Viral vector and use of TCR subunit

Abstract

The present invention relates to the field of genetic engineering, and in particular to a viral vector and a use of a TCR subunit. Provided is a lentiviral vector or a retroviral vector. The ability to infect a target cell by the lentiviral vector or the retroviral vector is reduced. Also disclosed is a use of a TCR subunit or a functional fragment thereof in the preparation of the lentiviral vector or the retroviral vector.

Description

Application of a viral vector and TCR subunit Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to applications of a viral vector and a TCR subunit. Background Art

[0002] In the field of genetic engineering and cell therapy, lentiviral vectors and retroviral vectors are commonly used nucleic acid vectors, which are used to carry shuttle genes encoding therapeutic fusion proteins or polypeptides such as chimeric antigen receptors (CARs).

[0003] During the packaging of lentiviral vectors and retroviral vectors, the CAR encoded by the shuttle gene and the antigen binding region contained therein are expressed on the cell membrane of packaging cells such as HEK-293T cells, resulting in the budding of the lentiviral vector or retroviral vector from the packaging cell line. The viral envelope of the lentiviral vector or retroviral vector derived from the cell membrane of the packaging cell also contains CAR.

[0004] Therefore, there is an unmet need for a method to reduce the amount of antigen-binding domain encoded by a portion of a shuttle gene contained in the viral envelope of a lentiviral or retroviral vector.

[0005] Summary of the Invention

[0006] The inventors of the present invention have discovered for the first time that lentiviral or retroviral vectors containing CARs in their viral envelopes can bind to corresponding antigens on the surface of target cells through the antigen-binding region of the CARs contained therein, thereby transfecting the target cells. This not only reduces the targeting of immune cells such as T cells transfected by the lentiviral or retroviral vectors, but also reduces the available antigens on the surface of the transfected target cells when used in in vivo CAR-T cell therapy or when transfecting the blood of patients with hematological cancers, thereby reducing the killing efficiency of engineered immune effector cells such as CAR-T cells.

[0007] At the same time, the transfected target cells will also express CAR and specifically bind to antigens on the surface of other target cells through the antigen binding region of CAR, further leading to the blocking / reduction of antigens available for binding on the surface of target cells, thereby further reducing the killing efficiency of engineered immune effector cells such as CAR-T cells.

[0008] In view of this, in order to solve at least one of the above technical problems, the present invention provides a lentiviral vector or a retroviral vector in one aspect.

[0009] (a) The lentiviral vector or retroviral vector carries a shuttle gene encoding a T cell receptor chimeric protein (TCell Receptor Chimeric Protein, "TCP"); the T cell receptor chimeric protein comprises: (1) a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; (2) an antigen binding region, wherein the antigen binding region is operably linked to the N-terminus of the TCR subunit or the functional fragment thereof, and the antigen binding region comprises one or more antibodies or antigen binding fragments thereof;

[0010] (b) the viral envelope of the lentiviral vector or retroviral vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to T cell endocytic receptors, wherein the antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof are not part of a viral envelope glycoprotein; and

[0011] (c) The envelope glycoprotein of the lentiviral vector or retroviral vector undergoes a first mutation, so that the ability of the envelope glycoprotein to specifically bind to a receptor is weakened or lost relative to before the first mutation occurs.

[0012] In some embodiments of the present invention, the viral envelope of the lentiviral vector or retroviral vector does not contain the TCP or the TCP is lowly distributed on the viral envelope of the lentiviral vector or retroviral vector.

[0013] Cells other than T cells do not contain TCR subunits such as TCRα, TCRβ, CD3γ, CD3ε, CD3γ, CD3ζ and CD3δ. Therefore, it is difficult for the TCP to effectively form a TCR / CD3 complex (T Cell Receptor / CD3 Complex) on the cell membrane and be anchored in cells other than T cells.

[0014] In some embodiments of the present invention, the T cell receptor chimeric protein comprises: (1) a TCR subunit, wherein the TCR subunit is selected from TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; and (2) an antigen binding region, wherein the antigen binding region is operably linked to the N-terminus of the TCR subunit, and the antigen binding region comprises one or more antibodies or antigen-binding fragments thereof.

[0015] In some embodiments of the present invention, the TCP forms a TCR / CD3 complex in T cells, is expressed outside the cell membrane and is anchored on the cell membrane of the T cells.

[0016] In some embodiments of the present invention, the TCP forms a TCR / CD3 complex on the cell membrane of T cells and is expressed on the cell membrane of T cells.

[0017] In some embodiments of the present invention, the TCP interacts with an endogenous TCR / CD3 complex or at least one endogenous TCR subunit or a functional fragment thereof, expressed on the cell membrane of a T cell; the interaction may occur within the T cell or on the cell membrane of the T cell.

[0018] In some embodiments of the present invention, the TCP is integrated into the endogenous TCR / CD3 complex or at least one endogenous TCR subunit or a functional fragment thereof, and expressed on the cell membrane of the T cell; the integration may occur within the T cell or on the cell membrane of the T cell.

[0019] In some embodiments of the present invention, the TCP comprising at least one TCR subunit or a functional fragment thereof is integrated into the endogenous TCR / CD3 complex or at least one endogenous TCR subunit or a functional fragment thereof to form a TCP-TCR complex; illustratively, the structure of the TCP-TCR complex is shown in Figure 1-b.

[0020] In some embodiments of the present invention, the low distribution of TCP on the viral envelope of the lentiviral vector or retroviral vector is relative to the distribution of CAR on the viral envelope of the lentiviral vector or retroviral vector carrying the CAR gene.

[0021] In some embodiments of the present invention, the TCP is expressed on the cell membrane of T cells, and is not expressed on the cell membrane of cells other than T cells or the expression efficiency is low.

[0022] In some embodiments of the present invention, the low expression efficiency of TCP in cells other than T cells is relative to the expression efficiency of CAR in cells other than T cells.

[0023] In some embodiments of the present invention, the TCR subunit is CD3γ or CD3ε.

[0024] In some embodiments of the present invention, the TCR subunit is CD3γ, and the CD3γ comprises the amino acid sequence shown in SEQ ID NO:5.

[0025] In some embodiments of the present invention, the TCR subunit further comprises a CD3γ variant, which comprises conservative amino acid modifications compared to native CD3γ, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative amino acid modifications.

[0026] In some embodiments of the present invention, the TCR subunit is CD3ε, and the CD3ε comprises the amino acid sequence shown in SEQ ID NO:19.

[0027] In some embodiments of the present invention, the TCR subunit further comprises a CD3ε variant, which comprises conservative amino acid modifications compared to native CD3ε, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative amino acid modifications.

[0028] In some embodiments of the present invention, the TCP comprises the TCR subunit.

[0029] In some embodiments of the present invention, the TCP comprises CD3ε or CD3γ.

[0030] In some embodiments of the present invention, the antigen binding region comprises one or more antibodies or antigen binding fragments thereof selected from full-length antibodies, half antibodies, Fab, Fab', F(ab')2, Fv, VHH (single domain antibodies) and scFv.

[0031] In some embodiments of the present invention, the antigen binding region specifically binds to one or more antigens associated with a disease or cancer (solid cancer and hematological cancer).

[0032] In some embodiments of the present invention, the TCP is monospecific, bispecific, or multispecific.

[0033] In some embodiments of the present invention, the disease or cancer is selected from the following diseases or cancers: acute myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal cancer, adrenocortical carcinoma, anal cancer, anaplastic astrocytoma, angiosarcoma, appendix cancer, astrocytoma, basal cell carcinoma, B cell lymphoma, bile duct cancer, bladder cancer, bone cancer, bone marrow cancer, intestinal cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphocytic leukemia, Baxteran leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, fallopian tube cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor, common germ cell tumor, gestational trophoblastic disease, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer Carcinoma, hemangioendothelioma, Hodgkin lymphoma, hypopharyngeal cancer, infiltrating ductal carcinoma, infiltrating lobular carcinoma, inflammatory breast cancer, colorectal cancer, intrahepatic bile duct cancer, invasive / infiltrating breast cancer, islet cell carcinoma, jaw cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, leukemia, lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal Chondrosarcoma, stromal tumor, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous neck cancer, mixed glioma, oral cancer, mucinous carcinoma, mucosal melanoma, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, nasal cancer, nasopharyngeal cancer, neck cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell carcinoma, eye cancer, ocular melanoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer,Ovarian epithelial cancer, ovarian germ cell tumor, primary ovarian peritoneal cancer, ovarian sex cord stromal tumor, Paget's disease, pancreatic cancer, papillary cancer, sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pineoblastoma, pituitary tumor, primary central nervous system, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, sarcoma, bone, sarcoma, soft tissue, sarcoma , uterus, sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cancer, spinal cancer, spinal cord cancer, spinal tumor, squamous cell carcinoma, gastric cancer, synovial sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymus cancer, thyroid cancer, tongue cancer, tonsil cancer, transitional cell carcinoma, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, renal tubular cancer, undiagnosed cancer, ureteral cancer, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer.

[0034] In some embodiments of the present invention, the antigen binding region specifically binds to at least one of the following antigens:

[0035] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD.2, GD.3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D;.

[0036] Preferably, the antigen binding region specifically binds to at least one of CD19, CD33, CEA and HER2.

[0037] In some embodiments of the present invention, the antigen binding region can also specifically bind to at least one of CD20, BCMA, CD79A, CD79B and GCC (guanylate cyclase).

[0038] In some embodiments of the present invention, the antigen specifically bound by the antigen binding region is an endocytic receptor antigen.

[0039] In some embodiments of the present invention, the endocytic receptor antigen is CD19.

[0040] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide;

[0041] Preferably, the connecting peptide comprises a flexible connecting peptide;

[0042] More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence of the connecting peptide 1 are shown in SEQ ID NO: 18; wherein n=1 to 4.

[0043] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via the connecting peptide.

[0044] In some embodiments of the present invention, the flexible connecting peptide is (G4S)3(connecting peptide 2), and the amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO:10.

[0045] In some embodiments of the present invention, the flexible connecting peptide is a connecting peptide 1 having an amino acid sequence as shown in SEQ ID NO: 18.

[0046] In some embodiments of the present invention, the shuttle gene further comprises a leader sequence;

[0047] Preferably, the leader sequence is selected from the group consisting of leader sequences encoding the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide;

[0048] More preferably, the leader sequence encodes a CD8α signal peptide.

[0049] In some embodiments of the present invention, the leader sequence is a nucleic acid encoding any one of the signal peptides.

[0050] In some embodiments of the present invention, the antigen binding region of TCP may further comprise one or more ligands or receptor binding fragments thereof.

[0051] In some embodiments of the present invention, the T cell endocytic receptor is CD7 or CD3.

[0052] In some embodiments of the present invention, the T cell endocytic receptor is CD7.

[0053] In some embodiments of the present invention, the T cell endocytic receptor is CD3.

[0054] In some embodiments of the present invention, the T cell endocytic receptor is CD5.

[0055] In some embodiments of the present invention, the T cell endocytic receptor is TCRα and / or TCRβ.

[0056] Endocytosis receptors refer to receptors that mediate endocytosis. Lymphocytes, such as NK cells, T cells, and B cells, express a variety of endocytosis receptors on their surfaces.

[0057] Endocytosis refers to the process by which substances enter cells. In endocytosis, the substance to be taken in is surrounded by a region of the plasma membrane. The plasma membrane then buds out from 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).

[0058] Receptor-mediated endocytosis, also known as clathrin-mediated endocytosis, is mediated by the production of small (approximately 100 nm in diameter) vesicles with a morphologically characteristic coat composed of the cytoplasmic protein clathrin (McMahon HT, Boucrot E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nature Reviews. Molecular Cell Biology. 2011).

[0059] Clathrin-coated vesicles (CCVs) are present in almost all cells and form plasma membrane domains known as "clathrin-coated pits." Clathrin-coated pits can concentrate extracellular macromolecules that bind to different receptors responsible for receptor-mediated endocytosis of ligands, such as low-density lipoprotein, transferrin, growth factors, antibodies, and many other substances (Marsh M, McMahon HT (July 1999). The structural era of endocytosis. Science. 285(5425):215-220.).

[0060] Therefore, one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to T cell endocytic receptors are constructed on the surface of any lentiviral vector or retroviral vector provided by the present invention. The lentiviral vector or retroviral vector can enter T cells through endocytosis triggered by binding to endocytic receptors on the surface of T cells, thereby improving the targeting of the lentiviral vector or retroviral vector in transfecting T cells.

[0061] In some embodiments of the present invention, the one or more antibodies or antigen-binding fragments thereof that specifically bind to T cell endocytic receptors are selected from full-length antibodies, half antibodies, Fab, Fab', F(ab')2, Fv, VHH and scFv.

[0062] Vesicular Stomatitis Virus strains, such as the envelope glycoprotein of the Indiana strain (VSV-G) or the envelope glycoprotein of the Cocal strain (Cocal-G), can specifically bind to the low-density lipoprotein receptor (LDL-R), which is widely present on the surface of various cells, and thus have broad transfection properties; therefore, lentiviral vectors or retroviral vectors often use VSV-G or Cocal-G to construct their envelope glycoproteins.

[0063] By causing the first mutation in VSV-G or Cocal-G, the ability of the mutated VSV-G or Cocal-G to specifically bind to LDL-R is reduced, and one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to T cell endocytosis receptors, such as CD7 or CD3, are constructed on the surface of the lentiviral vector or retroviral vector, thereby further improving the targeting of T cells transfected by the lentiviral vector or retroviral vector.

[0064] In some embodiments of the present invention, the envelope glycoprotein of the lentiviral vector or retroviral vector is selected from the following envelope glycoproteins and variants thereof: envelope glycoproteins of vesicular stomatitis virus strains and variants thereof, envelope glycoproteins of baboon endogenous retrovirus BaEV and variants thereof, envelope glycoproteins of feline endogenous retrovirus RD114 and variants thereof, and envelope glycoproteins of gibbon ape leukemia virus GALV and variants thereof;

[0065] The envelope glycoprotein of the vesicular stomatitis virus strain and its variants are selected from the following envelope glycoproteins and their variants: envelope glycoprotein of Indiana strain of vesicular stomatitis virus and its variants, envelope glycoprotein of Cocal strain of vesicular stomatitis virus and its variants, envelope glycoprotein of Maraba strain of vesicular stomatitis virus and its variants, envelope glycoprotein of Morreton strain of vesicular stomatitis virus and its variants, envelope glycoprotein of Alagoas strain of vesicular stomatitis virus and its variants, envelope glycoprotein of 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.

[0066] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus or a variant thereof;

[0067] The extracellular domain of the envelope glycoprotein of the Indiana strain 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;

[0068] Preferably, the first mutation reduces or loses the ability of the envelope glycoprotein to specifically bind to LDL-R; the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0069] (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;

[0070] (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 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;

[0071] More preferably, the first mutation includes a mutation in the amino acid sequence comprising at least one of the following amino acids: substitution of K47, deletion of K47, substitution of R354;

[0072] More preferably, the first mutation includes a mutation in the amino acid sequence comprising at least one of the following amino acids: substitution of amino acid 47 from lysine K to glutamine Q, substitution of amino acid 354 from arginine R to glutamine Q, and deletion of amino acid 47 from lysine;

[0073] Most preferably, the first mutation comprises a mutation in the amino acid sequence comprising the following amino acid: deletion of K47.

[0074] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus or a variant thereof;

[0075] The extracellular domain of the envelope glycoprotein of the Cocal strain 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;

[0076] Preferably, the first mutation reduces or loses the ability of the envelope glycoprotein to specifically bind to LDL-R; the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0077] (a) substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 2;

[0078] (b) after optimal global alignment with SEQ ID NO: 2, substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 2;

[0079] More preferably, the first mutation includes a mutation in the amino acid sequence comprising at least one of the following amino acids: substitution of K47, deletion of K47, substitution of R354;

[0080] More preferably, the first mutation includes a mutation in the amino acid sequence comprising at least one of the following amino acids: substitution of amino acid 47 from lysine K to glutamine Q, substitution of amino acid 354 from arginine R to glutamine Q, and deletion of amino acid 47 from lysine;

[0081] Most preferably, the first mutation comprises a mutation in the amino acid sequence comprising the following amino acid: deletion of K47.

[0082] In some embodiments of the present invention, the envelope glycoprotein of the lentiviral vector or retroviral vector undergoes a second mutation, thereby enhancing the ability of the envelope glycoprotein to antagonize inactivation by complement relative to before the second mutation occurs, or preventing the envelope glycoprotein from being inactivated by complement.

[0083] 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. After activation, it has enzymatic activity and undergoes a complex cascade reaction. The complement system is initiated through a series of enzymes (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 mediating 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 reactions. It is an effector system and effector method system with important biological functions in the body.

[0084] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus or a variant thereof; the extracellular domain of the envelope glycoprotein of the Indiana strain 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;

[0085] Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0086] (a) amino acid position 214 of SEQ ID NO: 1;

[0087] (b) after optimal global alignment with SEQ ID NO: 1, is located at amino acid position 214 corresponding to SEQ ID NO: 1;

[0088] (c) amino acid position 352 of SEQ ID NO: 1;

[0089] (d) after optimal global alignment with SEQ ID NO: 1, located at amino acid position equivalent to SEQ ID NO: 1 at position 352;

[0090] (e) amino acid position 50 of SEQ ID NO: 1;

[0091] (f) after optimal global alignment with SEQ ID NO: 1, located at amino acid position equivalent to 50 of SEQ ID NO: 1;

[0092] (g) amino acid position 146 of SEQ ID NO: 1;

[0093] (h) after optimal global alignment with SEQ ID NO: 1, located at amino acid position 146 corresponding to SEQ ID NO: 1;

[0094] More preferably, the amino acid mutation includes amino acid deletion, insertion or substitution;

[0095] More preferably, the amino acid sequence comprises at least one substitution of the following amino acids:

[0096] (a) amino acid position 214 of SEQ ID NO: 1;

[0097] (b) after optimal global alignment with SEQ ID NO: 1, is located at amino acid position 214 corresponding to SEQ ID NO: 1;

[0098] (c) amino acid position 352 of SEQ ID NO: 1;

[0099] (d) after optimal global alignment with SEQ ID NO: 1, located at amino acid position equivalent to SEQ ID NO: 1 at position 352;

[0100] (e) amino acid position 50 of SEQ ID NO: 1;

[0101] (f) after optimal global alignment with SEQ ID NO: 1, located at amino acid position equivalent to 50 of SEQ ID NO: 1;

[0102] (g) amino acid position 146 of SEQ ID NO: 1;

[0103] (h) After optimal global alignment with SEQ ID NO: 1, it is located at the amino acid position corresponding to SEQ ID NO: 146.

[0104] In some embodiments of the present invention, the second mutation includes one or more of the following site mutations in the amino acid sequence: substitution of T214, substitution of T352, substitution of K50, substitution of S146;

[0105] Preferably, the amino acid sequence comprises one or more of the following site mutations: amino acid 214 is substituted from threonine T to asparagine N, amino acid 352 is substituted from threonine T to alanine A, amino acid 50 is substituted from lysine K to threonine T, and amino acid 146 is substituted from serine S to threonine T;

[0106] Preferably, the amino acid sequence comprises a combination of any of the following site mutations:

[0107] (a) Replacement of T214 and T352;

[0108] (b) Replacement of T214, T352, K50 and S146;

[0109] More preferably, the amino acid sequence comprises a combination of any of the following site mutations:

[0110] (a) amino acid 214 is substituted from threonine T to asparagine N and amino acid 352 is substituted from threonine T to alanine A;

[0111] (b) The amino acid at position 214 was replaced by threonine T to asparagine N, the amino acid at position 352 was replaced by threonine T to alanine A, the amino acid at position 50 was replaced by lysine K to threonine T, and the amino acid at position 146 was replaced by serine S to threonine T.

[0112] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus or a variant thereof; the extracellular domain of the envelope glycoprotein of the Cocal strain 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 with the amino acid sequence as shown in SEQ ID NO: 2;

[0113] Preferably, the second mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0114] (a) amino acid position 214 of SEQ ID NO: 2;

[0115] (b) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 2;

[0116] (c) amino acid position 352 of SEQ ID NO: 2;

[0117] (d) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position 352 corresponding to SEQ ID NO: 2;

[0118] (e) amino acid position 50 of SEQ ID NO: 2;

[0119] (f) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position equivalent to SEQ ID NO: 2;

[0120] (g) amino acid position 146 of SEQ ID NO: 2;

[0121] (h) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position 146 corresponding to SEQ ID NO: 2;

[0122] More preferably, the amino acid mutation includes amino acid deletion, insertion or substitution;

[0123] More preferably, the amino acid sequence comprises at least one substitution of the following amino acids:

[0124] (a) amino acid position 214 of SEQ ID NO: 2;

[0125] (b) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 2;

[0126] (c) amino acid position 352 of SEQ ID NO: 2;

[0127] (d) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position equivalent to SEQ ID NO: 2 at position 352;

[0128] (e) amino acid position 50 of SEQ ID NO: 2;

[0129] (f) after optimal global alignment with SEQ ID NO: 2, is located at amino acid position equivalent to SEQ ID NO: 2;

[0130] (g) amino acid position 146 of SEQ ID NO: 2;

[0131] (h) After optimal global alignment with SEQ ID NO: 2, it is located at the amino acid position corresponding to SEQ ID NO: 2 at position 146.

[0132] In some embodiments of the present invention, the second mutation includes one or more of the following site mutations in the amino acid sequence: substitution of K214, substitution of T352, substitution of K50, substitution of S146;

[0133] Preferably, the amino acid sequence comprises one or more of the following site mutations: amino acid 214 is substituted from lysine K to asparagine N, amino acid 352 is substituted from threonine T to alanine A, amino acid 50 is substituted from lysine K to threonine T, and amino acid 146 is substituted from serine S to threonine T;

[0134] Preferably, the amino acid sequence comprises a combination of any of the following site mutations:

[0135] (a) Replacement of K214 and T352;

[0136] (b) Replacement of K214, T352, K50 and S146;

[0137] More preferably, the amino acid sequence comprises a combination of any of the following site mutations:

[0138] (a) amino acid 214 was substituted from lysine K to asparagine N and amino acid 352 was substituted from threonine T to alanine A;

[0139] (b) The amino acid at position 214 was replaced by lysine K to asparagine N, the amino acid at position 352 was replaced by threonine T to alanine A, the amino acid at position 50 was replaced by lysine K to threonine T, and the amino acid at position 146 was replaced by serine S to threonine T.

[0140] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 3 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: 3; relative to SEQ ID NO: 1, SEQ ID NO: 3 comprises a K47 deletion.

[0141] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 4, 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: 4; relative to SEQ ID NO: 1, SEQ ID NO: 4 comprises R354Q.

[0142] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 6, 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: 6; relative to SEQ ID NO: 1, SEQ ID NO: 6 comprises a K47 deletion, T214N and T352A.

[0143] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 7 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: 7; relative to SEQ ID NO: 1, SEQ ID NO: 7 comprises R354Q, T214N and T352A.

[0144] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 21 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: 21; relative to SEQ ID NO: 2, SEQ ID NO: 21 comprises a K47 deletion.

[0145] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 22 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: 22; relative to SEQ ID NO: 2, SEQ ID NO: 22 comprises R354Q.

[0146] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 23 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: 23; relative to SEQ ID NO: 2, SEQ ID NO: 23 comprises a K47 deletion, K214N and T352A.

[0147] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises an amino acid sequence as shown in SEQ ID NO: 24 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: 24; relative to SEQ ID NO: 2, SEQ ID NO: 24 comprises R354Q, K214N and T352A.

[0148] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein of the lentiviral vector or retroviral vector comprises the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

[0149] In some embodiments of the present invention, the viral envelope of the lentiviral vector or retroviral vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to T cell endocytic receptors, which are directly or indirectly connected to the transmembrane region and are contained on the surface of the viral envelope;

[0150] Preferably, the transmembrane region is selected from the transmembrane regions of the following proteins:

[0151] CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD 95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM), CD272( BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, K IR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70;

[0152] More preferably, the transmembrane region includes the transmembrane region of CD8α.

[0153] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof and / or ligand or receptor-binding fragment thereof that specifically binds to a T cell endocytic receptor is indirectly connected to the transmembrane region via a linker domain;

[0154] Preferably, the linker domain is selected from:

[0155] (i) an immunoglobulin hinge region selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0156] (ii) 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;

[0157] (iii) 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; and

[0158] (iv) a stalk domain of a type II C-lectin, wherein the type II C-lectin is selected from the stalk domains of CD23, CD69, CD72, CD94, NKG2A, and NKG2D;

[0159] More preferably, the connecting domain includes the hinge region of CD8α.

[0160] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof and / or ligand or receptor-binding fragment thereof that specifically binds to the T cell endocytic receptor is indirectly connected to the transmembrane region of CD8α through the hinge region of CD8α and is included on the surface of the viral envelope.

[0161] In another aspect, the present invention provides a lentiviral vector or a retroviral vector,

[0162] (a) The lentiviral vector or retroviral vector carries a shuttle gene encoding a T cell receptor chimeric protein; the T cell receptor chimeric protein comprises: (1) a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; (2) an antigen binding region, wherein the antigen binding region is operably linked to the N-terminus of the TCR subunit or the functional fragment thereof, and the antigen binding region comprises one or more antibodies or antigen binding fragments thereof and / or ligands or receptor binding fragments thereof;

[0163] (b) the viral envelope of the lentiviral vector or retroviral vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to T cell endocytic receptors, wherein the antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof are not part of a viral envelope glycoprotein; and

[0164] (c) The envelope glycoprotein of the lentiviral vector or retroviral vector undergoes a first mutation, so that the ability of the envelope glycoprotein to specifically bind to a receptor is weakened or lost relative to before the first mutation occurs.

[0165] In another aspect, the present invention also provides a method for transfecting T cells, comprising contacting T cells with any one of the lentiviral vectors or retroviral vectors provided by the present invention;

[0166] Preferably, said contacting occurs in vivo or in vitro in a subject;

[0167] When the contacting occurs outside the body of a subject, the subject is an individual to whom T cells transfected by the method for transfecting T cells are administered;

[0168] When the contacting occurs in the subject, administering the lentiviral vector or retroviral vector to the subject;

[0169] More preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion techniques.

[0170] In some embodiments of the present invention, the T cells are activated T cells and / or non-activated T cells.

[0171] In some embodiments of the present invention, the contact occurs outside the body of the subject, the T cells are inactivated T cells, and the lentiviral vector or retroviral vector is in contact with the inactivated T cells for no more than 2 days, no more than 47 hours, no more than 46 hours, no more than 45 hours, no more than 44 hours, no more than 43 hours, no more than 42 hours, no more than 41 hours, no more than 40 hours, no more than 39 hours, no more than 38 hours, no more than 37 hours, no more than 36 hours, no more than 35 hours, no more than 34 hours, no more than 33 hours, no more than 32 hours, no more than 31 hours, no more than 30 hours, no more than 29 hours, no more than 28 hours, no more than 27 hours. hours, no more than 26 hours, no more than 25 hours, no more than 24 hours, no more than 23 hours, no more than 22 hours, no more than 21 hours, no more than 20 hours, no more than 19 hours, no more than 18 hours, no more than 17 hours, no more than 16 hours, no more than 15 hours, no more than 14 hours, no more than 13 hours, no more than 12 hours, no more than 11 hours, no more than 10 hours, no more than 9 hours, no more than 8 hours, no more than 7 hours, no more than 6 hours, no more than 5 hours, no more than 4 hours, no more than 3 hours, no more than 2 hours, no more than 1 hour, no more than 59 minutes, no more than 58 minutes, no more than 57 minutes, no more than 56 minutes, No more than 55 minutes, no more than 54 minutes, no more than 53 minutes, no more than 52 minutes, no more than 51 minutes, no more than 50 minutes, no more than 49 minutes, no more than 48 minutes, no more than 47 minutes, no more than 46 minutes, no more than 45 minutes, no more than 44 minutes, no more than 43 minutes, no more than 42 minutes, no more than 41 minutes, no more than 40 minutes, no more than 39 minutes, no more than 38 minutes, no more than 37 minutes, no more than 36 minutes, no more than 35 minutes, no more than 34 minutes, no more than 33 minutes, no more than 32 minutes, no more than 31 minutes, no more than 30 minutes, no more than 29 minutes, no more than 28 minutes, no more than 27 minutes, no more than More than 26 minutes, no more than 25 minutes, no more than 24 minutes, no more than 23 minutes, no more than 22 minutes, no more than 21 minutes, no more than 20 minutes, no more than 19 minutes, no more than 18 minutes, no more than 17 minutes, no more than 16 minutes, no more than 15 minutes, no more than 14 minutes, no more than 13 minutes, no more than 12 minutes, no more than 11 minutes, no more than 10 minutes, no more than 9 minutes, no more than 8 minutes, no more than 7 minutes, no more than 6 minutes, no more than 5 minutes, no more than 4 minutes, no more than 3 minutes, no more than 2 minutes, no more than 1 minute, no more than 50 seconds, no more than 40 seconds, no more than 30 seconds, no more than 20 seconds or no more than 10 seconds.

[0172] In some embodiments of the present invention, when the contacting occurs outside the body of the subject, the T cells are contained in the blood of the subject.

[0173] In some embodiments of the present invention, when the contacting occurs outside the body of the subject, the T cells are contained in the peripheral blood of the subject.

[0174] In some embodiments of the present invention, when the contacting occurs outside the body of the subject, the T cells are contained in the umbilical cord blood of the subject.

[0175] In some embodiments of the present invention, when the contacting occurs outside the body of the subject, the T cells are contained in the subject's peripheral blood mononuclear cells ("PBMCs").

[0176] In some embodiments of the present invention, when the contacting occurs outside the body of a subject, the T cells are isolated from PBMCs of the subject.

[0177] In some embodiments of the present invention, when the contacting occurs outside the body of the subject, the T cells or PBMCs are collected by lymphocyte apheresis.

[0178] In some embodiments of the present invention, when the contact occurs outside the subject, the T cells include CD3 T cells isolated from PBMCs of the subject. + T cells.

[0179] In some embodiments of the invention, when the contacting occurs outside the subject, the T cells comprise non-activated T cells contained in non-activated PBMCs of the subject or an individual allogeneic to the subject.

[0180] In some embodiments of the present invention, the contacting occurs in a subject, and the lentiviral vector or retroviral vector is any of the lentiviral vectors or retroviral vectors provided by the present invention that has any of the first mutations.

[0181] In some embodiments of the present invention, the contacting occurs in a subject, and the lentiviral vector or retroviral vector is any of the lentiviral vectors or retroviral vectors provided by the present invention that has any of the first mutation and the second mutation.

[0182] In some embodiments of the present invention, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

[0183] In another aspect, the present invention also provides a method for reducing the extracellular expression of an antigen-binding region encoded by a partial shuttle gene on the cell membrane of packaging cells during packaging of a lentiviral vector or a retroviral vector, wherein the antigen-binding region comprises one or more antibodies or antigen-binding fragments thereof, and the method comprises operably linking the antigen-binding region to the N-terminus of a TCR subunit or a functional fragment thereof, wherein the TCR subunit or functional fragment thereof is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, and CD3ε; the reduction in extracellular expression of the antigen-binding region is relative to when the antigen-binding region is operably linked to a fusion protein, wherein the fusion protein is not any of the TCR subunits or functional fragments thereof;

[0184] Preferably, the TCR subunit is CD3γ or CD3ε.

[0185] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide;

[0186] Preferably, the connecting peptide comprises a flexible connecting peptide;

[0187] More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence of the connecting peptide 1 are shown in SEQ ID NO: 18; wherein n=1 to 4.

[0188] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via the connecting peptide.

[0189] In some embodiments of the present invention, the antigen binding region specifically binds to at least one of the following antigens:

[0190] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D. .

[0191] In some embodiments of the present invention, the shuttle gene further comprises a leader sequence encoding a signal peptide;

[0192] Preferably, the signal peptide is selected from the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide;

[0193] More preferably, the signal peptide is CD8α signal peptide.

[0194] In some embodiments of the present invention, the fusion protein comprises a transmembrane region and an intracellular signaling domain;

[0195] Preferably, the fusion protein further comprises a costimulatory signaling domain composed of one or more costimulatory molecules;

[0196] More preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein to form a chimeric antigen receptor (CAR);

[0197] More preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein via a hinge region to form a chimeric antigen receptor.

[0198] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the fusion protein via a flexible linker peptide to form a CAR.

[0199] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the fusion protein through a hinge region to form a CAR.

[0200] In some embodiments of the present invention, the hinge region of the CAR is selected from the hinge region of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154.

[0201] In some embodiments of the present invention, the hinge region of the CAR is the hinge region of CD8α.

[0202] In some embodiments of the present invention, the transmembrane region of the CAR 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, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4 , BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1.

[0203] In some embodiments of the present invention, the transmembrane region of the CAR includes the transmembrane region of CD8α.

[0204] In some embodiments of the present invention, the costimulatory signaling domain of the CAR comprises one or more costimulatory signaling domains of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAmL, CD244, CD100, ICOS, CD40, and MyD88.

[0205] In some embodiments of the present invention, the costimulatory signaling domain of the CAR comprises the costimulatory signaling domain of 4-1BB and / or CD28.

[0206] In some embodiments of the present invention, the intracellular signaling domain of the CAR is selected from the intracellular signaling domains of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FceRly, FceRβ, FcyRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12 and other proteins containing at least one ITAM intracellular signaling domain.

[0207] In some embodiments of the present invention, the intracellular signaling domain of the CAR is the intracellular signaling domain of CD3ζ.

[0208] In some embodiments of the present invention, the CAR comprises a signal peptide encoded by a leader sequence;

[0209] Preferably, the signal peptide is selected from the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide;

[0210] More preferably, the signal peptide is CD8α signal peptide.

[0211] Fusion protein refers to a recombinant protein or recombinant polypeptide encoded by connecting two or more genes that originally encode different proteins or their functional fragments.

[0212] The antigen binding region that can bind to the target cell surface antigen can be operably linked to the fusion protein to form a recombinant therapeutic protein or polypeptide that can specifically bind to the target cell surface antigen, such as CAR.

[0213] In cell therapy, CAR needs to be expressed on the cell membrane surface of the transfected T cells to form CAR-T cells; CAR-T cells specifically bind to antigens on the surface of target cells through the antigen binding region contained in their extracellular domain, thereby enabling CAR-T cells to effectively kill target cells; therefore, the shuttle gene encoding CAR usually needs to contain a leader sequence encoding a signal peptide.

[0214] However, when packaging a lentiviral vector or a retroviral vector, the CAR encoded by the shuttle gene containing a leader sequence encoding a signal peptide will also be expressed on the cell membrane of the packaging cell, and the viral envelope of the lentiviral vector or retroviral vector is derived from the cell membrane of the packaging cell; therefore, when the lentiviral vector or retroviral vector buds out of the packaging cell, the CAR expressed on the cell membrane of the packaging cell will also become part of the viral envelope of the lentiviral vector or retroviral vector, thereby allowing the lentiviral vector or retroviral vector to infect target cells through the antigen binding region of the CAR, thereby reducing the targeting of the lentiviral vector or retroviral vector to transfect T cells;

[0215] When T cells are transfected in an environment containing target cells, such as in the body of a patient or in the blood of a blood cancer patient, the lentiviral vector or retroviral vector can transfect the target cells, thereby reducing the antigens available for binding on the surface of the target cells; and the engineered immune effector T cells prepared by transfecting T cells with the lentiviral vector or retroviral vector, such as CAR-T cells, can also bind to antigens on the surfaces of other target cells through the antigen binding region of CAR, further leading to the blocking / reduction of antigens available for binding on the surface of the target cells, and ultimately resulting in a reduction in the killing efficiency of engineered immune effector cells such as CAR-T cells.

[0216] Unlike CARs that are normally expressed on the cell membrane of packaging cells, when the antigen-binding region is operably linked to a TCR subunit or a functional fragment thereof to form TCP, the TCP has low or no expression on the membrane in cells other than T cells, such as packaging cells, thereby causing the viral envelope of the packaged lentiviral vector or retroviral vector to not contain or contain a significantly reduced content of TCP and its antigen-binding region relative to the content containing CAR and its antigen-binding region, thereby weakening the ability of the lentiviral vector or retroviral vector to transfect target cells, effectively improving the targeting of T cells transfected by the lentiviral vector or retroviral vector, maintaining the abundance of antigen expression on the surface of target cells, and thereby improving the ability of the prepared TCP-T cells to kill target cells.

[0217] In some embodiments of the present invention, the antigen binding region of the TCP may further comprise one or more antibodies or antigen binding fragments thereof and / or ligands or receptor binding fragments thereof.

[0218] In another aspect, the present invention provides a method for reducing the content of an antigen-binding region encoded by a portion of a shuttle gene in the viral envelope of a lentiviral vector or a retroviral vector, wherein the antigen-binding region comprises one or more antibodies or antigen-binding fragments thereof, the method comprising operably linking the antigen-binding region to the N-terminus of a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, and CD3ε; the reduction in the content of the antigen-binding region is relative to when the antigen-binding region is operably linked to a fusion protein, wherein the fusion protein is not any of the TCR subunits or functional fragments thereof;

[0219] Preferably, the TCR subunit is CD3γ or CD3ε.

[0220] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide;

[0221] Preferably, the connecting peptide comprises a flexible connecting peptide;

[0222] More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence of the connecting peptide 1 are shown in SEQ ID NO: 18; wherein n=1 to 4.

[0223] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via the connecting peptide.

[0224] In some embodiments of the present invention, the antigen binding region specifically binds to at least one of the following antigens:

[0225] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D. .

[0226] In some embodiments of the present invention, the shuttle gene further comprises a leader sequence encoding a signal peptide;

[0227] Preferably, the signal peptide is selected from the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide;

[0228] More preferably, the signal peptide is CD8α signal peptide.

[0229] In some embodiments of the present invention, the fusion protein is any of the aforementioned fusion proteins.

[0230] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the fusion protein to form a CAR.

[0231] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the fusion protein via a flexible linker peptide to form a CAR.

[0232] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the fusion protein through a hinge region to form a CAR.

[0233] In some embodiments of the present invention, the CAR is any of the aforementioned CARs.

[0234] In some embodiments of the present invention, the antigen binding region may further comprise one or more antibodies or antigen binding fragments thereof and / or ligands or receptor binding fragments thereof.

[0235] In another aspect, the present invention provides a method for improving the targeting of a lentiviral vector or retroviral vector carrying a shuttle gene to transfect T cells and / or reducing the ability of the lentiviral vector or retroviral vector to transfect target cells other than T cells, the method comprising reducing the content of the antigen binding region encoded by a portion of the shuttle gene in the viral envelope of the lentiviral vector or retroviral vector;

[0236] Preferably, the method comprises reducing the expression of the antigen binding region encoded by a portion of the shuttle gene on the cell membrane of the packaging cell that packages the lentiviral vector or retroviral vector.

[0237] In some embodiments of the present invention, the method comprises operably linking the antigen binding region to the N-terminus of a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε;

[0238] Preferably, the TCR subunit is CD3γ or CD3ε.

[0239] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide;

[0240] Preferably, the connecting peptide comprises a flexible connecting peptide;

[0241] More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence of the connecting peptide 1 are shown in SEQ ID NO: 18; wherein n=1 to 4.

[0242] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via the connecting peptide.

[0243] In some embodiments of the present invention, the antigen binding region specifically binds to at least one of the following antigens:

[0244] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D. .

[0245] In some embodiments of the present invention, the shuttle gene further comprises a leader sequence encoding a signal peptide;

[0246] Preferably, the signal peptide is selected from the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide;

[0247] More preferably, the signal peptide is CD8α signal peptide.

[0248] In some embodiments of the present invention, the antigen binding region comprises one or more antibodies or antigen binding fragments thereof.

[0249] In some embodiments of the present invention, the antigen binding region comprises one or more ligands or receptor binding fragments thereof.

[0250] In some embodiments of the present invention, the antigen binding region comprises one or more antibodies or antigen binding fragments thereof and / or ligands or receptor binding fragments thereof.

[0251] In some embodiments of the present invention, the reduction in the content of the antigen binding region is relative to when the antigen binding region is operably linked to a fusion protein, and the fusion protein is not any of the TCR subunits or functional fragments thereof.

[0252] In some embodiments of the present invention, the fusion protein is any of the aforementioned fusion proteins.

[0253] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the fusion protein to form a CAR.

[0254] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the fusion protein via a flexible linker peptide to form a CAR.

[0255] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the fusion protein through a hinge region to form a CAR.

[0256] In some embodiments of the present invention, the CAR is any of the aforementioned CARs.

[0257] In another aspect, the present invention provides a TCR subunit or a functional fragment thereof

[0258] (a) reducing the use of antigen-binding regions encoded by a partial shuttle gene for expression on the cell membrane of packaging cells during the packaging of lentiviral vectors or retroviral vectors;

[0259] (b) reducing the content of antigen binding region encoded by a part of the shuttle gene in the viral envelope of a lentiviral vector or a retroviral vector;

[0260] (c) improving the targeting of a lentiviral vector or retroviral vector carrying a shuttle gene to transfect T cells, wherein the shuttle gene comprises a nucleic acid encoding an antigen binding region; or

[0261] (d) use of a lentiviral vector or retroviral vector carrying a shuttle gene to reduce the ability of the vector to transfect non-T cell target cells, wherein the shuttle gene comprises a nucleic acid encoding an antigen binding region;

[0262] The antigen-binding region comprises one or more antibodies or antigen-binding fragments thereof, and the application comprises operably linking the antigen-binding region to the N-terminus of a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, and CD3ε; the reduction in membrane expression of the antigen-binding region, the reduction in the content of the antigen-binding region, the improvement in targeting, or the reduction in transfection ability are relative to when the antigen-binding region is operably linked to a fusion protein, and the fusion protein is not any of the TCR subunits or functional fragments thereof;

[0263] Preferably, the TCR subunit is CD3γ or CD3ε.

[0264] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide;

[0265] Preferably, the connecting peptide comprises a flexible connecting peptide;

[0266] More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence of the connecting peptide 1 are shown in SEQ ID NO: 18; wherein n=1 to 4.

[0267] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via the connecting peptide.

[0268] In some embodiments of the present invention, the antigen binding region specifically binds to at least one of the following antigens:

[0269] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D. .

[0270] In some embodiments of the present invention, the shuttle gene further comprises a leader sequence encoding a signal peptide;

[0271] Preferably, the signal peptide is selected from the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide;

[0272] More preferably, the signal peptide is CD8α signal peptide.

[0273] In some embodiments of the present invention, the fusion protein is any of the aforementioned fusion proteins.

[0274] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the fusion protein to form a CAR.

[0275] In some embodiments of the present invention, the antigen binding region is operably linked to the N-terminus of the fusion protein via a flexible linker peptide to form a CAR.

[0276] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the fusion protein through a hinge region to form a CAR.

[0277] In some embodiments of the present invention, the CAR is any of the aforementioned CARs.

[0278] In some embodiments of the present invention, the antigen binding region may further comprise one or more antibodies or antigen binding fragments thereof and / or ligands or receptor binding fragments thereof.

[0279] In another aspect, the present invention provides an engineered T cell, which is prepared by any one of the methods for transfecting T cells provided by the present invention.

[0280] In another aspect, the present invention provides a composition comprising any one of the lentiviral vectors or retroviral vectors provided by the present invention and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary components.

[0281] In some embodiments of the present invention, the composition comprises any lentiviral vector or retroviral vector having any of the first and second mutations in the envelope glycoprotein provided by the present invention and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservatives, and other auxiliary components.

[0282] In another aspect, the present invention provides a method for maintaining the abundance of target cell surface antigen expression in a subject's body or blood before engineered T cells kill target cells in the subject's body or blood, the method comprising administering any one of the lentiviral vectors or retroviral vectors provided by the present invention or any one of the compositions provided by the present invention to the subject or the subject's blood;

[0283] Preferably, the administration is at least one of intravenous, intratumoral, subcutaneous, intramuscular, sternal and infusion techniques.

[0284] In some embodiments of the present invention, the maintenance of the abundance of target cell surface antigen expression is relative to before the engineered T cells (CAR-T cells) prepared by the lentiviral vector or retroviral vector carrying the CAR gene are administered to the subject or the subject's blood to kill the target cells in the subject's body or blood.

[0285] In some embodiments of the present invention, the maintenance of target cell surface antigen expression abundance is relative to administering a control group lentiviral vector or retroviral vector carrying a CAR gene to the subject or the subject's blood.

[0286] In some embodiments of the present invention, the control group lentiviral vector or retroviral vector has the same technical features as any of the lentiviral vectors or retroviral vectors provided by the present invention, except that it carries the CAR gene instead of any of the TCP genes.

[0287] In another aspect, the present invention provides use of any one of the lentiviral vectors or retroviral vectors or any one of the compositions provided by the present invention in the preparation of a drug for treating and / or preventing a disease;

[0288] Preferably, the disease is selected from at least one of blood cancer and solid cancer;

[0289] More preferably, the disease is at least one selected from blood cancers;

[0290] More preferably, the blood cancer is at least one selected from acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), primary mediastinal large B-cell lymphoma (PMBCL), multiple myeloma (MM), Hodgkin lymphoma (HL), and non-Hodgkin lymphoma (NHL).

[0291] More preferably, the disease is at least one selected from solid cancers;

[0292] More preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

[0293] In some embodiments of the present invention, the blood cancer includes relapsed or refractory blood cancer.

[0294] In some embodiments of the present invention, the blood cancer is selected from at least one of mantle cell lymphoma (MCL) and follicular lymphoma (FL).

[0295] In another aspect, the present invention provides a method for treating cancer in a subject or killing cancer cells in a subject, characterized by administering any one of the lentiviral vectors or retroviral vectors or any one of the compositions provided by the present invention to the subject;

[0296] Preferably, the disease is selected from at least one of blood cancer and solid cancer;

[0297] More preferably, the disease is at least one selected from blood cancers;

[0298] More preferably, the blood cancer is at least one selected from acute lymphoblastic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma;

[0299] More preferably, the disease is at least one selected from solid cancers;

[0300] More preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

[0301] In some embodiments of the present invention, a therapeutically effective amount of the lentiviral vector or retroviral vector or the composition is administered to a subject.

[0302] In some embodiments of the present invention, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

[0303] In another aspect, the present invention provides a shuttle gene, wherein the shuttle gene encodes a T cell receptor chimeric protein, wherein the T cell receptor chimeric protein comprises: (1) a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; (2) an antigen binding region, wherein the antigen binding region is operably linked to the N-terminus of the TCR subunit or the functional fragment thereof, wherein the antigen binding region comprises one or more antibodies or antigen binding fragments thereof;

[0304] Preferably, the TCR subunit is CD3γ or CD3ε.

[0305] In some embodiments of the present invention, the connecting peptide comprises a flexible connecting peptide;

[0306] Preferably, the flexible connecting peptide is selected from the group consisting of a (G4S)n connecting peptide and a connecting peptide 1 having an amino acid sequence as shown in SEQ ID NO: 18; wherein n=1 to 4.

[0307] In some embodiments of the present invention, the C-terminus of the antigen binding region is operably linked to the N-terminus of the TCR subunit or its functional fragment via the connecting peptide.

[0308] In some embodiments of the present invention, the antigen binding region specifically binds to at least one of the following antigens:

[0309] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D. .

[0310] In some embodiments of the present invention, the shuttle gene further comprises a leader sequence;

[0311] Preferably, the leader sequence is selected from the group consisting of leader sequences encoding the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide;

[0312] More preferably, the signal peptide is CD8α signal peptide.

[0313] In some embodiments of the present invention, the antigen binding region may further comprise one or more antibodies or antigen binding fragments thereof and / or ligands or receptor binding fragments thereof.

[0314] In another aspect, the present invention provides a T cell receptor chimeric protein (TCP), wherein the TCP is encoded by any one of the shuttle genes provided by the present invention.

[0315] In another aspect, the present invention provides a nucleic acid vector carrying any one of the shuttle genes provided by the present invention.

[0316] In some embodiments of the present invention, the nucleic acid vector is selected from lipid nanoparticles (Lipid Nanoparticles, "LNPs"), virus-like particles (Virus-Like Particles, "VLPs"), plasmids and viruses.

[0317] In some embodiments of the present invention, the nucleic acid carrier is LNPs.

[0318] In some embodiments of the present invention, the nucleic acid vector is VLPs.

[0319] In some embodiments of the present invention, the nucleic acid vector is a plasmid.

[0320] In some embodiments of the present invention, the nucleic acid vector is a virus; the virus is selected from viruses with a viral envelope and viruses without a viral envelope.

[0321] In some embodiments of the present invention, the virus without a viral envelope is selected from adenovirus and adeno-associated virus.

[0322] In some embodiments of the present invention, the virus with a viral envelope is a lentiviral vector or a retroviral vector.

[0323] The beneficial effects of the present invention include:

[0324] The lentiviral vector or retroviral vector, shuttle gene, method for reducing the expression of antigen binding regions encoded by some shuttle genes on the cell membrane of packaging cells, method for reducing the content of antigen binding regions encoded by some shuttle genes contained in the viral envelope of lentiviral vectors or retroviral vectors, method for improving the targeting of lentiviral vectors or retroviral vectors in transfecting T cells, reducing the ability of lentiviral vectors or retroviral vectors to transfect non-T cell target cells, and the use of TCR subunits in the aforementioned methods can effectively solve technical problems such as lentiviral vectors or retroviral vectors carrying shuttle genes such as CAR genes can effectively infect target cells through the antigen binding regions of CAR contained in their viral envelopes, thereby effectively improving the targeting of lentiviral vectors or retroviral vectors in transfecting T cells (especially in the patient's body or blood), maintaining the expression abundance of target cell surface antigens, and the efficiency of the prepared engineered immune effector T cells in killing target cells.

[0325] In this article:

[0326] "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).

[0327] 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 + After expressing TCP and binding to target tumor cells, T cells produce IL-2, TFN, TNF or a combination thereof. In some embodiments of the present invention, CD8 + T cells lyse antigen-specific target cells after expressing TCP and binding to target cells.

[0328] "T Cell Receptor Chimeric Protein": T Cell Receptor Chimeric Protein is a recombinant protein. The TCP includes various polypeptides such as TCR subunits or functional fragments thereof that constitute the TCR / CD3 complex, and an antigen binding region that can specifically bind to at least one antigen; the TCP is generally capable of binding to antigens on the surface of target cells through the antigen binding region it contains. In some embodiments of the present invention, the antigen binding region is operably linked to the TCR subunit or its functional fragment; illustratively, the antigen binding region can be operably linked to the TCR subunit or its functional fragment via a flexible connecting peptide. In some embodiments of the present invention, the TCP is also operably linked to a signal peptide encoded by a leader sequence. In some embodiments of the present invention, the TCP comprises a TCR subunit.

[0329] "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. For commonly used flexible linker peptides, see 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.

[0330] "Non-activated T cells" refer to T cells that are not proliferating, not differentiating, in a resting state, do not recognize antigens, and have not been activated or stimulated by co-stimulatory molecules, such as T cells in the G0 phase of the cell cycle, resting / quiescent T cells, or immature T cells. T cells.

[0331] "T cell activation molecules or co-stimulatory molecules": In conventional technical methods for preparing CAR-T cells in vitro, additional exogenous activators are usually required, such as anti-CD3 antibody magnetic beads and / or anti-CD28 antibody magnetic beads to stimulate and activate T cells. Various T cell activation molecules or co-stimulatory molecules are known in the art, including but not limited to anti-CD3 antibodies or antigen-binding fragments thereof, CD28 ligands (CD28L) or receptor-binding fragments thereof, anti-CD28 antibodies or antigen-binding fragments thereof, and 4-1BB ligands (4-1BBL or CD137L) or receptor-binding fragments thereof.

[0332] "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.

[0333] 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.

[0334] 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.

[0335] The terms "VHH domain," "nanoantibody," 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.

[0336] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0337] 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.

[0338] Illustratively, 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-stabilized antibodies (dsFv), heavy chain variable regions (VH) or light chain variable regions (VL) of antibodies, Fd fragments consisting of VH and CH1 domains, linear antibodies, heavy chain antibodies, and nanobodies (VHH).

[0339] In some embodiments of the present invention, there is no particular limitation on the order of VH and VL in the scFv 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, such as (G4S) n , where n = 1 to 4.

[0340] "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.

[0341] "Receptor binding fragment" refers to a fragment that does not possess the entire structure of a complete ligand but only contains a portion or a partial variant of the complete ligand, wherein the portion or partial variant has the ability to bind to the receptor. For example, "receptor binding fragment" herein includes, but is not limited to, the extracellular domain and variable region of the ligand.

[0342] "Chimeric Antigen Receptor": Chimeric Antigen Receptor (CAR) refers to a recombinant artificial cell surface receptor that is modified to be expressed on immune effector cells such as lymphocytes and specifically binds to an antigen; CAR does not include TCR subunits such as CD3γ or CD3; CAR includes at least (1) an extracellular antigen binding region, such as scFv or VHH; (2) a transmembrane region that anchors CAR to the cell membrane of immune effector cells such as T cells, and (3) an intracellular signal transduction domain; The extracellular structure of CAR may further include a hinge region; The intracellular structure of CAR may further include one or more costimulatory molecules to form a costimulatory signal transduction domain. CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner using the extracellular antigen binding region. In some embodiments of the present invention, the CAR further includes a signal peptide encoded by a leader sequence.

[0343] "Shuttle gene": Transgene, also known as "transgene", includes exogenous target genes / nucleic acids carried by vectors such as lentiviral vectors or retroviral vectors.

[0344] "Nucleic acid" refers to any compound and / or substance including a polymer containing nucleotides, such as a polynucleotide. 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 from 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. Moreover, the "nucleic acids" described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues.

[0345] "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.

[0346] "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).

[0347] "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 encapsulated in a lipid bilayer, and they transfect target cells by fusing the viral envelope with the cell membrane.

[0348] "Retrovirus" and "retroviral vector": Retrovirus and retroviral vector. A retrovirus is a virus that can integrate a DNA copy of its RNA genome into the DNA of a host cell it infects, thereby altering the host cell genome. An overview of available packaging systems is provided in JM Coffin, SM Hughes et al., Cold Spring Harbor Laboratory Press, 1997, page 447, which is incorporated herein by reference in its entirety.

[0349] "Lentivirus": Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain additional genes with regulatory or structural functions. This increased complexity allows the virus to regulate its life cycle, as it does during latent infection. Some examples of lentiviruses include human immunodeficiency viruses (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV).

[0350] "Lentiviral vector": also known as Lentiviral Vector, which is produced by multiple attenuation of HIV virulence genes through gene editing, genetic engineering and other technical means. For example, the genes env, vif, vpr, vpu and nef are deleted, making the lentiviral vector biosafe.

[0351] Lentiviral vectors can stably integrate target genes, such as shuttle genes, into the chromosomes of target cells, allowing them to express the delivered shuttle genes long-term, offering significant advantages for gene therapy. 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 have a relatively large cloning capacity, sufficient for most anticipated clinical applications.

[0352] 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., J. Virol. 72:8463-8471 (1998), U.S. Pat. No. 6,013,516 and U.S. Pat. No. 5,994,136, each of which is herein incorporated by reference in its entirety.

[0353] Lentiviral vectors are generally packaged in packaging cell lines using a lentiviral vector system. For example, the process and method of packaging lentiviral vectors are described in Merten OW, et al., Production of lentiviral vectors. Mol Ther Methods Clin Dev. 2016, which is incorporated herein by reference in its entirety.

[0354] Commonly used lentiviral vector systems include so-called third-generation lentiviral vector systems. The third-generation lentiviral vector system includes four plasmids. "Transfer plasmid" (Transfer Vector, "TV"; in some embodiments of the present invention, also referred to as "master plasmid") contains the lentiviral vector backbone genome and the polynucleotide / nucleic acid delivered to the target cell by the lentiviral vector, such as target genes such as shuttle genes. Transfer plasmids typically have one or more target gene (shuttle gene) sequences flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequence into the host genome. For safety reasons, transfer plasmids are typically designed to render the resulting vector replication ineffective. For example, the transfer plasmid lacks the genetic elements necessary to produce infectious lentiviral particles in host cells. In addition, the transfer plasmid can be designed to lack the 3'LTR, thereby rendering the virus "self-inactivating" (SIN). See Dull, et al., J. Virol. 72:8463-71 (1998); Miyoshi, et al., J. Virol. 72:8150-57 (1998).

[0355] Third-generation lentiviral vector systems typically also include two "packaging plasmids" and an "envelope plasmid." The "envelope plasmid" typically carries the gene encoding VSV-G, which is operably linked to a promoter, typically the CMV promoter. Third-generation lentiviral vector systems use two packaging plasmids, one encoding the genes gag and pol, and the other encoding the gene rev as a further safety feature, an improvement over the single packaging plasmid of so-called second-generation systems. Exemplary packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0356] The use of lentiviral vector systems relies on a "packaging cell line". Generally speaking, a packaging cell line is a cell line whose cells are capable of producing lentiviral vectors that do not have the ability to self-replicate and can infect / transfect target cells when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cells. Exemplarily, a transfection method including a chemically mediated transfection method, a physically mediated transfection method, or a biologically mediated transfection method can be used to introduce the plasmid into the packaging cell line. For example, chemically mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran, or PEI (Polyethylenimine, polyethyleneimine transfection reagent), and physically mediated transfection methods include transfection methods such as electroporation.

[0357] "Packaging cell line": Generally speaking, a packaging cell line is a cell line that, when a transfer plasmid, one or more packaging plasmids, and an envelope plasmid are introduced into the cell line, produces a lentiviral vector capable of infecting / transfecting host cells. Various methods for introducing plasmids into cells can be used, including but not limited to transfection using chemical reagents such as calcium phosphate, DEAE-dextran, and PEI, or electroporation.

[0358] The packaging cell line is genetically engineered to improve the immunological properties of the lentiviral vectors and retroviral vectors disclosed herein and / or to facilitate transfection of target cells by the lentiviral vectors and retroviral vectors in other ways; such other ways include, but are not limited to, adding genes, deleting genes, and introducing point mutations into genes.

[0359] In some embodiments of the present invention, the packaging cell line includes but is not limited to at least one of the following cell lines: NS0 cell line, Vero cell line, HeLa cell line, COS cell line, CHO cell line, HEK cell line, BHK cell line and MDCKⅡ cell line.

[0360] "Target cells" are cells that express disease-associated antigens.

[0361] "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.

[0362] "Subject": As used herein, "subject" or "individual" are used synonymously and include, but are not limited to, mammals, such as humans or non-human mammals, such as domestic, agricultural, or wild animals, as well as birds and aquatic animals.

[0363] A "patient" is a subject suffering from, at risk for developing, or otherwise in need of any of the lentiviral or retroviral vectors, TCP-T cells, compositions, or methods provided herein.

[0364] "Administering": The lentiviral or retroviral vectors disclosed herein can be administered by any route, including oral, nasal, intravenous, intraarterial, intramuscular, or intraperitoneal. In some embodiments, administering comprises at least one of intravenous, intratumoral, subcutaneous, intramuscular, intrasternal, and infusion techniques.

[0365] “And / or”: should be understood to mean one or two alternatives.

[0366] "About": In this specification, when "about" is used to describe a numerical value "X", "about X" includes the numerical value "X" itself or varies within the range of 1%-15% above or below the numerical value "X"; for example, "about 50%" means including 50% itself or varies within the range of 1%-15% above or below 50%.

[0367] Unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the stated range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0368] "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.

[0369] "Embodiments": Reference throughout this specification to "some embodiments," "some embodiments," "embodiments," "related embodiments," "an 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, 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.

[0370] "Treatment": As used herein, "treatment" includes any beneficial or desired effect associated with treatment. "Treatment" does not necessarily indicate complete eradication or cure of a disease or condition, or its associated symptoms.

[0371] "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.

[0372] "Stable integration": also known as "stable transfection" or "stable expression" (Stable Gene Expression), refers to the integration of exogenous nucleic acids into the host cell genome after introduction into the host cell, and their long-term stable expression in the host cell.

[0373] "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 nonspecific 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.

[0374] "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. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity." 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.

[0375] "Signal peptide": Signal peptide, sometimes also called signal sequence, targeting signal, localization signal, localization sequence, transit peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), including the leader signal peptide encoded by the leader sequence (nucleic acid); signal peptides are present at the N-terminus of most newly synthesized proteins that enter the secretory pathway (occasionally non-classically 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). The role of signal peptides includes prompting cells to transfer proteins, usually to the cell membrane.

[0376] In some embodiments of the present invention, the leader sequence contained in the shuttle gene is selected from the leader sequences encoding the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide.

[0377] In some embodiments of the present invention, the leader sequence is a nucleic acid encoding a CD8α signal peptide.

[0378] "MOI": Multiplicity of Infection (MOI) refers to the number of viral particles added to each cell during transfection. For example, when one million viral particles are added to one million cells, MOI = 1.

[0379] "Operably linked": One nucleic acid is "operably linked" to another nucleic acid when they are placed in a functional relationship. For example, operably linked nucleic acid sequences can be adjacent to each other and, for example, in the case of joining two protein coding regions, in the same reading frame. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if the DNA is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if the site is positioned so as to promote translation.

[0380] "Autologous": As used herein, the term "autologous" means any material derived from the same individual that is subsequently reintroduced into that individual.

[0381] "Allogeneic": As used herein, "allogeneic" refers to a transplant derived from a different individual of the same species.

[0382] "Transfection": As used herein, the term "transfection," "transformation," or "transduction" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell, packaging cell, or other cell. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include primary cells and their progeny.

[0383] "Therapeutically effective amount": As used herein, a "therapeutically effective amount" is an amount of a composition or its active substance sufficient to provide a beneficial effect or otherwise reduce harmful, non-beneficial events when administered to an individual. "Therapeutically effective dose" herein means a dose that produces one or more desired or expected (e.g., beneficial) effects as a result of its administration, which is performed one or more times over a specified time period. The exact dosage will depend on the purpose of the treatment and can be determined by one skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Volumes 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0384] All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety, to the same extent as if each individual publication, document, or patent not specifically and individually indicated as incorporated by reference in its entirety were incorporated by reference in its entirety. In the event of a conflict, the present application, including any definitions herein, will control.

[0385] 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

[0386] Figure 1: Figure 1-a is a schematic diagram of the structure of the TCP comprising the connecting peptide 1 or connecting peptide 2, the antigen binding region targeting CD19, and a TCR subunit; Figure 1-b is a schematic diagram of the structure of the TCP comprising different TCR subunits and the TCR / CD3 complex when combined to form a TCP-TCR;

[0387] Figure 2 is a schematic diagram of the structure of the CAR-19;

[0388] FIG3 shows the expression of CAR-19, TCP-E19-L1 or TCP-E19-L2 in three groups of HEK-293T cells after packaging the lentiviral vectors G1A7-CAR19, G1A7-E19-L1 and G1A7-E19-L2.

[0389] Figure 4: The lentiviral vectors G1A7-CAR19, G1A7-E19-L1 and G1A7-E19-L2 were respectively transfected with CD19 + CD7 - Nalm6 cells, CD19 - CD7 + Jurkat cells and CD19 - CD7 + After the Mock T cells were grown, the expression of CAR-19, TCP-E19-L1 or TCP-E19-L2 in each group of cells was detected;

[0390] Figure 5: The lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2 were transfected into human non-activated PBMCs to prepare TCP-T cells, and the ability of the TCP-T cells in each group to kill cancer cells CD19 in vitro was detected. + Figure 2 shows the results of the killing efficiency test of Nalm6 cells;

[0391] Figure 6: is the plasmid map of the master plasmid TCP-E19-L1;

[0392] FIG7 shows the expression of CAR-19, TCP-G19-L1 or TCP-G19-L2 in each group of packaging cells after packaging the lentiviral vectors G2A7-CAR19, G2A7-G19-L1 and G2A7-G19-L2, respectively.

[0393] FIG8 shows the results of transfecting Nalm6 cells, Mock T cells and Jurkat cells with the lentiviral vectors G2A7-CAR19, G2A7-G19-L1 and G2A7-G19-L2, respectively, to detect the expression of CAR-19, TCP-G19-L1 or TCP-G19-L2 in each group of cells;

[0394] Figure 9: The lentiviral vectors G2A7-G19-L1 and G2A7-G19-L2 were transfected into human non-activated PBMCs to prepare TCP-T cells, and the TCP-T cells in each group were tested for killing CD19 in vitro + Figure 2 shows the results of the killing efficiency test of Nalm6 cells;

[0395] Figure 10 shows the results of transfecting Nalm6 cells, Mock T cells and Jurkat cells with the lentiviral vectors G2A3-G19-L1, G2A3-G19-L2, G2A3-E19-L1 and G2A3-E19-L2, respectively, and detecting the expression of TCP-G19-L1, TCP-G19-L2, TCP-E19-L1 or TCP-E19-L2 in each group of cells;

[0396] Figure 11: The lentiviral vectors G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1 and G2A3-G19-L2 were transfected into human non-activated PBMCs to prepare TCP-T cells, and the ability of the TCP-T cells in each group to kill CD19 in vitro was detected. + Figure 2 shows the results of the killing efficiency test of Nalm6 cells;

[0397] FIG12 is a graph showing the growth curve of Raji cells in each group of Raji-Luci model mice;

[0398] Figure 13: TCP-T cells were prepared by transfecting non-activated human PBMCs with the lentiviral vector G4A7-E19-L1, and the ability of the TCP-T cells to kill CD19 in vitro was tested. + Figure 2 shows the results of the killing efficiency test of Nalm6 cells;

[0399] Figure 14 is a plasmid map of the envelope plasmid 1;

[0400] FIG15 : TCP-T-E33 cells were prepared by transfecting human non-activated PBMCs with the lentiviral vector G2A7-E33 in vitro, and the killing ability of the TCP-T-E33 cells on CD33 cells was detected in vitro. + Figure 2 shows the detection results of the killing efficiency of MOLM13 cells;

[0401] Figure 16: TCP-T-EHER2 cells were prepared by transfecting human non-activated PBMCs with the lentiviral vector G2A7-EHER2 in vitro, and the ability of the TCP-T-EHER2 cells to kill HER2 in vitro was tested. + Figure 2 shows the detection results of the killing efficiency of OVCAR-3 cells;

[0402] FIG17 : TCP-T-ECEA cells were prepared by transfecting human non-activated PBMCs with the lentiviral vector G2A7-ECEA in vitro, and the killing ability of the TCP-T-ECEA cells on CEA was detected in vitro. + Figure 3 shows the detection results of the killing efficiency of T84 cells. DETAILED DESCRIPTION

[0403] Example 1

[0404] The lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2 were packaged.

[0405] 1. Design of membrane-based expression of anti-CD7 antibodies

[0406] In this embodiment, the membrane-expressing anti-CD7 antibody comprises an anti-CD7 antibody, a CD8α hinge region, and a CD8α transmembrane region. The anti-CD7 antibody is indirectly connected to the CD8α transmembrane region through the CD8α hinge region and is contained in the viral envelope of the lentiviral vector, thereby improving the transfection efficiency of the lentiviral vector into CD7. + Targeting of cells, such as T cells;

[0407] The structure of the membrane-expressed anti-CD7 antibody from N-terminus to C-terminus is: CD8α signal peptide, TH-69 heavy chain variable region (VH), the connecting peptide 2, TH-69 light chain variable region (VL), CD8α hinge region, and CD8α transmembrane region.

[0408] (1) The CD8α signal peptide comprises the amino acid sequence shown in SEQ ID NO: 8;

[0409] (2) the VH region of TH-69 comprises the amino acid sequence shown in SEQ ID NO: 16;

[0410] (3) the connecting peptide 2 comprises the amino acid sequence shown in SEQ ID NO: 10;

[0411] (4) the VL region of TH-69 comprises the amino acid sequence shown in SEQ ID NO: 17;

[0412] (5) the hinge region of CD8α comprises the amino acid sequence shown in SEQ ID NO: 12;

[0413] (6) The transmembrane region of CD8α comprises the amino acid sequence shown in SEQ ID NO: 13.

[0414] 2. Construction of T cell receptor chimeric protein containing CD3ε

[0415] In this example, a TCP (TCP-E19) comprising an antigen binding region targeting CD19 and CD3ε was constructed. The structure of the TCP-E19 from the N-terminus to the C-terminus was: the CD8α signal peptide, the antigen binding region targeting CD19, the connecting peptide 1 or the connecting peptide 2, and CD3ε; the antigen binding region included a scFv that specifically binds to CD19, and the VH and VL regions of the FMC-63 antibody. Exemplarily, the schematic structural diagram of the TCP-E19 is shown in FIG1-a ;

[0416] (1) The amino acid sequence of the VH region of FMC-63 is shown in SEQ ID NO: 9;

[0417] (2) The amino acid sequence of the VL region of FMC-63 is shown in SEQ ID NO: 11; the VH region and the VL region are connected by the connecting peptide 2;

[0418] (3) the amino acid sequence of CD3ε is shown in SEQ ID NO: 19;

[0419] (4) The amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO: 18;

[0420] (5) The amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO: 10;

[0421] The C-terminus of the antigen binding region of TCP-E19 can be operably linked to the N-terminus of CD3ε through the connecting peptide 1 (TCP-E19-L1);

[0422] The C-terminus of the antigen-binding region of TCP-E19 can be operably linked to the N-terminus of CD3ε via the linker peptide 2 (TCP-E19-L2).

[0423] 3. Packaging of Lentiviral Vectors G1A7-E19-L1 and G1A7-E19-L2

[0424] A. Prepare the following four plasmids: an envelope plasmid carrying a nucleic acid encoding a mutant VSV-G1 and a nucleic acid encoding the membrane-expressing anti-CD7 antibody (envelope plasmid 1), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a master plasmid carrying a shuttle gene encoding TCP-E19-L1 / 2 (master plasmid TCP-E19-L1 / 2). The envelope plasmid 1 and the master plasmid TCP-E19-L1 / 2 are synthesized by conventional molecular cloning methods. Exemplarily, the plasmid maps of the master plasmid TCP-E19-L1 and the envelope plasmid 1 are shown in Figures 6 and 14, respectively.

[0425] The extracellular domain of the mutant VSV-G1 comprises the amino acid sequence shown in SEQ ID NO: 4; relative to SEQ ID NO: 1, SEQ ID NO: 4 comprises R354Q;

[0426] The full-length protein of the wild-type VSV-G (including the signal peptide) comprises the amino acid sequence shown in SEQ ID NO: 32:

[0427] Wherein, the amino acid sequence shown at positions 1 to 16 of SEQ ID NO: 32:

[0428] MKCLLYLAFLFIGVNC is the amino acid sequence of the signal peptide of wild-type VSV-G.

[0429] B. Packaging lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2: Mix the four plasmids and transfect them into packaging cells HEK-293T cells using PEI reagent. The specific steps are as follows:

[0430] Day 0, 9ug of the main plasmid TCP-E19-L1 / 2, 4ug of the pMDLg / pRRE packaging plasmid, 2ug of the pRSV-REV packaging plasmid, and 2ug of the envelope plasmid 1 were added to 1mL of Opti-MEM medium, shaken, and then 64uL of PEI reagent was added. After pipetting evenly, the mixture was allowed to stand for 10 minutes, and then added to the HEK-293T cell culture medium. The culture medium was renewed after 6 hours. The culture supernatant was collected 48 hours after transfection, filtered through a 0.45um filter membrane, centrifuged at 50,000g for 2.5h, and the supernatant was discarded. The lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2 were resuspended in 200uL of F12 medium and frozen at -80°C.

[0431] The viral envelope of the lentiviral vector G1A7-E19-L1 comprises the mutant VSV-G1 and the membrane-type anti-CD7 antibody-expressing shuttle gene encoding TCP-E19-L1;

[0432] The viral envelope of the lentiviral vector G1A7-E19-L2 comprises the mutant VSV-G1 and the membrane-type anti-CD7 antibody-expressing shuttle gene encoding TCP-E19-L2;

[0433] On Day 2, flow cytometry was used to detect the expression of TCP-E19-L1 or TCP-E19-L2 in the two groups of packaging cells HEK-293T cells. The results are shown in Figure 3-C and Figure 3-D, respectively.

[0434] Opti-MEM alpha Reduced Serum Medium, Brand: GIBCO, Catalog Number: #SP0272;

[0435] HEK-293T cell line culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog Number: #C12430500BT; FBS: Brand: EXCELL, Catalog Number: #FSP500;

[0436] F12 culture medium: Brand: GIBCO, catalog number: #C11330500BT;

[0437] Syringe filter: Brand: SORFA, item number: #622120.

[0438] 4. Packaging of the control lentiviral vector G1A7-CAR19 carrying the nucleic acid encoding CAR

[0439] A. Design of Chimeric Antigen Receptor CAR-19

[0440] In this embodiment, a CAR targeting CD19 (CAR-19) is designed. The structure of the CAR-19 from N-terminus to C-terminus is: the CD8α signal peptide, the CD19-targeting antigen binding region, the CD8α hinge region, the CD8α transmembrane region, the 4-1BB co-stimulatory signaling domain, and the CD3ζ intracellular signaling domain; the antigen binding region is the scFv that specifically binds to CD19, the FMC-63 antibody; illustratively, the structure of the CAR-19 is shown in FIG2 ;

[0441] (1) the 4-1BB costimulatory signaling domain comprises the amino acid sequence shown in SEQ ID NO: 14;

[0442] (2) The intracellular signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO:15.

[0443] B. Packaging of Lentiviral Vector G1A7-CAR19

[0444] (1) preparing the following four plasmids: the envelope plasmid 1, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, and the main plasmid carrying the nucleic acid encoding the CAR-19;

[0445] (2) Packaging of lentiviral vector G1A7-CAR19: Day 0, packaging of lentiviral vector G1A7-CAR19 according to the above-mentioned method for packaging the lentiviral vector G1A7-E19-L1 / 2;

[0446] The viral envelope of the lentiviral vector G1A7-CAR19 comprises the mutant VSV-G1 and the membrane-type expressing anti-CD7 antibody and carries the shuttle gene encoding the CAR-19.

[0447] On Day 2, flow cytometry was used to detect the expression of CAR-19 in the packaging cells HEK-293T cells. The results are shown in Figure 3-B.

[0448] As can be seen from Figure 3, the expression of TCP-E19-L1 and TCP-E19-L2 in HEK-293T cells was significantly lower than that of CAR-19 in HEK-293T cells, which proves that compared with CAR-19, TCP-E19-L1 / 2 is not expressed outside the membrane in packaging cells or the efficiency of membrane expression is significantly lower; this may be because TCP-E19-L1 / 2 is difficult to effectively form TCR / CD3 complex membrane expression in HEK-293T cells.

[0449] Antibody used in flow cytometry: Anti-FMC63, brand: ACROBIOSYSTEM; product number: #FM3-PY54A2-25tests.

[0450] Example 2

[0451] The lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2 were used to transfect different target cells (MOI=1).

[0452] 1. Preparation of Mock T Cells

[0453] Take 1×10 7 Non-activated PBMCs (healthy individuals) were resuspended in 10 mL of PBMCs culture medium (PBMCs culture system), wherein the PBMCs culture medium consisted of 1640 medium + 10% FBS.

[0454] 1 μg / mL (final concentration) of anti-CD3 antibody and 1 μg / mL (final concentration) of anti-CD28 antibody were added to the PBMCs culture system to stimulate and activate the human non-activated PBMCs to prepare Mock T cells.

[0455] 2. Each group of lentiviral vectors transfects different target cells

[0456] On Day 0, at an MOI of 1, the lentiviral vectors G1A7-E19-L1, G1A7-E19-L2, and G1A7-CAR19 were used to co-culture with 1×10 5 CD19 - CD7 + Mock T cells, 1×10 5 CD19 - CD7 + Jurkat cells and 1×10 5 CD19 + CD7 - Nalm6 cells were mixed, infected at room temperature, and cultured in vitro in an incubator at 37°C and a CO2 concentration of 5% for each group of cells; the culture medium for each group of cells was 1 mL of 1640 medium containing 10% FBS, and the culture medium for Mock T cells also contained IL-2 with a final concentration of 1000 IU / mL; on Day 2, 10 mL of DPBS buffer was added, mixed, and then centrifuged at 500 g for 3 minutes, and the supernatant was discarded; on Day 5, the expression of the CAR-19 or TCP-E19-L1 / 2 in the above-mentioned cells was detected by flow cytometry, and the results are shown in Figure 4.

[0457] As shown in Figure 4, the CD19 mixed with the lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2 + CD7 - Nalm6 cells barely expressed TCP-E19-L1 / 2, whereas Nalm6 cells mixed with the lentiviral vector G1A7-CAR19 expressed CAR-19 in a relatively large amount;

[0458] This is because the viral envelope of the lentiviral vector G1A7-CAR19 contains a large amount of CAR-19 derived from the cell membrane of the packaging cell HEK-293T cell. Therefore, the lentiviral vector G1A7-CAR19 can specifically bind to CD19 through the antigen binding region of CAR-19 and the FMC-63 antibody. +The antigen CD19 on the surface of Nalm6 cells is then transfected into the Nalm6 cells, and the nucleic acid encoding CAR-19 is stably integrated into the genome of the Nalm6 cells, so that the CAR-19 is expressed in large quantities on the cell membrane of the Nalm6 cells;

[0459] However, since the TCP-E19-L1 / 2 is not expressed or its expression efficiency is significantly lower in HEK-293T packaging cells, the viral envelope of the lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2 does not contain or almost does not contain the TCP-E19-L1 / 2 derived from the cell membrane of HEK-293T packaging cells, making it difficult to effectively transfect CD19. + CD7 - Nalm6 cells.

[0460] At the same time, as shown in Figure 4, the lentiviral vectors G1A7-E19-L1, G1A7-E19-L2 and G1A7-CAR19 can all specifically bind to CD7 through the anti-CD7 antibodies contained in their viral envelopes. + Jurkat cells and CD7 + The endocytic receptor CD7 on the surface of the Mock T cells is effectively transfected into the Jurkat cells and the Mock T cells through endocytosis;

[0461] Since both the Jurkat cells and the Mock T cells contain TCR subunits, namely CD3ε, the TCP-E19-L1 / 2 can effectively combine with the endogenous TCR / CD3 complex in both Jurkat cells (human T lymphoid leukemia cells derived from T cells) and Mock-T cells, effectively forming a TCR / CD3 complex that is expressed on the cell membranes of Jurkat cells and Mock T cells.

[0462] DPBS buffer: Brand: Seville, Product No.: #G4200-500ML;

[0463] IL-2: Beijing Sihuan Pharmaceutical, product number: S200040008.

[0464] Example 3

[0465] Detect the effect of TCP-T-E19 cells in killing target cells.

[0466] At an MOI of 5, 1×10 6 For personal non-activated PBMCs (healthy individuals), the specific steps are as follows:

[0467] Day 0, take 1×10 6 The revived human inactivated PBMCs were resuspended in 500 uL of normal saline. The method for resuscitating frozen PBMCs is well known to those skilled in the art. The lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2 were added to the human inactivated PBMCs at an MOI of 5 and mixed evenly. After transfection at room temperature for 10 minutes, 10 mL of DPBS buffer was added and mixed evenly. The mixture was then centrifuged at 500 g for 3 minutes, the supernatant was discarded, and 1 mL of XVT medium containing 20 ng / ml IL-7 and 20 ng / ml IL-15 was added to resuspend the transfected PBMCs. In vitro cell culture was performed in an incubator at 37°C and a CO2 concentration of 5% to prepare TCP-T-E19 cells.

[0468] On Day 3, according to the effector-target ratio E:T=3:1, the transfected PBMCs in each group were mixed with 1×10 5 Target cell CD19 + Nalm6 cells (human B lymphoid leukemia cells) were mixed and cultured in vitro in an incubator at 37°C and 5% CO2 concentration;

[0469] On Day 6, flow cytometry was used to detect the expression of CD19 in each group of mixed cells to detect the killing effect of two groups of TCP-T-E19 cells prepared by transfecting human non-activated PBMCs with the lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2, respectively, on Nalm6 cells. The results are shown in Figures 5-B and 5-C, respectively.

[0470] The control group was a mixture of human non-activated PBMCs and target cells Nalm6 cells for killing at an effector-target ratio E:T=3:1. The results are shown in FIG5-A .

[0471] As shown in Figure 5, TCP-T-E19 cells prepared by transfecting human non-activated PBMCs with the lentiviral vectors G1A7-E19-L1 and G1A7-E19-L2 respectively can effectively kill target cells Nalm6 cells, and the killing effect is significantly better than the killing effect of human non-activated PBMCs on Nalm6 cells.

[0472] XVT culture medium: Brand: IRVINE, Trade name: PRIME-XV T cell CDM, Catalog number: #91154;

[0473] IL-7: Brand: Sino Biological, Trade Name: IL-7 Protein, Human, Recombinant, Catalog Number: #11821-HNAE;

[0474] IL-15: Brand: Sino Biological, Trade Name: IL-15 Protein, Human, Recombinant (His Tag), Catalog Number: #10360-H07E.

[0475] Example 4

[0476] 1. Construction of a T cell receptor chimeric protein containing CD3γ

[0477] A TCP (TCP-G19) comprising an antigen binding region targeting CD19 and CD3γ was constructed, wherein the TCP-G19 comprises the connecting peptide 1 (TCP-G19-L1) or the connecting peptide 2 (TCP-G19-L2).

[0478] In this embodiment, the structure of TCP-G19-L1 / 2 from N-terminus to C-terminus is: the CD8α signal peptide, the antigen binding region targeting CD19, the connecting peptide 1 or the connecting peptide 2, CD3γ;

[0479] The amino acid sequence of CD3γ is shown in SEQ ID NO: 5;

[0480] The C-terminus of the antigen-binding region is operably linked to the N-terminus of CD3γ through the connecting peptide 1 or the connecting peptide 2.

[0481] 2. Packaging of Lentiviral Vectors G2A7-G19-L1 and G2A7-G19-L2

[0482] Day 0: Referring to the method for packaging the lentiviral vector G1A7-E19-L1, the following four plasmids were transfected into HEK-293T packaging cells using PEI reagent: an envelope plasmid carrying a nucleic acid encoding the membrane-expressing anti-CD7 antibody and encoding the mutant VSV-G2 (envelope plasmid 2), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a main plasmid carrying the shuttle gene encoding the TCP-G19-L1 / 2, to package the lentiviral vectors G2A7-G19-L1 and G2A7-G19-L2, respectively.

[0483] The extracellular domain of the mutant VSV-G2 comprises the amino acid sequence shown in SEQ ID NO: 3, and relative to SEQ ID NO: 1, SEQ ID NO: 3 comprises a K47 deletion;

[0484] The viral envelope of the lentiviral vector G2A7-G19-L1 comprises the mutant VSV-G2 and the membrane-type anti-CD7 antibody-expressing shuttle gene encoding TCP-G19-L1;

[0485] The viral envelope of the lentiviral vector G2A7-G19-L2 comprises the mutant VSV-G2 and the membrane-type anti-CD7 antibody-expressing shuttle gene encoding the TCP-G19-L2.

[0486] On Day 2, flow cytometry was used to detect the expression of TCP-G19-L1 and TCP-G19-L2 in the two groups of packaging cells HEK-293T cells. The results are shown in Figure 7-C and Figure 7-D, respectively.

[0487] 3. Packaging of the control group lentiviral vector G2A7-CAR19

[0488] Day 0: Referring to the method for packaging the control group lentiviral vector G1A7-CAR19, the following four plasmids: the envelope plasmid 2, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, and the main plasmid CAR-19 were transfected into HEK-293T cells using PEI reagent to package the control group lentiviral vector G2A7-CAR19;

[0489] The viral envelope of the lentiviral vector G2A7-CAR19 comprises the mutant VSV-G2 and the membrane-type anti-CD7 antibody-expressing shuttle gene encoding the CAR-19;

[0490] On Day 2, flow cytometry was used to detect the expression of CAR-19 in the packaging cells HEK-293T cells. The results are shown in Figure 7-B.

[0491] As can be seen from Figure 7, the expression efficiency of TCP-G19-L1 and TCP-G19-L2 in packaging cells is significantly lower than the expression efficiency of CAR-19 in packaging cells. TCP constructed using CD3γ to connect the antigen binding region is not expressed on the membrane in cells other than T cells, such as HEK-293T cells, or its expression efficiency is significantly lower than that of fusion proteins such as CAR.

[0492] Example 5

[0493] The lentiviral vectors G2A7-CAR19, G2A7-G19-L1 and G2A7-G19-L2 were used to transfect Mock T cells, Jurkat cells, Nalm6 cells and human non-activated PBMCs cells (healthy people), respectively.

[0494] 1. Transfection of Nalm6 cells and Mock T cells (MOI = 5)

[0495] On Day 0, at an MOI of 5, the lentiviral vector G1A7-E19-L1 was used to transfect Mock T cells and Nalm6 cells. The lentiviral vectors G2A7-CAR19, G2A7-G19-L1, and G2A7-G19-L2 were respectively co-transfected with 1×10 5 CD19 - CD7 + Mock T cells, 1×10 5 CD19 + CD7 - On Day 5, the expression of CAR-19, TCP-G19-L1 or TCP-G19-L2 in each group of cells was detected by flow cytometry, and the results are shown in FIG8 .

[0496] As shown in Figure 8, the lentiviral vectors G2A7-CAR19, G2A7-G19-L1 and G2A7-G19-L2 can effectively transfect CD7 through the anti-CD7 antibodies contained in their viral envelopes. + Mock T cells;

[0497] However, compared with Nalm6 cells mixed with the lentiviral vector G2A7-G19-L1 or G2A7-G19-L2, the expression of CD19 on the surface of Nalm6 cells mixed with the lentiviral vector G2A7-CAR19 was significantly reduced and the cells began to express the CAR-19.

[0498] This demonstrates that the lentiviral vector G2A7-CAR19 can effectively transfect CD19 through the antigen binding region (FMC-63) of the CAR-19 derived from the cell membrane of the packaging cell contained in its viral envelope. + CD7 - Nalm6 cells; and the viral envelope of the lentiviral vector G2A7-G19-L1 or G2A7-G19-L2 does not contain or hardly contains the TCP-G19-L1 / 2 and the antigen binding region (FMC-63) contained therein, so it is difficult to effectively transfect CD19 + CD7 - Nalm6 cells.

[0499] 2. Transfection of Jurkat cells (MOI = 0.2)

[0500] On Day 0, referring to the transfection method of the lentiviral vector G1A7-E19-L1 to transfect Jurkat cells, at an MOI of 0.2, 1×10 5CD19 - CD7 + Jurkat cells; Day 5, flow cytometry was used to detect the expression of CAR-19, TCP-G19-L1 or TCP-G19-L2 in each group of Jurkat cells; the results are shown in Figure 8;

[0501] As shown in Figure 8, the lentiviral vectors G2A7-CAR19, G2A7-G19-L1 and G2A7-G19-L2 can effectively transfect CD7 through the anti-CD7 antibodies contained in their viral envelopes. + Jurkat cells.

[0502] 3. Transfection of non-activated human PBMCs (MOI = 5)

[0503] Day 0, at an MOI of 5, referring to the method for transfecting non-activated human PBMCs with the lentiviral vector G1A7-E19-L1, non-activated human PBMCs were transfected with the lentiviral vectors G2A7-G19-L1 and G2A7-G19-L2, respectively, to prepare engineered immune effector T cells (TCP-T-G19 cells) expressing the TCP-G19-L1 or TCP-G19-L2;

[0504] On Day 2, transfected PBMCs in each group were mixed with target Nalm6 cells at an effector-to-target ratio of 3:1 and cultured in vitro in a 37°C, 5% CO2 incubator. A control group was treated with a mixture of non-activated human PBMCs and target Nalm6 cells at an effector-to-target ratio of 3:1.

[0505] On Day 7, flow cytometry was used to detect and record the expression of CD19 in each group of mixed cells to detect the killing effect of TCP-G19 cells expressing the TCP-G19-L1 or the TCP-G19-L2 prepared by transfecting human non-activated PBMCs with the lentiviral vectors G2A7-G19-L1 and G2A7-G19-L2, respectively, on Nalm6 cells. The results are shown in Figure 9.

[0506] As shown in Figure 9, TCP-T-G19 cells prepared by transfecting human non-activated PBMCs with the lentiviral vectors G2A7-G19-L1 and G2A7-G19-L2 respectively can effectively kill target cells Nalm6 cells, and the killing effect is significantly better than the killing effect of human non-activated PBMCs on Nalm6 cells.

[0507] Example 6

[0508] Construct a lentiviral vector G2A3-E19 / G19-L1 / L2 targeting the endocytic receptor CD3.

[0509] 1. Construction of membrane-bound anti-CD3 antibody expression

[0510] Constructing a membrane-expressing anti-CD3 antibody, wherein the structure of the membrane-expressing anti-CD3 antibody from N-terminus to C-terminus is: the CD8α signal peptide, UCHT1-scFv, the CD8α hinge region, and the CD8α transmembrane region;

[0511] The UCHT1-scFv comprises the amino acid sequence shown in SEQ ID NO: 20.

[0512] 2. Packaging of Lentiviral Vector G2A3-E19 / G19-L1

[0513] Day 0, referring to the method for packaging the lentiviral vector G1A7-E19-L1, the following four plasmids were transfected into HEK-293T packaging cells using PEI reagent: an envelope plasmid carrying a nucleic acid encoding the mutant VSV-G2 and a nucleic acid encoding the membrane-bound anti-CD3 antibody (envelope plasmid 3), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a main plasmid carrying a nucleic acid encoding the TCP-E19-L1 / 2 or TCP-G19-L1 / 2, to package the lentiviral vectors G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1, and G2A3-G19-L2, whose viral envelopes contained the mutant VSV-G2 and the membrane-bound anti-CD3 antibody;

[0514] The lentiviral vector G2A3-E19-L1 comprises a shuttle gene encoding the TCP-E19-L1;

[0515] The lentiviral vector G2A3-E19-L2 comprises a shuttle gene encoding the TCP-E19-L2;

[0516] The lentiviral vector G2A3-G19-L1 comprises a shuttle gene encoding the TCP-G19-L1;

[0517] The lentiviral vector G2A3-G19-L2 comprises a shuttle gene encoding the TCP-G19-L2.

[0518] 3. Transfect Mock T cells and Nalm6 cells (MOI=5)

[0519] On Day 0, at an MOI of 5, the lentiviral vector G1A7-E19-L1 was used to transfect Mock T cells and Nalm6 cells. The lentiviral vectors G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1, and G2A3-G19-L2 were respectively transfected with 1×10 5 CD19 - CD3 + Mock T cells and 1×10 5 CD19 + CD3 - Nalm6 cell mixture;

[0520] On Day 5, flow cytometry was used to detect the expression of TCP-E19-L1 / 2 (CD3E-A3 group) or TCP-G19-L1 / 2 (CD3G-A3 group) in each group of cells. The results are shown in FIG10 .

[0521] 4. Transfection of Jurkat cells (MOI = 0.2)

[0522] On Day 0, referring to the transfection method of the lentiviral vector G1A7-E19-L1 to transfect Jurkat cells, at an MOI of 0.2, 1×10 5 CD19 - CD3 + Jurkat cells; Day 5, flow cytometry was used to detect the expression of TCP-E19-L1 / 2 or TCP-G19-L1 / 2 in each group of Jurkat cells; the results are shown in Figure 10.

[0523] As shown in Figure 10, the lentiviral vectors G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1 and G2A3-G19-L2 can effectively transfect CD3 through the anti-CD3 antibodies contained in their viral envelopes. + Mock T cells and CD3 + Jurkat cells;

[0524] However, CD19 mixed with the lentiviral vectors G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1 or G2A3-G19-L2 + CD3 -The expression of CD19 on the surface of Nalm6 cells remained almost unchanged; this proves that the viral envelope of the lentiviral vector G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1 or G2A3-G19-L2 does not contain or hardly contains TCP-E19-L1 / 2 or TCP-G19-L1 / 2, making it difficult to effectively transfect CD19 + CD3 - Nalm6 cells.

[0525] Example 7

[0526] Human non-activated PBMCs (healthy subjects) were transfected using the lentiviral vectors G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1, and G2A3-G19-L2, respectively.

[0527] Day 0, at an MOI of 5, referring to the method for transfecting non-activated human PBMCs with the lentiviral vector G1A7-E19-L1, and transfecting non-activated human PBMCs with the lentiviral vectors G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1, and G2A3-G19-L2, respectively;

[0528] On Day 2, according to the effector-target ratio E:T=1:1, the transfected PBMCs in each group were mixed with 1×10 5 Nalm6 cells were mixed and cultured in vitro at 37°C in an incubator with a CO2 concentration of 5%;

[0529] On Day 7, flow cytometry was used to detect and record the expression of CD19 in the mixed cells of each group to detect whether the TCP-T-E19 and TCP-T-G19 cells prepared by transfecting human non-activated PBMCs with the lentiviral vectors G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1 and G2A3-G19-L2 respectively killed CD19. + The killing effect of Nalm6 cells is shown in Figure 11.

[0530] In the control group, human non-activated PBMCs and target cells Nalm6 were mixed for killing at an effector-target ratio E:T=1:1. The results are shown in FIG11 .

[0531] As can be seen from Figure 11, TCP-T-E19 and TCP-T-G19 cells prepared by transfecting human non-activated PBMCs with the lentiviral vectors G2A3-E19-L1, G2A3-E19-L2, G2A3-G19-L1 and G2A3-G19-L2, respectively, can efficiently kill target cells Nalm6 cells, and the killing effects are significantly better than the killing effects of human non-activated PBMCs on Nalm6 cells.

[0532] Example 8

[0533] 1. Packaging of Lentiviral Vector G3A7-E19-L1

[0534] Referring to the method for packaging the lentiviral vector G1A7-E19-L1, the following four plasmids were transfected into packaging cells HEK-293T cells using PEI reagent: an envelope plasmid (envelope plasmid 4) carrying a nucleic acid encoding a mutant VSVG-3 and a nucleic acid encoding the membrane-expressing anti-CD7 antibody, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a main plasmid carrying a nucleic acid encoding the TCP-E19-L1. The viral envelope contained a mutant VSV-G3 and the membrane-expressing anti-CD7 antibody and carried the nucleic acid encoding the TCP-E19-L1. Lentiviral vector G3A7-E19-L1;

[0535] The extracellular domain of the mutant VSV-G3 comprises the amino acid sequence shown in SEQ ID NO: 6; relative to SEQ ID NO: 1, SEQ ID NO: 6 comprises K47 deletion, T214N and T352A.

[0536] 2. Packaging of the control group lentiviral vector G3A7-CAR19

[0537] Referring to the method for packaging the lentiviral vector G1A7-E19-L1, the following four plasmids: the envelope plasmid 4, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid and the CAR-19 main plasmid were transfected into the packaging cell HEK-293T cells using PEI reagent, and the packaging virus envelope contained the mutant VSV-G3 and the membrane-type anti-CD7 antibody-expressing lentiviral vector G3A7-CAR19 carrying the nucleic acid encoding the CAR-19.

[0538] 3. In vivo killing assay in model mice

[0539] On Day 0, 1×10 6 Raji (CD19 +The mouse was injected with 200 μL of 15 mg / mL D-luciferin sodium salt working solution into the peritoneal cavity. In vivo imaging showed that the model was successfully established.

[0540] On Day 0, 1×10 7 Human non-activated PBMCs (healthy people); 2 hours later, at an MOI of 5, the two groups of membrane-forming mice that had been injected with human non-activated PBMCs were injected with the virus solution of the lentiviral vectors G3A7-CAR19 and G3A7-E19-L1 through the tail vein respectively;

[0541] By means of in vivo imaging, the changes in the number of Raji-Luci cells in each group of mice were recorded. The growth curves of Raji cells in each group of mice are shown in FIG12 .

[0542] As shown in Figure 12, the lentiviral vector G3A7-E19-L1 was transfected into PBMCs in model mice to prepare TCP-T cells that killed CD19 + The killing efficiency of Raji cells was significantly better than that of CAR-T cells prepared by transfecting PBMCs with the lentiviral vector G3A7-CAR19 in model mice to kill CD19. + The killing efficiency of Raji cells.

[0543] This is because the lentiviral vector G3A7-CAR19 can transfect CD19 through the CD19-targeting antigen binding region (FMC63 antibody) of CAR-19 contained in its viral envelope and derived from the cell membrane of the packaging cell. + This will not only reduce the efficiency of the lentiviral vector G3A7-CAR19 in targeting and transfecting T cells in human PBMCs, thereby reducing the efficiency of preparing CAR-T cells; it will also lead to a decrease in the expression abundance of CD19 on the surface of Raji cells in the model mice, further reducing the efficiency of CAR-T cells in killing Raji cells by binding to CD19 on the surface of Raji cells;

[0544] However, the viral envelope of the lentiviral vector G3A7-E19-L1 does not contain or hardly contains the TCP-E19-L1 and its antigen binding region targeting CD19, making it difficult to effectively transfect CD19 in model mice. + Raji cells; therefore, the efficiency of the lentiviral vector G3A7-E19-L1 in targeting and transfecting T cells in human PBMCs in model mice is significantly higher, and the expression abundance of CD19 on the surface of Raji cells in mice can also be relatively maintained, and the efficiency of the prepared TCP-T cells in killing Raji cells is also significantly higher.

[0545] D-Luciferin sodium salt working solution: Brand: Yisheng Biological, Trade name: D-Luciferin, Sodium Salt, Item number: #40901ES10.

[0546] Example 9

[0547] 1. Packaging of Lentiviral Vector G4A7-E19-L1

[0548] Referring to the method for packaging the lentiviral vector G1A7-E19-L1, the following four plasmids were transfected into packaging cells HEK-293T cells using PEI reagent: an envelope plasmid carrying a nucleic acid encoding a mutant Cocal-G1 and a nucleic acid encoding the membrane-expressing anti-CD7 antibody (envelope plasmid 5), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a main plasmid carrying a nucleic acid encoding the TCP-E19-L1. The viral envelope contained the mutant Cocal-G1 and the membrane-expressing anti-CD7 antibody and carried the nucleic acid encoding the TCP-E19-L1. Lentiviral vector G4A7-E19-L1;

[0549] The extracellular domain of wild-type Cocal-G comprises the amino acid sequence shown in SEQ ID NO: 2;

[0550] The extracellular domain of the mutant Cocal-G1 comprises the amino acid sequence shown in SEQ ID NO: 21; relative to SEQ ID NO: 2, SEQ ID NO: 21 comprises a K47 deletion.

[0551] The sequence of the full-length protein of wild-type Cocal-G (including the signal peptide) is shown in SEQ ID NO: 25;

[0552] Wherein, the sequence shown in positions 1 to 17 of SEQ ID NO: 25:

[0553] MNFLLLTFIVLPLCSHA is the amino acid sequence of the signal peptide of wild-type Cocal-G:

[0554] 2. Transfect PBMCs using the lentiviral vector G4A7-E19-L1

[0555] Day 0, at an MOI of 5, the lentiviral vector G4A7-E19-L1 was used to transfect human non-activated PBMCs, referring to the method for transfecting human non-activated PBMCs with the lentiviral vector G1A7-E19-L1;

[0556] On Day 2, transfected PBMCs were mixed with target Nalm6 cells at an effector-to-target ratio of 3:1 and cultured in vitro in a 37°C, 5% CO2 incubator. A control group consisted of a mixture of non-activated human PBMCs and target Nalm6 cells at an effector-to-target ratio of 3:1.

[0557] On Day 7, flow cytometry was used to detect and record the expression of CD19 in each group of mixed cells to detect the killing effect of TCP-T-E19 cells prepared by transfecting human non-activated PBMCs with the lentiviral vector G4A7-E19-L1 on Nalm6 cells. The results are shown in Figure 13.

[0558] As shown in Figure 13, TCP-T-E19 cells prepared by transfecting human non-activated PBMCs with the lentiviral vector G4A7-E19-L1 can efficiently kill target cells Nalm6 cells, and the killing effect is significantly better than the killing effect of human non-activated PBMCs on Nalm6 cells.

[0559] Example 10

[0560] 1. Build TCP-E33

[0561] A TCP (TCP-E33) comprising an antigen binding region targeting CD33 and CD3ε was constructed. The structure of the TCP-E33 from N-terminus to C-terminus was: the CD8α signal peptide, the antigen binding region targeting CD33, the connecting peptide 1, and the CD3ε. The antigen binding region comprised a scFv (scFv-33) targeting CD33. The scFv-33 comprised a heavy chain variable region (VH) and a light chain variable region (VL) derived from the antibody Gemtuzumab. The VH region of the scFv-33 was connected to the VL region of the scFv-33 via the connecting peptide 2.

[0562] The VH region of the scFv-33 comprises the amino acid sequence shown in SEQ ID NO: 26;

[0563] The VL region of the scFv-33 comprises the amino acid sequence shown in SEQ ID NO: 27.

[0564] 2. Packaging of Lentiviral Vector G2A7-E33

[0565] Referring to the method for packaging the lentiviral vector G1A7-E19-L1, the envelope plasmid 2, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the main plasmid carrying the nucleic acid encoding the TCP-E33 were transfected into packaging cell HEK-293T cells using PEI reagent, and the viral envelope contained the membrane-expressing anti-CD7 antibody and the mutant VSV-G2 and contained the nucleic acid encoding the TCP-E33. The lentiviral vector G2A7-E33 was packaged and collected.

[0566] 3. Transfecting human non-activated PBMCs to prepare CD33-targeted TCP-T-E33 cells

[0567] On Day 0, at an MOI of 5, the method for transfecting non-activated human PBMCs with the lentiviral vector G1A7-E19-L1 was used to transfect 1×10 6 Individual non-activated PBMCs were used to prepare TCP-T-E33 cells expressing the TCP-E33;

[0568] On Day 2, the transfected PBMCs were mixed with 1×10 5 CD33 + MOLM13 cells (human acute myeloid leukemia cells, brand: Punosai, catalog number: #CL-0681) were mixed; the control group was 3×10 5 Non-activated human PBMCs that were not transfected with the virus were mixed with 1×10 5 A mixture of MOLM13 cells (effector-target ratio E:T = 3:1);

[0569] On Day 7, flow cytometry was used to detect and record the expression of CD33 in each group of mixed cells to detect the killing effect of TCP-T-E33 cells prepared by transfecting human non-activated PBMCs with the lentiviral vector G2A7-E33 on Nalm6 cells. The results are shown in Figure 15.

[0570] As shown in FIG15 , the TCP-T-E33 cells prepared by transfecting human non-activated PBMCs in vitro with the lentiviral vector G2A7-E33 can efficiently kill CD33 + The killing effect of PBMCs on MOLM13 cells was significantly better than that of non-activated human PBMCs on MOLM13 cells.

[0571] Example 11

[0572] 1. Build TCP-EHER2

[0573] A TCP (TCP-EHER2) comprising an antigen binding region targeting HER2 and CD3ε was constructed, wherein the structure of the TCP-EHER2 from the N-terminus to the C-terminus was: the CD8α signal peptide, the antigen binding region targeting HER2, the connecting peptide 1, and the CD3ε; the antigen binding region comprised an scFv (scFv-HER2) targeting HER2, the scFv-HER2 comprised a heavy chain variable region and a light chain variable region derived from the antibody Pertuzumab, and the VH region of the scFv-HER2 was connected to the VL region of the scFv-HER2 via the connecting peptide 2.

[0574] The VH region of the scFv-HER2 comprises the amino acid sequence shown in SEQ ID NO: 28;

[0575] The VL region of the scFv-HER2 comprises the amino acid sequence shown in SEQ ID NO: 29.

[0576] 2. Packaging of Lentiviral Vector G2A7-EHER2

[0577] Referring to the method for packaging the lentiviral vector G1A7-E19-L1, the envelope plasmid 2, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the main plasmid carrying the nucleic acid encoding the TCP-EHER2 were transfected into packaging cells HEK-293T cells using PEI reagent, and the lentiviral vector G2A7-EHER2 containing the membrane-expressing anti-CD7 antibody and the mutant VSV-G2 and the nucleic acid encoding the TCP-EHER2 was packaged and collected.

[0578] 3. Transfecting non-activated human PBMCs to prepare HER2-targeted TCP-T-EHER2 cells

[0579] On Day 0, at an MOI of 5, the method for transfecting non-activated human PBMCs with the lentiviral vector G1A7-E19-L1 was used to transfect 1×10 6 Individual non-activated PBMCs were used to prepare TCP-T-EHER2 cells expressing the TCP-EHER2;

[0580] On Day 2, the transfected PBMCs were mixed with 1×10 5 HER2 + OVCAR-3 cells (human ovarian cancer cells, brand: Punosai, catalog number: #CL-0178) were mixed; the control group was 3×10 5 Non-activated human PBMCs that were not transfected with the virus were mixed with 1×10 5A mixture of 100 OVCAR-3 cells (effector-target ratio E:T = 3:1);

[0581] On Day 7, flow cytometry was used to detect and record the expression of HER2 in each group of mixed cells to detect the killing effect of TCP-T-EHER2 cells prepared by transfecting human non-activated PBMCs with the lentiviral vector G2A7-EHER2 on Nalm6 cells. The results are shown in Figure 16.

[0582] As shown in Figure 16, the TCP-T-EHER2 cells prepared by transfecting human non-activated PBMCs in vitro with the lentiviral vector G2A7-EHER2 can effectively kill HER2 + The killing effect of PBMCs on OVCAR-3 cells was significantly better than that of non-activated human PBMCs on OVCAR-3 cells.

[0583] Example 12

[0584] 1. Build TCP-ECEA

[0585] A TCP (TCP-ECEA) comprising an antigen binding region targeting CEA (Carcinoembryonic Antigen, "CEA") and CD3ε is constructed, wherein the structure of the TCP-ECEA from N-terminus to C-terminus is: the CD8α signal peptide, the antigen binding region targeting CEA, the connecting peptide 1, and the CD3ε; the antigen binding region comprises a scFv targeting CEA (scFv-CEA), wherein the VH region of the scFv-CEA is connected to the VL region of the scFv-CEA via the connecting peptide 2.

[0586] The VH region of the scFv-CEA comprises the amino acid sequence shown in SEQ ID NO: 30;

[0587] The VL region of the scFv-CEA comprises the amino acid sequence shown in SEQ ID NO: 31.

[0588] 2. Packaging of Lentiviral Vector G2A7-ECEA

[0589] Referring to the method for packaging the lentiviral vector G1A7-E19-L1, the envelope plasmid 2, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid and the main plasmid carrying the nucleic acid encoding the TCP-ECEA were transfected into packaging cells HEK-293T cells using PEI reagent, and the lentiviral vector G2A7-ECEA whose viral envelope contained the membrane-expressing anti-CD7 antibody and the mutant VSV-G2 and contained the nucleic acid encoding the TCP-ECEA was packaged and collected.

[0590] 3. Transfecting human non-activated PBMCs to prepare CEA-targeted TCP-T-ECEA cells

[0591] On Day 0, at an MOI of 5, the method for transfecting non-activated human PBMCs with the lentiviral vector G1A7-E19-L1 was used to transfect 1×10 6 Individual non-activated PBMCs were used to prepare TCP-T-ECEA cells expressing the TCP-ECEA;

[0592] On Day 2, the transfected PBMCs were mixed with 1×10 5 CEA + T84 cells (human colon adenocarcinoma lung metastasis cells, brand: Punosai, catalog number: #CL-0229) were mixed; the control group was 3×10 5 Non-activated human PBMCs that were not transfected with the virus were mixed with 1×10 5 A mixture of 10 T84 cells (effector-target ratio E:T=3:1);

[0593] On Day 7, flow cytometry was used to detect and record the expression of CEA in each group of mixed cells to detect the killing effect of TCP-T-ECEA cells prepared by transfecting human non-activated PBMCs with the lentiviral vector G2A7-ECEA on Nalm6 cells. The results are shown in Figure 17.

[0594] As shown in Figure 17, the TCP-T-ECEA cells prepared by transfecting human non-activated PBMCs in vitro with the lentiviral vector G2A7-ECEA can effectively kill CEA. + The killing effect of PBMCs on T84 cells was significantly better than that of non-activated human PBMCs on T84 cells.

Claims

1. A lentiviral vector or a retroviral vector, characterized in that: (a) The lentiviral vector or retroviral vector carries a shuttle gene encoding a T cell receptor chimeric protein ("TCP"); the T cell receptor chimeric protein comprises: (1) a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; (2) an antigen binding region, wherein the antigen binding region is operably linked to the N-terminus of the TCR subunit or the functional fragment thereof, and the antigen binding region comprises one or more antibodies or antigen binding fragments thereof; (b) the viral envelope of the lentiviral vector or retroviral vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof that specifically bind to T cell endocytic receptors, and the antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof are not part of the viral envelope glycoprotein; and (c) The envelope glycoprotein of the lentiviral vector or retroviral vector undergoes a first mutation, so that the ability of the envelope glycoprotein to specifically bind to a receptor is weakened or lost relative to before the first mutation occurs.

2. The lentiviral vector or retroviral vector according to claim 1, characterized in that The viral envelope of the lentiviral vector or retroviral vector does not contain the T cell receptor chimeric protein or the T cell receptor chimeric protein is lowly distributed on the viral envelope of the lentiviral vector or retroviral vector.

3. The lentiviral vector or retroviral vector according to any one of claims 1 or 2, characterized in that The TCR subunit is CD3γ or CD3ε.

4. The lentiviral vector or retroviral vector according to any one of claims 1 to 3, characterized in that The antigen binding region specifically binds to at least one of the following antigens: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D. .

5. The lentiviral vector or retroviral vector according to any one of claims 1 to 4, characterized in that The antigen binding region is operably connected to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide; Preferably, the connecting peptide comprises a flexible connecting peptide; More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence are as shown in the connecting peptide 1 of SEQ ID NO: 18; wherein n=1 to 4.

6. The lentiviral vector or retroviral vector according to any one of claims 1 to 5, characterized in that The shuttle gene also includes a leader sequence; Preferably, the leader sequence is selected from the group consisting of leader sequences encoding the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide; More preferably, the leader sequence encodes a CD8α signal peptide.

7. The lentiviral vector or retroviral vector according to any one of claims 1 to 6, characterized in that The T cell endocytic receptor is CD7 or CD3.

8. The lentiviral vector or retroviral vector according to any one of claims 1 to 7, characterized in that The envelope glycoprotein of the lentiviral vector or retroviral vector is selected from the following envelope glycoproteins and variants thereof: envelope glycoproteins of vesicular stomatitis virus strains and variants thereof, envelope glycoproteins of baboon endogenous retrovirus BaEV and variants thereof, envelope glycoproteins of feline endogenous retrovirus RD114 and variants thereof, and envelope glycoproteins of gibbon ape leukemia virus GALV and variants thereof; The envelope glycoprotein and variants thereof of the vesicular stomatitis virus strain are selected from 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 glycoproteins of Jersey strain and variants thereof, the envelope glycoproteins of Carajas strain and variants thereof, the envelope glycoproteins of Chandipura strain and variants thereof, the envelope glycoproteins of Eptesicus strain and variants thereof, the envelope glycoproteins of Isfahan strain and variants thereof, the envelope glycoproteins of Jurona strain and variants thereof, the envelope glycoproteins of Malpais strain and variants thereof, the envelope glycoproteins of Perinet strain and variants thereof, the envelope glycoproteins of Piry strain and variants thereof, the envelope glycoproteins of Radi strain and variants thereof, the envelope glycoproteins of Rhinolopus strain and variants thereof, and the envelope glycoproteins of Yug Bogdanovac strain and variants thereof.

9. The lentiviral vector or retroviral vector according to claim 8, characterized in that The envelope glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus or a variant thereof; The extracellular domain of the envelope glycoprotein of the Indiana strain comprises an amino acid sequence as set forth in SEQ ID NO:1 or at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO:1; Preferably, the first mutation reduces or loses the ability of the envelope glycoprotein to specifically bind to LDL-R; 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 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, Amino acids 321-374 are missing, amino acids 331-364 are missing, amino acids 344-354 are missing, and amino acids 345-353 are missing; (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 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; More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: substitution of K47, deletion of K47, substitution of R354; More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: the 47th amino acid is replaced by lysine K to glutamine Q, the 354th amino acid is replaced by arginine R to glutamine Q, and the 47th amino acid lysine is deleted; Most preferably, the first mutation comprises a mutation in which the amino acid sequence comprises the following amino acid: a deletion of K47.

10. The lentiviral vector or retroviral vector according to claim 8, characterized in that The envelope glycoprotein is the envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus or a variant thereof; The extracellular domain of the envelope glycoprotein of the Cocal strain 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; Preferably, the first mutation reduces or loses the ability of the envelope glycoprotein to specifically bind to LDL-R; the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, 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: 2; (b) after optimal global alignment with SEQ ID NO:2, substitution of Q8, substitution of S9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of D183, substitution of A179, substitution of T180, substitution of V182, substitution of T184, substitution of Y209, substitution of I347, substitution of S350, 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:2; More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: substitution of K47, deletion of K47, substitution of R354; More preferably, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: the 47th amino acid is replaced by lysine K to glutamine Q, the 354th amino acid is replaced by arginine R to glutamine Q, and the 47th amino acid lysine is deleted; Most preferably, the first mutation comprises a mutation in which the amino acid sequence comprises the following amino acid: a deletion of K47.

11. The lentiviral vector or retroviral vector according to any one of claims 1 to 10, characterized in that The envelope glycoprotein of the lentiviral vector or retroviral vector undergoes a second mutation, so that the ability of the envelope glycoprotein to antagonize inactivation by complement is enhanced compared with that before the second mutation, or the envelope glycoprotein will not be inactivated by complement.

12. The lentiviral vector or retroviral vector according to claim 11, characterized in that The envelope glycoprotein is an envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus or a variant thereof; the extracellular domain of the envelope glycoprotein of the Indiana strain comprises an amino acid sequence as shown in SEQ ID NO:1 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:1; Preferably, the second mutation comprises 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; (b) after optimal global alignment with SEQ ID NO: 1, is located at the 214th amino acid corresponding to SEQ ID NO: 1; (c) amino acid position 352 of SEQ ID NO: 1; (d) after optimal global alignment with SEQ ID NO: 1, located at the amino acid position equivalent to 352 of SEQ ID NO: 1; (e) amino acid position 50 of SEQ ID NO: 1; (f) after optimal global alignment with SEQ ID NO: 1, is located at the 50th amino acid corresponding to SEQ ID NO: 1; (g) amino acid position 146 of SEQ ID NO: 1; (h) After optimal global alignment with SEQ ID NO: 1, the position corresponding to the 146th position of SEQ ID NO: 1 amino acid; More preferably, the amino acid mutation includes deletion, insertion or substitution of amino acids; Still more preferably, the amino acid sequence comprises at least one of the following amino acid substitutions: (a) amino acid position 214 of SEQ ID NO: 1; (b) after optimal global alignment with SEQ ID NO: 1, is located at the 214th amino acid corresponding to SEQ ID NO: 1; (c) amino acid position 352 of SEQ ID NO: 1; (d) after optimal global alignment with SEQ ID NO: 1, located at the amino acid position equivalent to 352 of SEQ ID NO: 1; (e) amino acid position 50 of SEQ ID NO: 1; (f) after optimal global alignment with SEQ ID NO: 1, is located at the 50th amino acid corresponding to SEQ ID NO: 1; (g) amino acid position 146 of SEQ ID NO: 1; (h) After optimal global alignment with SEQ ID NO: 1, it is located at the 146th amino acid corresponding to SEQ ID NO:

1.

13. The lentiviral vector or retroviral vector according to claim 12, characterized in that: The second mutation includes that the amino acid sequence contains one or more of the following site mutations: substitution of T214, substitution of T352, substitution of K50, substitution of S146; Preferably, the amino acid sequence comprises one or more of the following site mutations: amino acid 214 is replaced by threonine T to asparagine N, amino acid 352 is replaced by threonine T to alanine A, amino acid 50 is replaced by lysine K to threonine T, and amino acid 146 is replaced by serine S to threonine T; Preferably, the amino acid sequence comprises any combination of the following site mutations: (a) Replacement of T214 and T352; (b) Replacement of T214, T352, K50 and S146; More preferably, the amino acid sequence comprises a combination of any of the following site mutations: (a) amino acid 214 is replaced by threonine T to asparagine N and amino acid 352 is replaced by threonine T to alanine A; (b) The amino acid at position 214 is replaced by threonine T to asparagine N, the amino acid at position 352 is replaced by threonine T to alanine A, the amino acid at position 50 is replaced by lysine K to threonine T, and the amino acid at position 146 is replaced by serine S to threonine T.

14. The lentiviral vector or retroviral vector according to claim 11, characterized in that The envelope glycoprotein is an envelope glycoprotein of the Cocal strain of the vesicular stomatitis virus genus or a variant thereof; the extracellular domain of the envelope glycoprotein of the Cocal strain comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identity with the amino acid sequence as shown in SEQ ID NO:2; Preferably, the second mutation comprises 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: 2; (b) after optimal global alignment with SEQ ID NO: 2, is located at the 214th amino acid corresponding to SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 2, is located at the amino acid position corresponding to SEQ ID NO: 2 at position 352; (e) amino acid position 50 of SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 2, is located at the 50th amino acid corresponding to SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 2; (h) after optimal global alignment with SEQ ID NO: 2, is located at the amino acid position corresponding to 146 of SEQ ID NO: 2; More preferably, the amino acid mutation includes deletion, insertion or substitution of amino acids; Still more preferably, the amino acid sequence comprises at least one of the following amino acid substitutions: (a) amino acid position 214 of SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 2, is located at the 214th amino acid corresponding to SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 2, is located at the amino acid position corresponding to SEQ ID NO: 2 at position 352; (e) amino acid position 50 of SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 2, is located at the 50th amino acid corresponding to SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 2; (h) After optimal global alignment with SEQ ID NO: 2, it is located at the 146th amino acid corresponding to SEQ ID NO:

2.

15. The lentiviral vector or retroviral vector according to claim 14, characterized in that The second mutation includes that the amino acid sequence contains one or more of the following site mutations: substitution of K214, substitution of T352, substitution of K50, substitution of S146; Preferably, the amino acid sequence comprises one or more of the following mutations: amino acid 214 Lysine K was replaced by asparagine N, the 352nd amino acid was replaced by threonine T by alanine A, the 50th amino acid was replaced by lysine K by threonine T, and the 146th amino acid was replaced by serine S by threonine T; Preferably, the amino acid sequence comprises any combination of the following site mutations: (a) Replacement of K214 and T352; (b) Replacement of K214, T352, K50 and S146; More preferably, the amino acid sequence comprises a combination of any of the following site mutations: (a) amino acid 214 is replaced by lysine K to asparagine N and amino acid 352 is replaced by threonine T to alanine A; (b) The amino acid at position 214 is replaced by lysine K to asparagine N, the amino acid at position 352 is replaced by threonine T to alanine A, the amino acid at position 50 is replaced by lysine K to threonine T, and the amino acid at position 146 is replaced by serine S to threonine T.

16. The lentiviral vector or retroviral vector according to any one of claims 1 to 15, characterized in that The antibody or antigen-binding fragment thereof and / or ligand or receptor-binding fragment thereof that specifically binds to a T-cell endocytic receptor is directly or indirectly connected to a transmembrane region and is included on the surface of the viral envelope; Preferably, the transmembrane region is selected from the transmembrane regions of the following proteins: CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD 86. CD95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HV EM), CD272(BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, KIR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5, and Zap70; More preferably, the transmembrane region includes the transmembrane region of CD8α.

17. The lentiviral vector or retroviral vector according to claim 16, characterized in that The antibody or antigen-binding fragment thereof and / or ligand or receptor-binding fragment thereof that specifically binds to a T cell endocytic receptor is indirectly connected to the transmembrane region via a connecting domain; Preferably, the linking domain is selected from: (i) 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; (ii) a hinge region selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154; (iii) 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; and (iv) a stem region of a type II C-lectin, wherein the type II C-lectin is selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; More preferably, the connecting domain includes the hinge region of CD8α.

18. A method for transfecting T cells, characterized in that: The method comprises contacting a T cell with a lentiviral vector or a retroviral vector as described in any one of claims 1 to 17; Preferably, the contacting occurs in vivo or in vitro in a subject; When the contacting occurs outside the body of a subject, the subject is an individual to whom T cells transfected by the method for transfecting T cells are administered; When the contact occurs in a subject, the lentiviral vector or reverse transcriptase is administered to the subject. Viral vectors; More preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

19. The method according to claim 18, characterized in that The contact occurs outside the subject, the T cells are inactivated T cells, and the lentiviral vector or retroviral vector is in contact with the inactivated T cells for no more than 2 days, no more than 47 hours, no more than 46 hours, no more than 45 hours, no more than 44 hours, no more than 43 hours, no more than 42 hours, no more than 41 hours, no more than 40 hours, no more than 39 hours, no more than 38 hours, no more than 37 hours, no more than 36 hours, no more than 35 hours, no more than 34 hours, no more than 33 hours, no more than 32 hours, no more than 31 hours, no more than 30 hours, no more than 29 hours, no more than 28 hours, no more than 27 hours, no more than 26 hours hours, no more than 25 hours, no more than 24 hours, no more than 23 hours, no more than 22 hours, no more than 21 hours, no more than 20 hours, no more than 19 hours, no more than 18 hours, no more than 17 hours, no more than 16 hours, no more than 15 hours, no more than 14 hours, no more than 13 hours, no more than 12 hours, no more than 11 hours, no more than 10 hours, no more than 9 hours, no more than 8 hours, no more than 7 hours, no more than 6 hours, no more than 5 hours, no more than 4 hours, no more than 3 hours, no more than 2 hours, no more than 1 hour, no more than 59 minutes, no more than 58 minutes, no more than 57 minutes, no more than 56 minutes, no more than 55 minutes, no more than 54 minutes, no more than 53 minutes, no more than 52 minutes, no more than 51 minutes, no more than 50 minutes, no more than 49 minutes, no more than 48 minutes, no more than 47 minutes, no more than 46 minutes, no more than 45 minutes, no more than 44 minutes, no more than 43 minutes, no more than 42 minutes, no more than 41 minutes, no more than 40 minutes, no more than 39 minutes, no more than 38 minutes, no more than 37 minutes, no more than 36 minutes, no more than 35 minutes, no more than 34 minutes, no more than 33 minutes, no more than 32 minutes, no more than 31 minutes, no more than 30 minutes, no more than 29 minutes, no more than 28 minutes, no more than 27 minutes, no more 26 minutes, no more than 25 minutes, no more than 24 minutes, no more than 23 minutes, no more than 22 minutes, no more than 21 minutes, no more than 20 minutes, no more than 19 minutes, no more than 18 minutes, no more than 17 minutes, no more than 16 minutes, no more than 15 minutes, no more than 14 minutes, no more than 13 minutes, no more than 12 minutes, no more than 11 minutes, no more than 10 minutes, no more than 9 minutes, no more than 8 minutes, no more than 7 minutes, no more than 6 minutes, no more than 5 minutes, no more than 4 minutes, no more than 3 minutes, no more than 2 minutes, no more than 1 minute, no more than 50 seconds, no more than 40 seconds, no more than 30 seconds, no more than 20 seconds or no more than 10 seconds.

20. A method for reducing the expression of antigen binding regions encoded by a partial shuttle gene on the cell membrane of packaging cells during packaging of a lentiviral vector or a retroviral vector, wherein the antigen binding region comprises one or more antibodies or antigen binding fragments thereof, characterized in that: The method comprises operably linking the antigen binding region to the N-terminus of a TCR subunit or a functional fragment thereof, wherein the TCR subunit or the functional fragment thereof is selected from TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; the reduction of the membrane expression of the antigen binding region is relative to when the antigen binding region is operably linked to a fusion protein, and the fusion protein is not any of the TCR subunits or the functional fragment thereof; Preferably, the TCR subunit is CD3γ or CD3ε.

21. The method according to claim 20, characterized in that The antigen binding region is operably connected to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide; Preferably, the connecting peptide comprises a flexible connecting peptide; More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence are as shown in the connecting peptide 1 of SEQ ID NO: 18; wherein n=1 to 4.

22. The method according to claim 20 or 21, characterized in that The antigen binding region specifically binds to at least one of the following antigens: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D. .

23. The method according to any one of claims 20 to 22, characterized in that The shuttle gene also comprises a leader sequence encoding a signal peptide; Preferably, the signal peptide is selected from the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide; More preferably, the signal peptide is CD8α signal peptide.

24. The method according to any one of claims 20 to 23, characterized in that The fusion protein comprises a transmembrane region and an intracellular signaling domain; Preferably, the fusion protein further comprises a co-stimulatory signal transduction domain composed of one or more co-stimulatory molecules; More preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein to form a chimeric antigen receptor ("CAR"); More preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein via a hinge region to form a chimeric antigen receptor.

25. A method for reducing the content of antigen binding regions encoded by a portion of a shuttle gene in the viral envelope of a lentiviral vector or a retroviral vector, wherein the antigen binding regions comprise one or more antibodies or antigen binding fragments thereof, characterized in that: The method comprises operably linking the antigen binding region to the N-terminus of a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; the reduction in the content of the antigen binding region is relative to when the antigen binding region is operably linked to a fusion protein, and the fusion protein is not any of the TCR subunits or functional fragments thereof; Preferably, the TCR subunit is CD3γ or CD3ε.

26. The method according to claim 25, characterized in that The antigen binding region is operably connected to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide; Preferably, the connecting peptide comprises a flexible connecting peptide; More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence are as shown in the connecting peptide 1 of SEQ ID NO: 18; wherein n=1 to 4.

27. The method according to claim 25 or 26, characterized in that The antigen binding region specifically binds to at least one of the following antigens: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD 44. CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248 , VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4 , MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 mutants, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2 ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, and NKG2D.

28. The method according to any one of claims 25 to 27, characterized in that The shuttle gene also comprises a leader sequence encoding a signal peptide; Preferably, the signal peptide is selected from the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide; More preferably, the signal peptide is CD8α signal peptide.

29. The method according to any one of claims 25 to 28, characterized in that The fusion protein comprises a transmembrane region and an intracellular signaling domain; Preferably, the fusion protein further comprises a co-stimulatory signal transduction domain composed of one or more co-stimulatory molecules; More preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein to form a chimeric antigen receptor; More preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein via a hinge region to form a chimeric antigen receptor.

30. A method for improving the targeting of a lentiviral vector or retroviral vector carrying a shuttle gene to transfect T cells and / or reducing the ability of the lentiviral vector or retroviral vector to transfect target cells other than T cells, characterized in that: The method comprises reducing the content of the antigen binding region encoded by a portion of the shuttle gene included in the viral envelope of the lentiviral vector or retroviral vector; Preferably, the method comprises reducing the expression of a portion of the antigen binding region encoded by the shuttle gene on the cell membrane of the packaging cell that packages the lentiviral vector or retroviral vector.

31. The method according to claim 30, characterized in that The method comprises operably linking the antigen binding region to the N-terminus of a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; Preferably, the TCR subunit is CD3γ or CD3ε.

32. The method according to claim 30 or 31, characterized in that The antigen binding region is operably connected to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide; Preferably, the connecting peptide comprises a flexible connecting peptide; More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence are as shown in the connecting peptide 1 of SEQ ID NO: 18; wherein n=1 to 4.

33. The method according to any one of claims 30 to 32, characterized in that The antigen binding region specifically binds to at least one of the following antigens: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6 CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D.

34. The method according to any one of claims 30 to 33, characterized in that The shuttle gene also comprises a leader sequence encoding a signal peptide; Preferably, the signal peptide is selected from the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide; More preferably, the signal peptide is CD8α signal peptide.

35. The method according to any one of claims 30 to 34, characterized in that The antigen binding region comprises one or more antibodies or antigen binding fragments thereof.

36. The method according to any one of claims 30 to 35, characterized in that The reduction in the membrane expression of the antigen binding region is relative to when the antigen binding region is operably linked to a fusion protein, and the fusion protein is not any of the TCR subunits or functional fragments thereof; Preferably, the fusion protein comprises a transmembrane region and an intracellular signaling domain; More preferably, the fusion protein further comprises a co-stimulatory signaling domain composed of one or more co-stimulatory molecules; Still further preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein to form a chimeric antigen receptor; Still further preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein via a hinge region to form a chimeric antigen receptor. 37.TCR subunits or their functional fragments (a) reducing the use of antigen binding regions encoded by a partial shuttle gene expressed on the cell membrane of packaging cells during the packaging of lentiviral vectors or retroviral vectors; (b) reducing the content of antigen binding region encoded by a part of shuttle gene in the viral envelope of lentiviral vector or retroviral vector; (c) use of a lentiviral vector or a retroviral vector carrying a shuttle gene to improve the targeting of T cells, wherein the shuttle gene comprises a nucleic acid encoding an antigen binding region; or (d) use of a lentiviral vector or a retroviral vector carrying a shuttle gene to reduce the ability of the vector to transfect non-T cell target cells, wherein the shuttle gene comprises a nucleic acid encoding an antigen binding region; The antigen binding region comprises one or more antibodies or antigen binding fragments thereof, characterized in that: The use comprises operably linking the antigen binding region to the N-terminus of a TCR subunit or a functional fragment thereof. The TCR subunit is selected from TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; the reduction of the membrane expression of the antigen binding region, the reduction of the content of the antigen binding region, the improvement of the targeting or the reduction of the transfection ability are relative to when the antigen binding region is operably linked to a fusion protein, and the fusion protein is not any of the TCR subunits or functional fragments thereof; Preferably, the TCR subunit is CD3γ or CD3ε.

38. The use according to claim 37, characterized in that The antigen binding region is operably connected to the N-terminus of the TCR subunit or its functional fragment via a connecting peptide; Preferably, the connecting peptide comprises a flexible connecting peptide; More preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence are as shown in the connecting peptide 1 of SEQ ID NO: 18; wherein n=1 to 4.

39. The use according to claim 37 or 38, characterized in that The antigen binding region specifically binds to at least one of the following antigens: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D.

40. The use according to any one of claims 37 to 39, characterized in that The shuttle gene also comprises a leader sequence encoding a signal peptide; Preferably, the signal peptide is selected from the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide; More preferably, the signal peptide is CD8α signal peptide.

41. The use according to any one of claims 37 to 40, characterized in that The fusion protein comprises a transmembrane region and an intracellular signaling domain; Preferably, the fusion protein further comprises a co-stimulatory signal transduction domain composed of one or more co-stimulatory molecules; More preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein to form a chimeric antigen receptor; More preferably, the antigen binding region is operably linked to the N-terminus of the fusion protein via a hinge region to form a chimeric antigen receptor.

42. An engineered T cell, characterized in that The engineered T cells are prepared by the method for transfecting T cells according to claim 18 or 19.

43. A composition, characterized in that The composition comprises the lentiviral vector or retroviral vector according to any one of claims 1 to 17 and at least one of the following components: pharmaceutically acceptable additives, carriers, diluents, excipients, preservation solutions and other auxiliary components.

44. A method for maintaining the abundance of target cell surface antigen expression in a subject's body or blood before engineered T cells kill target cells in the subject's body or blood, characterized in that: The method comprises administering the lentiviral vector or retroviral vector of any one of claims 1 to 17 or the composition of claim 43 to the subject or the subject's blood; Preferably, the administration is at least one of intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection and infusion technique.

45. Use of the lentiviral vector or retroviral vector according to any one of claims 1 to 17 or the composition according to claim 43 in the preparation of a drug for treating and / or preventing a disease; Preferably, the disease is selected from at least one of blood cancer and solid cancer; More preferably, the disease is at least one selected from blood cancers; More preferably, the blood cancer is selected from acute lymphoblastic leukemia (Acute Lymphoblastic Leukemia, "ALL"), diffuse large B-cell lymphoma (Diffuse Large B-cell Lymphoma, "DLBCL"), primary mediastinal large B-cell lymphoma (Primary Mediastinal At least one of B-cell Lymphoma, "PMBCL"), Multiple Myeloma (MM"), Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL); More preferably, the disease is selected from at least one of solid cancers; More preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

46. ​​A method for treating cancer in a subject or killing cancer cells in a subject, characterized in that: Administering the lentiviral vector or retroviral vector of any one of claims 1 to 17 or the composition of claim 43 to the subject; Preferably, the disease is selected from at least one of blood cancer and solid cancer; More preferably, the disease is at least one selected from blood cancers; More preferably, the blood cancer is selected from at least one of acute lymphoblastic leukemia, diffuse large B-cell lymphoma, primary mediastinal large B-cell lymphoma, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma; More preferably, the disease is selected from at least one of solid cancers; More preferably, the solid cancer is selected from at least one of colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer and ovarian cancer.

47. The method according to claim 46, characterized in that A therapeutically effective amount of the lentiviral vector or retroviral vector of any one of claims 1-17 or the composition of claim 43 is administered to the subject.

48. A shuttle gene, characterized in that The shuttle gene encodes a T cell receptor chimeric protein, which comprises: (1) a TCR subunit or a functional fragment thereof, wherein the TCR subunit is selected from TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ and CD3ε; (2) an antigen binding region, wherein the antigen binding region is operably linked to the N-terminus of the TCR subunit or the functional fragment thereof, and the antigen binding region comprises one or more antibodies or antigen binding fragments thereof; Preferably, the TCR subunit is CD3γ or CD3ε.

49. The shuttle gene according to claim 48, characterized in that The connecting peptide comprises a flexible connecting peptide; Preferably, the flexible connecting peptide is selected from (G4S) n The connecting peptide and the amino acid sequence are as shown in the connecting peptide 1 of SEQ ID NO: 18; wherein n=1 to 4.

50. The shuttle gene according to claim 48 or 49, characterized in that The antigen binding region specifically binds to at least one of the following antigens: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD37, CD38, CD40, CD40L, CD 44. CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248 , VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4 , MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, 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 hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, and NKG2D.

51. The shuttle gene according to any one of claims 48 to 50, characterized in that The shuttle gene also includes a leader sequence; Preferably, the leader sequence is selected from the group consisting of leader sequences encoding the following signal peptides: CD8α signal peptide, TCRα signal peptide, TCRβ signal peptide, TCRγ signal peptide, TCRδ signal peptide, CD3γ signal peptide, CD3δ signal peptide and CD3ε signal peptide; More preferably, the signal peptide is CD8α signal peptide.

52. A T cell receptor chimeric protein, characterized in that The T cell receptor chimeric protein is encoded by the shuttle gene of any one of claims 48-51.

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