.

98. The method according to claim 97, wherein The 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's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cell leukemia, precursor B-lymphocytic leukemia, non-Hodgkin's lymphoma, high-grade B-cell lymphoma, and multiple myeloma.

99. The method according to claim 97, characterized in that The blood cancer is selected from CD19 + Blood Cancers and CD33 + Blood cancer; Preferably, CD19 + a blood cancer 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; CD33 + The blood cancer is selected from the group consisting of multiple myeloma (MM), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and acute monocytic leukemia (AMoL).

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