Viral vector packaging system and use thereof

Abstract

Provided are a packaging system for packaging a lentiviral vector or a retroviral vector, and a use of the packaging system. Specifically, the provided packaging system comprises a polynucleotide encoding a CAR binding molecule, wherein the CAR binding molecule can bind to a CAR. Pseudotransduction is reduced when a host cell is transduced by the lentiviral vector or the retroviral vector packaged by the provided packaging system, and the ability to transduce a target cell expressing an antigen decreases, thereby significantly improving the targeting performance of the lentiviral vector or the retroviral vector in the transduction of immune cells such as T cells.

Description

A viral vector packaging system and its application Technical Field

[0001] The present invention relates to the field of viral vectors, and in particular to a viral vector packaging system and applications thereof. Background Art

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

[0003] LVV and RVV are widely used in in vitro and in vivo transduction of T cells to prepare CAR-T cells because they can integrate target genes such as chimeric antigen receptor genes (CAR) into the host cell genome, allowing the target genes to be stably expressed in the host cells.

[0004] However, during the packaging of LVV or RVV, the CAR molecule can be expressed on the cell membrane of the packaging cell under the action of a leading signal peptide (such as a CD8α signal peptide), and buds out of the packaging cell as the LVV or RVV buds out of the packaging cell, becoming part of the viral envelope of the LVV or RVV.

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

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

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

[0008] In addition, LVV or RVV can also bind to antigens expressed on the surface of disease cells such as cancer cells through the antigen binding region of the CAR molecules contained in its viral envelope, such as single-chain antibody scFv, and then transduce the cancer cells, reducing the amount of antigens on the surface of the transduced cancer cells available for binding to the antigen binding regions of engineered immune cells such as CAR-T cells; more importantly, the transduced cancer cells will also express CAR molecules, and bind to their own antigens through the antigen binding regions of the CAR molecules and then transduce each other, causing the amount of antigens on the surface of cancer cells available for binding to the antigen binding regions of the CAR molecules to be further reduced, making it impossible for them to be recognized by engineered immune cells such as CAR-T cells, thereby escaping the tumor killing effect (antigen escape), seriously affecting engineered immune cell therapies such as CAR-T cells, especially the efficacy of in vivo CAR-T cell therapies.

[0009] Therefore, compared to LVV or RVV whose viral envelope contains CAR molecules and antigen-binding regions derived from the cell membrane of the packaging cells, a viral envelope that does not contain or contains a significantly reduced content of CAR molecules and antigen-binding regions that can bind to the antigen is the key to reducing false transduction when using LVV or RVV to transduce immune cells such as T cells, and weakening the ability of LVV or RVV to transduce disease cells such as cancer cells (off-target effect), thereby improving the efficacy of cell therapy. Summary of the Invention

[0010] In view of this, in order to solve at least one of the above technical problems, one aspect of the present invention discloses a packaging system for packaging LVV or RVV.

[0011] (a) the shuttle gene (Transgene) contained in the packaging system comprises a polynucleotide encoding a chimeric antigen receptor (CAR); and

[0012] (b) The packaging system comprises a polynucleotide (CAR binding molecule gene) encoding a CAR binding molecule (CAR-Binding Molecule), and the CAR binding molecule comprises a CAR binding region (CAR-Binding Domain), and the CAR binding region can bind to the CAR.

[0013] In some embodiments of the present invention, the CAR binding region can be combined with the CAR to reduce the availability of the antigen-binding CAR for expression on the cell membrane of the packaging cell when the packaging system packages LVV or RVV in the packaging cell.

[0014] In some embodiments of the present invention, the CAR comprises an antigen binding region, a transmembrane region, and an intracellular signaling domain.

[0015] In some embodiments of the present invention, the CAR binding region of the CAR binding molecule can bind to the CAR in the cytoplasm and / or on the cell membrane of the packaging cell containing the packaging system, and assemble into a complex molecule.

[0016] In some embodiments of the present invention, the antigen binding region of the CAR comprises an antibody or an antigen binding fragment thereof, and the antibody or the antigen binding fragment thereof comprises an antibody epitope.

[0017] In some embodiments of the present invention, the CAR binding region comprises an antigen, and the antigenic epitope of the antigen can bind to the antibody epitope.

[0018] In some embodiments of the present invention, the antigen of the CAR binding region is selected from at least one of a full-length antigen, an antigen extracellular domain, an antigen variable region, and an antigen epitope.

[0019] In some embodiments of the present invention, the antigen of the CAR binding region comprises (a) a full-length antigen or (b) a functional fragment of an antigen, wherein the functional fragment of the antigen at least comprises the antigen epitope;

[0020] Optionally, the functional fragment of the antigen comprises at least the extracellular domain of the antigen;

[0021] Optionally, the functional fragment of the antigen comprises the extracellular domain and the transmembrane region of the antigen.

[0022] In some embodiments of the present invention, the CAR binding region comprises a first anti-antibody, which can bind to at least one CDR region (Complementarity-Determining Region) of the antibody epitope.

[0023] In some embodiments of the present invention, the first anti-antibody can bind to at least the HCDR3 and / or LCDR3 region of the antibody epitope.

[0024] In some embodiments of the present invention, the CAR binding region comprises (a) the antigen and (b) the first anti-antibody.

[0025] In some embodiments of the present invention, the CAR binding region comprises (a) the antigen and (b) the first anti-antibody; the antigen is connected to the first anti-antibody via a polypeptide linker.

[0026] In some embodiments of the present invention, the CAR binding region comprises (a) the antigen and / or (b) the antibody epitope of the antibody or antigen-binding fragment thereof that can specifically bind to the antigen binding region of the CAR.

[0027] In some embodiments of the present invention, the CAR binding molecule does not comprise a leader signal peptide.

[0028] In some embodiments of the present invention, the CAR binding molecule further comprises a leader signal peptide, wherein the leader signal peptide is located at the N-terminal end of the CAR binding region;

[0029] Preferably, the leader signal peptide is selected from any one of the following signal peptides: a natural leader signal peptide of an antigen, a CD8α signal peptide, a CD28 signal peptide, an IgG signal peptide, and an HLA-A signal peptide;

[0030] More preferably, the leader signal peptide is the natural leader signal peptide of the antigen or the CD8α signal peptide.

[0031] In some embodiments of the present invention, the C-terminus of the leader signal peptide is operably linked to the N-terminus of the CAR binding region.

[0032] In some embodiments of the present invention, the leader signal peptide is a natural leader signal peptide of an antigen or a CD8α signal peptide.

[0033] In some embodiments of the present invention, the CD8α signal peptide is human CD8α signal peptide (human CD8α, UniProtKB number: P01732).

[0034] In some embodiments of the present invention, the CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, and the CAR binding region comprises (i) the antigen and / or (ii) the first anti-antibody.

[0035] In some embodiments of the present invention, the CAR binding molecule comprising the leader signal peptide further comprises an endoplasmic reticulum retention signal peptide, and the endoplasmic reticulum retention signal peptide is located at the C-terminal end of the CAR binding region.

[0036] In some embodiments of the present invention, the N-terminus of the endoplasmic reticulum retention signal peptide is operably linked to the C-terminus of the CAR binding region.

[0037] In some embodiments of the present invention, the endoplasmic reticulum retention signal peptide comprises the amino acid sequence shown in SEQ ID NO: 51, and the SEQ ID NO: 51 comprises the amino acid sequence KDEL (lysine-aspartic acid-glutamic acid-leucine).

[0038] In some embodiments of the present invention, the amino acid sequence of the endoplasmic reticulum retention signal peptide is shown as SEQ ID NO: 51 (KDEL).

[0039] In some embodiments of the present invention, the CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, and (c) the endoplasmic reticulum retention signal peptide.

[0040] In some embodiments of the present invention, the CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, (c) a polypeptide linker, and (d) the endoplasmic reticulum retention signal peptide;

[0041] Preferably, the polypeptide linker is selected from:

[0042] (a) an immunoglobulin hinge region selected from the group consisting of wild-type and modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions;

[0043] (b) a hinge region selected from the wild-type and 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;

[0044] (c) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain;

[0045] (d) 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; and

[0046] (e) flexible linker peptide;

[0047] More preferably, the flexible connecting peptide is selected from (G4S) n Connector peptide, Connector peptide 1 and Connector peptide 3; wherein n=1 to 4;

[0048] Connector peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23)

[0049] Connector peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 137).

[0050] In some embodiments of the present invention, the polypeptide linker is the flexible connecting peptide (G4S)4 connecting peptide, and the amino acid sequence of the (G4S)4 connecting peptide is shown in SEQ ID NO:133.

[0051] In some embodiments of the present invention, the CAR binding molecule further comprises (a) a leader signal peptide, (b) the CAR binding region and (c) an endoplasmic reticulum retention signal peptide; the CAR binding region comprises the antigen or the first anti-antibody; the leader signal peptide is located at the N-terminal direction of the CAR binding region, and the endoplasmic reticulum retention signal peptide is located at the C-terminal direction of the CAR binding region; preferably, the C-terminus of the leader signal peptide is operably connected to the N-terminus of the CAR binding region, and the N-terminus of the endoplasmic reticulum retention signal peptide is operably connected to the C-terminus of the CAR binding region.

[0052] In some embodiments of the present invention, the CAR binding molecule further comprises (a) the leader signal peptide, (b) the CAR binding region, and (c) the endoplasmic reticulum retention signal peptide;

[0053] The CAR binding region comprises a second anti-antibody, which can bind to a region of the CAR that does not comprise the antibody epitope (non-antibody epitope region);

[0054] Preferably, the endoplasmic reticulum retention signal peptide comprises the amino acid sequence (KDEL) as shown in SEQ ID NO: 51.

[0055] In some embodiments of the present invention, the leader signal peptide is located at the N-terminus of the second anti-antibody, and the endoplasmic reticulum retention signal peptide is located at the C-terminus of the second anti-antibody.

[0056] In some embodiments of the present invention, the C-terminus of the leader signal peptide is operably linked to the N-terminus of the second anti-antibody, and the N-terminus of the endoplasmic reticulum retention signal peptide is operably linked to the C-terminus of the second anti-antibody.

[0057] In some embodiments of the present invention, the leader signal peptide linked to the second anti-antibody is any of the aforementioned leader signal peptides.

[0058] In some embodiments of the present invention, the leader signal peptide linked to the second anti-antibody is the CD8α signal peptide.

[0059] In some embodiments of the present invention, the second anti-antibody can bind to the transmembrane region or the intracellular region of the CAR.

[0060] In some embodiments of the invention, the second anti-antibody can bind to the hinge region of the CAR.

[0061] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR is selected from the group consisting of: immunoglobulin (full-length antibody), half antibody, Fab, Fab', F(ab')2, Fv fragment, single-chain variable region fragment (scFv), disulfide bond-stabilized antibody (dsFv), antibody heavy chain variable region (VH) or light chain variable region (VL), Fd fragment consisting of VH and CH1 domains, linear antibody and single-domain antibody (nanoantibody VHH) One or more.

[0062] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof in the antigen-binding region of the CAR is scFv.

[0063] In some embodiments of the present invention, the VH region of the scFv is connected to the VL region of the scFv via a connecting peptide.

[0064] In some embodiments of the present invention, the connecting peptide contained in the scFv is a flexible connecting peptide.

[0065] In some embodiments of the present invention, the connecting peptide contained in the scFv is (G4S) n Connecting peptide, wherein n=1 to 4; preferably, n=3.

[0066] In some embodiments of the present invention, the second anti-antibody can bind to anti-(G4S) n Connector peptide.

[0067] In some embodiments of the present invention, the second anti-antibody is KPI-4, and the KPI-4 is anti-(G4S) n Linked peptide antibodies.

[0068] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof in the antigen-binding region of the CAR is VHH.

[0069] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR is the VH region or VL region.

[0070] In some embodiments of the present invention, (a) the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR binds to an antigen selected from the group consisting of: and / or (b) the antigen binding region of the CAR binds to an antigen selected from the group consisting of:

[0071] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5TAC, 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-3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, uPAR, GCC, EPCAM, Nectin4, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2(CLDN18.2), Prostase, PAP, ELF2M, Ephrin B2, IGF-Ⅰ receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut At least one of hsp70-2, CD79α, CD79β, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, and NKG2D;

[0072] Preferably, the antibody or antigen binding fragment thereof of the antigen binding region of the CAR binds to and / or (b) the antigen of the CAR binding region is selected from: at least one of CD19, CD20, CD33, MSLN, CD79β, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2, B7H3 and GUCY2C.

[0073] In some embodiments of the present invention, the antigen binding region of the CAR binds to at least one selected from the group consisting of: CD19, CD20, CD33, MSLN, CD79β, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2, B7H3 and GUCY2C.

[0074] In some embodiments of the present invention, the antigen binding region of the CAR binds to CD19.

[0075] In some embodiments of the present invention, the antigen binding region of the CAR binds to CD33.

[0076] In some embodiments of the present invention, the antigen binding region of the CAR binds to BCMA.

[0077] In some embodiments of the present invention, the antigen binding region of the CAR binds to Claudin18.2.

[0078] In some embodiments of the present invention, the antigen binding region of the CAR binds to B7H3.

[0079] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR comprises an anti-CD19 antibody or antigen-binding fragment thereof that can bind to CD19;

[0080] Preferably, the CD19 is human CD19 (UniProtKB No.: P15391).

[0081] In some embodiments of the present invention, the CAR binding region comprises the antigen CD19 and / or an anti-anti-CD19 antibody or an antigen-binding fragment thereof (first anti-antibody, anti-anti-CD19 antibody).

[0082] In some embodiments of the present invention, the antigen CD19 of the CAR binding region comprises its extracellular domain and transmembrane region.

[0083] In some embodiments of the present invention, the antigen CD19 of the CAR binding region comprises its extracellular domain, transmembrane region and intracellular domain.

[0084] In some embodiments of the present invention, the antigen CD19 in the CAR binding region is the full-length antigen CD19 including its signal peptide.

[0085] In some embodiments of the present invention, the amino acid sequence of the full-length antigen CD19 including its signal peptide is shown in SEQ ID NO: 52.

[0086] In some embodiments of the present invention, the amino acid sequence of the CD19 signal peptide is shown in SEQ ID NO: 68.

[0087] In some embodiments of the present invention, the amino acid sequence of the transmembrane region of the antigen CD19 of the CAR binding region is shown in SEQ ID NO: 134.

[0088] In some embodiments of the present invention, the anti-CD19 antibody or antigen-binding fragment thereof contained in the antigen-binding region of the CAR is a scFv (FMC63-scFv) derived from the monoclonal antibody FMC-63, and the amino acid sequences of the HCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 94-96, respectively, and the amino acid sequences of the LCDR1-3 regions of the anti-CD19 antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 97-99, respectively.

[0089] In some embodiments of the present invention, the amino acid sequences of the HCDR1-3 regions of the anti-anti-CD19 antibody are shown in SEQ ID NOs: 102-104, respectively, and the amino acid sequences of the LCDR1-3 regions of the anti-anti-CD19 antibody are shown in SEQ ID NOs: 105-107, respectively.

[0090] In some embodiments of the present invention, the transmembrane region of the CAR is derived from the transmembrane region of at least one of the following proteins:

[0091] CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1;

[0092] Preferably, the transmembrane region of the CAR is derived from the transmembrane region of CD8α or CD28;

[0093] More preferably, the transmembrane region of the CAR is derived from the transmembrane region of CD8α.

[0094] In some embodiments of the present invention, the CD8α transmembrane region is a human CD8α transmembrane region, and the amino acid sequence of the CD8α transmembrane region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:15.

[0095] In some embodiments of the present invention, the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of at least one of the following proteins:

[0096] CD3ε, CD3γ, CD3δ, CD3ζ, CD79α, CD79β, FcεRlγ, FcεRβ, FcγRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12, and other intracellular signaling domains of proteins containing at least one ITAM;

[0097] Preferably, the intracellular signaling domain is derived from the intracellular signaling domain of CD3ζ.

[0098] In some embodiments of the present invention, the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of CD3ζ; preferably the intracellular signaling domain of human CD3ζ.

[0099] In some embodiments of the present invention, the amino acid sequence of the intracellular signaling domain of human CD3ζ is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:50.

[0100] In some embodiments of the present invention, the CAR further comprises a costimulatory signaling domain;

[0101] Preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of at least one of the following proteins:

[0102] CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRlγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-l, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88;

[0103] More preferably, the costimulatory signaling domain of the CAR is derived from the costimulatory signaling domain of 4-1BB and / or CD28.

[0104] In some embodiments of the present invention, the costimulatory signaling domain of the CAR is the costimulatory signaling domain of 4-1BB; preferably the costimulatory signaling domain of human 4-1BB.

[0105] Human 4-1BB (CD137), UniProtKB code: Q07011.

[0106] In some embodiments of the present invention, the amino acid sequence of the costimulatory signaling domain of human 4-1BB is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 49.

[0107] In some embodiments of the present invention, the CAR further comprises a hinge region, which sequentially connects the antigen binding region and the transmembrane region of the CAR.

[0108] In some embodiments of the present invention, the hinge region of the CAR is derived from the hinge region of at least one of the following proteins:

[0109] CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154;

[0110] Preferably, the hinge region is derived from the hinge region of at least one of the following proteins:

[0111] CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154;

[0112] More preferably, the hinge region is derived from the hinge region of CD8α or CD28.

[0113] In some embodiments of the present invention, the CD8α hinge region is a human CD8α hinge region, and the amino acid sequence of the human CD8α hinge region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:14.

[0114] In some embodiments of the present invention, the hinge region of the CAR is derived from the hinge region of CD8α.

[0115] In some embodiments of the present invention, the region of the CAR that does not contain an antigen binding region (non-antigen binding region) contains a TCR / CD3 complex subunit-related polypeptide (TCR / CD3 Complex Subunit Related Peptide, "TSP") derived from a TCR / CD3 complex subunit, and the TSP contains at least one of a TCR / CD3 complex subunit, a TCR / CD3 complex subunit functional fragment, a TCR / CD3 complex subunit variant, and a variant of a TCR / CD3 complex subunit functional fragment; the antigen binding region is located in the N-terminal direction of the TSP.

[0116] The TSP provided by the present invention can be expressed in T cells by (1) being incorporated into the endogenous TCR / CD3 complex or endogenous TCR / CD3 complex subunit or its functional fragment; and / or (2) functionally interacting with the endogenous TCR / CD3 complex or endogenous TCR / CD3 complex subunit or its functional fragment, for example, forming a TCR / CD3 complex or its functional fragment with an endogenous CD3 subunit or its functional fragment, and then being expressed outside the membrane and anchored on the cell membrane of the T cell.

[0117] In some embodiments of the present invention, the TCR / CD3 complex subunit is selected from at least one of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ζ and CD3ε.

[0118] In some embodiments of the present invention, the functional fragment of the TCR / CD3 complex subunit comprises at least one of the full-length protein, extracellular region, transmembrane region, intracellular region, variable region and constant region of the TCR / CD3 complex subunit.

[0119] In some embodiments of the present invention, the TSP comprises CD3γ or a functional fragment thereof.

[0120] In some embodiments of the present invention, the TSP comprises (a) the transmembrane region and the intracellular region of CD3γ; or (b) the constant region of CD3γ.

[0121] In some embodiments of the present invention, the TSP comprises (a) the transmembrane region and intracellular region of CD3γ and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3ζ and CD3δ; or (b) the constant region of CD3γ and the variable region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3ζ and CD3δ.

[0122] In some embodiments of the present invention, the TSP comprises (a) the transmembrane and intracellular regions of CD3γ and the extracellular region of CD3δ; or (b) the constant region of CD3γ and the variable region of CD3δ.

[0123] In some embodiments of the present invention, (a) the C-terminus of the extracellular region of CD3δ is located towards the N-terminus of the transmembrane region of CD3γ, and the C-terminus of the transmembrane region of CD3γ is located towards the N-terminus of the intracellular region of CD3γ; or (b) the C-terminus of the variable region of CD3δ is located towards the N-terminus of the constant region of CD3γ.

[0124] In some embodiments of the present invention, (a) the C-terminus of the extracellular region of CD3δ is linked to the N-terminus of the transmembrane region of CD3γ, and the C-terminus of the transmembrane region of CD3γ is linked to the N-terminus of the intracellular region of CD3γ; or (b) the C-terminus of the variable region of CD3δ is linked to the N-terminus of the constant region of CD3γ.

[0125] In some embodiments of the present invention, the TSP comprises CD3ε or a functional fragment thereof.

[0126] In some embodiments of the present invention, the TSP comprises (a) CD3γ or a functional fragment thereof or (b) CD3ε or a functional fragment thereof.

[0127] In some embodiments of the present invention, the TSP comprises (a) the transmembrane region and the intracellular region of CD3ε; or (b) the constant region of CD3ε.

[0128] In some embodiments of the present invention, the TSP comprises (a) the transmembrane region and intracellular region of CD3ε and the extracellular region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3ζ and CD3δ; or (b) the constant region of CD3ε and the variable region of at least one of the following proteins: TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3ζ and CD3δ.

[0129] In some embodiments of the present invention, the TSP comprises (a) the transmembrane and intracellular regions of CD3ε and the extracellular region of CD3γ; or (b) the constant region of CD3ε and the variable region of CD3γ.

[0130] In some embodiments of the present invention, (a) the C-terminus of the extracellular region of CD3γ is located towards the N-terminus of the transmembrane region of CD3ε, and the C-terminus of the transmembrane region of CD3ε is located towards the N-terminus of the intracellular region of CD3ε; or (b) the C-terminus of the variable region of CD3γ is located towards the N-terminus of the constant region of CD3ε.

[0131] In some embodiments of the present invention, (a) the C-terminus of the extracellular region of CD3γ is connected to the N-terminus of the transmembrane region of CD3ε, and the C-terminus of the transmembrane region of CD3ε is connected to the N-terminus of the intracellular region of CD3ε; or (b) the C-terminus of the variable region of CD3γ is connected to the N-terminus of the constant region of CD3ε.

[0132] In some embodiments of the present invention, the TSP comprises TCRα or a functional fragment thereof and TCRβ or a functional fragment thereof.

[0133] In some embodiments of the present invention, the TSP comprises the constant region of TCRα and the constant region of TCRβ.

[0134] In some embodiments of the present invention, the constant region of the TCRα is mutated, and the mutation includes a cysteine ​​substitution in the TCRα constant region; the mutation can enhance disulfide bond-based interchain interactions.

[0135] In some embodiments of the present invention, the TCRα constant region is derived from a human or mouse TCRα constant region, the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 39, and the mouse TCRα constant region comprises the amino acid sequence shown in SEQ ID NO: 40;

[0136] The mutation includes replacing the 47th amino acid Threonine T of the human TCRα constant region with Cysteine ​​C (human TCRα constant region variant 1) or replacing the 47th amino acid Threonine T of the mouse TCRα constant region with Cysteine ​​C (mouse TCRα constant region variant 1);

[0137] The human TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:41, and the mouse TCRα constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:42.

[0138] In some embodiments of the present invention, the TCRβ constant region is mutated, and the mutation includes a cysteine ​​substitution in the TCRβ constant region; the mutation can enhance disulfide bond-based interchain interactions.

[0139] In some embodiments of the present invention, the TCRβ constant region is derived from a human TCRβ constant region, and the human TCRβ constant region comprises the amino acid sequence shown in SEQ ID NO:43 (hTRBC1) or SEQ ID NO:44 (hTRBC2), and the mutation includes replacing the 56th amino acid serine S of the human TCRβ constant region with cysteine ​​C (human TCRβ constant region variant 1), and the human TCRβ constant region variant 1 comprises the amino acid sequence shown in SEQ ID NO:45 or SEQ ID NO:46.

[0140] In some embodiments of the present invention, the TCRα constant region undergoes a mutation, and the mutation includes replacing at least one uncharged amino acid in the TCRα constant region with a hydrophobic amino acid; the mutation increases the hydrophobicity of the TCRα transmembrane region, offsets the instability caused by the positive charge carried by the TCRα transmembrane region, and enables the TCRα and its dimer formed with TCRβ to be more stably expressed on the T cell membrane, thereby obtaining better function.

[0141] In some embodiments of the present invention, the TCRα constant region is derived from a human TCRα constant region, and the human TCRα constant region comprises an amino acid sequence as shown in SEQ ID NO: 39, and the mutation includes at least one of amino acids 115, 118, and 119 of the human TCRα constant region being replaced by a hydrophobic amino acid.

[0142] In some embodiments of the present invention, the mutation includes that the human TCRα constant region contains at least one of the following mutations: amino acid serine S at position 115 is replaced by leucine L, amino acid glycine G at position 118 is replaced by valine V, and amino acid phenylalanine F at position 119 is replaced by leucine L.

[0143] In some embodiments of the present invention, the mutation includes replacing the 115th amino acid serine S of the human TCRα constant region with leucine L, the 118th amino acid glycine G with valine V, and the 119th amino acid phenylalanine F with leucine L (human TCRα constant region variant 2), and the human TCRα constant region variant 2 comprises the amino acid sequence shown in SEQ ID NO:47.

[0144] In some embodiments of the present invention, the TCRα constant region is derived from a human TCRα constant region, and the human TCRα constant region comprises the amino acid sequence shown in SEQ ID NO:39; the human TCRα constant region undergoes a mutation, and the mutation includes replacing the 47th amino acid threonine T of the human TCRα constant region with cysteine ​​C, replacing the 115th amino acid serine S with leucine L, replacing the 118th amino acid glycine G with valine V, and replacing the 119th amino acid phenylalanine F with leucine L (human TCRα constant region variant 3), and the human TCRα constant region variant 3 comprises the amino acid sequence shown in SEQ ID NO:48.

[0145] In some embodiments of the present invention, the TCRα constant region and the TCRβ constant region are derived from a human TCRα constant region and a human TCRβ constant region; the human TCRα constant region comprises the amino acid sequence as shown in SEQ ID NO: 39, and the human TCRβ constant region comprises the amino acid sequence as shown in SEQ ID NO: 43 or SEQ ID NO: 44; the human TCRα constant region and the human TCRβ constant region are mutated, and the mutations include substitution of amino acid 47 of the human TCRα constant region with cysteine, amino acid serine S at position 115 with leucine L, amino acid glycine G at position 118 with valine V, and amino acid phenylalanine F at position 119 with leucine L (human TCRα constant region variant 3), and substitution of amino acid serine S at position 56 of the human TCRβ constant region with cysteine ​​C (human TCRβ constant region variant 1); the human TCRα constant region variant 3 comprises the amino acid sequence as shown in SEQ ID NO: 48, and the human TCRβ constant region variant 1 comprises the amino acid sequence as shown in SEQ ID NO: NO:45 or the amino acid sequence shown in SEQ ID NO:46.

[0146] In some embodiments of the present invention, the single-chain antibody scFv is located at the N-terminal end of the TCRα constant region or its variant or the TCRβ constant region or its variant.

[0147] In some embodiments of the present invention, the single-chain antibody scFv is linked to the N-terminus of the TCRα constant region or its variant or the TCRβ constant region or its variant.

[0148] In some embodiments of the present invention, the VH region or VL region of the antibody is located at the N-terminal end of the TCRα constant region or its variant; the VH region or VL region of the antibody is located at the N-terminal end of the TCRβ constant region or its variant.

[0149] In some embodiments of the present invention, the VH region or VL region of the antibody is linked to the N-terminus of the TCRα constant region or its variant; the VH region or VL region of the antibody is linked to the N-terminus of the TCRβ constant region or its variant.

[0150] In some embodiments of the present invention, (a) when the VH region is located at the N-terminal direction of the TCRα constant region or its variant, the VL region is located at the N-terminal direction of the TCRβ constant region or its variant; or (b) when the VL region is located at the N-terminal direction of the TCRα constant region or its variant, the VH region is located at the N-terminal direction of the TCRβ constant region or its variant.

[0151] In some embodiments of the present invention, (a) when the VH region is linked to the N-terminus of the TCRα constant region or its variant, the VL region is linked to the N-terminus of the TCRβ constant region or its variant; or (b) when the VL region is linked to the N-terminus of the TCRα constant region or its variant, the VH region is linked to the N-terminus of the TCRβ constant region or its variant.

[0152] In some embodiments of the present invention, the VH region and VL region are derived from the same antibody or antigen-binding fragment thereof.

[0153] In some embodiments of the present invention, the antigen binding region is linked to the N-terminus of any of the aforementioned TSPs via a linker peptide;

[0154] Preferably, the connecting peptide is selected from a flexible connecting peptide and a hinge region of a protein;

[0155] More preferably, the flexible connecting peptide is selected from (G4S) n A connecting peptide and a connecting peptide 1 comprising the amino acid sequence shown in SEQ ID NO: 23; wherein n=1 to 4.

[0156] In some embodiments of the present invention, the flexible connecting peptide is the connecting peptide 1.

[0157] In some embodiments of the present invention, any of the aforementioned CARs comprising the TSP further comprises a costimulatory signaling domain.

[0158] In some embodiments of the present invention, the costimulatory signaling domain is any of the aforementioned costimulatory signaling domains.

[0159] In some embodiments of the present invention, the costimulatory signaling domain is located at the C-terminus of the CAR comprising the TSP.

[0160] In some embodiments of the present invention, the costimulatory signaling domain is linked to the C-terminus of the TSP.

[0161] In some embodiments of the present invention, any of the aforementioned CARs comprising the TSP, when expressed in T cells, (a) is incorporated into the endogenous TCR / CD3 complex or TCR / CD3 complex subunit or a functional fragment thereof; or (b) functionally interacts with the endogenous TCR / CD3 complex or an endogenous TCR / CD3 complex subunit or a functional fragment thereof.

[0162] In some embodiments of the present invention, any of the aforementioned CARs comprising the TSP is not expressed on the membrane in cells other than T cells, or the efficiency of membrane expression of the CAR in cells other than T cells is lower than the efficiency of membrane expression of the CAR in T cells.

[0163] In some embodiments of the present invention, any of the aforementioned CARs does not comprise a leader signal peptide.

[0164] In some embodiments of the present invention, any of the aforementioned CARs comprises a leader signal peptide;

[0165] Preferably, the leader signal peptide is selected from the following signal peptides: CD8α signal peptide, CD28 signal peptide, IgG signal peptide, HLA-A signal peptide, CD3γ signal peptide, CD3δ signal peptide, CD3ζ signal peptide and CD3ε signal peptide;

[0166] More preferably, the leader signal peptide is CD8α signal peptide.

[0167] In some embodiments of the present invention, the leading signal peptide of CAR is a CD8α signal peptide.

[0168] In some embodiments of the present invention, the antigen binding region of the CAR is monospecific, bispecific, or multispecific.

[0169] In some embodiments of the present invention, the antigen binding region of the CAR is bispecific.

[0170] In some embodiments of the present invention, the antigen binding region of the CAR can specifically bind to CD19 and CD20.

[0171] In some embodiments of the present invention, the antigen binding region of the CAR can specifically bind to CD19 and BCMA.

[0172] In some embodiments of the present invention, the antigen binding region of the CAR can specifically bind to CD20 and BCMA.

[0173] In some embodiments of the present invention, the antigen binding region of the CAR can specifically bind to BCMA and GPRC5D.

[0174] In some embodiments of the present invention, any of the aforementioned packaging systems further comprises a polynucleotide encoding an immune cell targeting molecule, wherein the immune cell is selected from one or more of B cells, T cells, NK cells and macrophages.

[0175] In some embodiments of the present invention, the immune cell is a T cell, the immune cell targeting molecule is a T cell targeting molecule, and the T cell targeting molecule can bind to a T cell surface receptor.

[0176] In some embodiments of the present invention, the T cell targeting molecule binds to CD3, CD5, CD7, CD28, TCRγ, TCRδ, TCRα or TCRβ.

[0177] In some embodiments of the present invention, the T cell targeting molecule binds to CD5 or CD7.

[0178] In some embodiments of the present invention, the T cell targeting molecule binds to CD7, and the T cell targeting molecule comprises at least one selected from (a) an anti-CD7 antibody or an antigen-binding fragment thereof and (b) a CD7 ligand or a binding fragment thereof;

[0179] Preferably, the CD7 is human CD7 (UniProtKB No.: P09564);

[0180] More preferably, the T cell targeting molecule comprises anti-CD7 scFv and / or VHH;

[0181] Further preferably, the T cell targeting molecule includes an anti-CD7 scFv, which is derived from the monoclonal antibody TH-69 (TH69-scFv), and the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 87-89, respectively; the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 90-92, respectively.

[0182] In some embodiments of the present invention, any of the aforementioned packaging systems further comprises a polynucleotide encoding an immune cell activation signaling molecule, wherein the immune cell is selected from one or more of B cells, T cells, NK cells and macrophages.

[0183] In some embodiments of the present invention, the immune cell is a T cell, and the immune cell activation signal molecule is a T cell activation signal molecule.

[0184] In some embodiments of the present invention, the T cell activation signaling molecule includes a T cell activation primary signaling molecule.

[0185] In some embodiments of the present invention, the T cell activation primary signal molecule can bind to at least one selected from TCR / CD3 complex subunits and TCR / CD3 complex subunit functional fragments; the TCR / CD3 complex subunits are selected from at least one of CD3ε, CD3γ, CD3δ, CD3ζ, TCRγ, TCRδ, TCRα and TCRβ.

[0186] In some embodiments of the present invention, the T cell activation primary signal molecule binds to CD3;

[0187] Preferably, the T cell activation primary signal molecule can bind to human CD3, and the human CD3 is selected from human CD3ε / CD3E (UniProtKB No.: P07766), CD3γ / CD3G (UniProtKB No.: P09693), CD3δ / CD3D (UniProtKB No.: P04234) and CD3ζ / CD3Z (UniProtKB No.: P20963).

[0188] In some embodiments of the present invention, the T cell activation primary signal molecule comprises an anti-CD3 antibody or an antigen-binding fragment thereof;

[0189] Preferably, the anti-CD3 antibody or antigen-binding fragment thereof is an anti-CD3 scFv and / or VHH.

[0190] In some embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof is selected from at least one of OKT3, UCHT1, YTH12.5 and TR66.

[0191] In some embodiments of the present invention, the anti-CD3 antibody is selected from UCHT1 or a variant or derivative thereof, OKT3 or a variant or derivative thereof, SP34 or a variant or derivative thereof, HuM291 or a variant or derivative thereof, and TR66 or a variant or derivative thereof;

[0192] Preferably, the anti-CD3 antibody is an anti-CD3ε scFv, and the anti-CD3ε scFv is derived from the monoclonal antibody UCHT1 (UCHT1-scFv); the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NOs: 72-74, respectively, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NOs: 75-77, respectively.

[0193] In some embodiments of the present invention, the amino acid sequence of the UCHT1-scFv is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 13.

[0194] In some embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof is UCHT1.

[0195] In some embodiments of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof binds to CD3ε.

[0196] In some embodiments of the present invention, the T cell activation signal molecule further includes a T cell activation secondary signal molecule.

[0197] In some embodiments of the present invention, the T cell activation secondary signaling molecule binds to CD28;

[0198] Preferably, the T cell activation secondary signaling molecule can bind to human CD28 (UniProtKB No.: P10747).

[0199] In some embodiments of the present invention, the T cell activation secondary signaling molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof;

[0200] Preferably, the CD28 ligand or its receptor binding fragment comprises CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.

[0201] In some embodiments of the present invention, the CD28 ligand or its receptor binding fragment includes CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.

[0202] In some embodiments of the present invention, the receptor-binding fragment of CD80 comprises its extracellular domain; the receptor-binding fragment of CD86 comprises its extracellular domain.

[0203] In some embodiments of the present invention, the CD80 is human CD80, UniProtKB number: P33681.

[0204] In some embodiments of the present invention, the CD86 is human CD86, UniProtKB number: P42081.

[0205] In some embodiments of the present invention, the T cell activation secondary signaling molecule comprises an anti-CD28 antibody or an antigen-binding fragment thereof;

[0206] Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is an anti-CD28 scFv; the anti-CD28 scFv is derived from the monoclonal antibody 15E8 (15E8-scFv); the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 80-82, respectively; the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 83-85, respectively.

[0207] In some embodiments of the present invention, the amino acid sequence of the 15E8-scFv is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:17.

[0208] In some embodiments of the present invention, the anti-CD28 antibody or antigen-binding fragment thereof is selected from at least one of CD28.2, 10F3 and TGN1412.

[0209] In some embodiments of the present invention, the T cell activation secondary signal molecule includes CD28 ligand or its receptor binding fragment and / or anti-CD28 antibody or its antigen binding fragment, and may also include at least one ligand or its receptor binding fragment selected from ICOS (inducible costimulator, "ICOS") ligand (ICOSL) or its receptor binding fragment, 4-1BB ligand (4-1BBL) or its receptor binding fragment and OX40 ligand (OX40L) or its receptor binding fragment.

[0210] In some embodiments of the present invention, the T cell activation secondary signaling molecule can also bind to at least one of CD27, HVEM, LIGHT, CD40, DR3, GITR, CD30, TIM1, SLAM, CD2 and CD226.

[0211] In some embodiments of the present invention, the T cell activation secondary signaling molecule may further include at least one selected from B7-H2, CD70, LIGHT, HVEM, CD40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155 and CD112.

[0212] In some embodiments of the present invention, the T cell targeting molecule further comprises an adhesion molecule; preferably, the adhesion molecule can bind to CD2; more preferably, the adhesion molecule comprises CD58 or its extracellular domain or a functional fragment thereof.

[0213] In some embodiments of the present invention, the T cell targeting molecule and / or T cell activation signaling molecule further comprises a transmembrane domain, and the transmembrane domain is anchored on the surface of the LVV or RVV packaged in the packaging cell by the packaging system.

[0214] In some embodiments of the present invention, the transmembrane domain is selected from the transmembrane regions of the following proteins:

[0215] CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1;

[0216] Preferably, the transmembrane domain is the CD8α transmembrane region;

[0217] More preferably, the CD8α transmembrane region is a human CD8α transmembrane region, and the amino acid sequence of the human CD8α transmembrane region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 15.

[0218] In some embodiments of the present invention, the T cell targeting molecule and / or T cell activation signaling molecule further comprises a connecting domain, which connects the extracellular domain of the T cell targeting molecule and / or T cell activation signaling molecule and the transmembrane domain.

[0219] In some embodiments of the present invention, the linker domain comprised by the T cell targeting molecule and / or T cell activation signaling molecule is selected from:

[0220] (a) immunoglobulin hinge region,

[0221] (b) the immunoglobulin hinge region is selected from wild-type and modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions;

[0222] (c) a hinge region selected from the wild-type and 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;

[0223] (d) all or a portion of an Fc domain, wherein the Fc domain is selected from at least one of a CH1 domain, a CH2 domain, and a CH3 domain;

[0224] (e) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and

[0225] (f) flexible linker peptide;

[0226] Preferably, the connecting domain is the CD8α hinge region;

[0227] More preferably, the CD8α hinge region is a human CD8α hinge region, and the amino acid sequence of the human CD8α hinge region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO: 14.

[0228] In some embodiments of the present invention, the packaging system comprises a polynucleotide encoding an envelope glycoprotein.

[0229] In some embodiments of the present invention, the polynucleotide encoding the envelope glycoprotein contained in the packaging system encodes any one of the following envelope glycoproteins: the envelope glycoprotein of the vesicular stomatitis virus strain and its variants, the envelope glycoprotein of the baboon endogenous retrovirus BaEV and its variants, the envelope glycoprotein RD114 of the feline endogenous retrovirus and its variants, and the envelope glycoprotein GALV of the gibbon ape leukemia virus and its variants.

[0230] In some embodiments of the present invention, the envelope glycoprotein is selected from the group consisting of glycoproteins of vesicular stomatitis virus strains, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoproteins, rabies virus glycoproteins (RVG), gibbon ape leukemia virus glycoproteins (GaLV), amphibious murine leukemia virus glycoproteins (MLV-A), feline endogenous virus (RD114) glycoproteins, fowl plague virus (FPV) glycoproteins, Ebola virus (EboV) glycoproteins, and T-cell choriomeningitis virus (LCMV) glycoproteins.

[0231] In some embodiments of the present invention, the envelope glycoprotein is selected from any one of the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof;

[0232] Preferably, the envelope glycoprotein of the vesicular stomatitis virus strain and its variants include the following envelope glycoproteins and their variants: envelope glycoprotein of the vesicular stomatitis virus Indiana strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Cocal strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Maraba strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Morreton strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Alagoas strain and its variants, envelope glycoprotein of the vesicular stomatitis virus New The envelope glycoprotein of Jersey strain and its variants, the envelope glycoprotein of Carajas strain and its variants, the envelope glycoprotein of Chandipura strain and its variants, the envelope glycoprotein of Eptesicus strain and its variants, the envelope glycoprotein of Isfahan strain and its variants, the envelope glycoprotein of Jurona strain and its variants, the envelope glycoprotein of Malpais strain and its variants, the envelope glycoprotein of Perinet strain and its variants, the envelope glycoprotein of Piry strain and its variants, the envelope glycoprotein of Radi strain and its variants, the envelope glycoprotein of Rhinolopus strain and its variants, and the envelope glycoprotein of Yug Bogdanovac strain and its variants.

[0233] In some embodiments of the present invention, the envelope glycoprotein is the envelope glycoprotein of the Indiana strain of vesicular stomatitis virus (VSV-G) or a variant thereof or the envelope glycoprotein of the Cocal strain (Cocal-G) or a variant thereof;

[0234] The extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0235] In some embodiments of the present invention, any of the aforementioned envelope glycoproteins undergoes a first mutation, which reduces or loses the ability of the envelope glycoprotein to bind to a glycoprotein receptor relative to before the first mutation occurs.

[0236] In some embodiments of the present invention, the envelope glycoprotein is selected from the envelope glycoprotein of a vesicular stomatitis virus strain and variants thereof.

[0237] In some embodiments of the present invention, the envelope glycoprotein is VSV-G or Cocal-G; the envelope glycoprotein receptor is a low-density lipoprotein receptor (LDL-R); the VSV-G or Cocal-G undergoes a first mutation, such that the ability of the VSV-G or Cocal-G to bind to LDL-R is reduced or lost relative to before the first mutation occurs;

[0238] The extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0239] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0240] (a) substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 209, substitution or deletion of amino acid at position 347 in SEQ ID NO: 1 or SEQ ID NO: 2. 354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, deletion of amino acids 345-353; and

[0241] (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, substitution or deletion of amino acid 8, substitution or deletion of amino acid 9, substitution or deletion of amino acid 10, substitution or deletion of amino acid 47, substitution or deletion of amino acid 50, substitution or deletion of amino acid 51, substitution or deletion of amino acid 183, substitution or deletion of amino acid 179, substitution or deletion of amino acid 180, substitution or deletion of amino acid 182, substitution or deletion of amino acid 184, substitution or deletion of amino acid 209, substitution or deletion of amino acid 347, substitution or deletion of amino acid 358, substitution or deletion of amino acid 360, substitution or deletion of amino acid 361, substitution or deletion of amino acid 362, substitution or deletion of amino acid 364, substitution or deletion of amino acid 365, substitution or deletion of amino acid 366, substitution or deletion of amino acid 367, substitution or deletion of amino acid 368, substitution or deletion of amino acid 369, substitution or deletion of amino acid 370, substitution or deletion of amino acid 371, substitution or deletion of amino acid 372, substitution or deletion of amino acid 373, substitution or deletion of amino acid 374, substitution or deletion of amino acid 375, substitution or deletion of amino acid 376, substitution or deletion of amino acid 377, substitution or deletion of amino acid 378, substitution or deletion of amino acid 379, substitution or deletion of amino acid 371, substitution or deletion of amino acid 377 the substitution or deletion of the amino acid at position 350, the substitution or deletion of the amino acid at position 352, the substitution or deletion of the amino acid at position 353, the substitution of the amino acid at position 354, the deletion of amino acids at positions 1-18, the deletion of amino acids at positions 19-36, the deletion of amino acids at positions 37-51, the deletion of amino acids at positions 314-384, the deletion of amino acids at positions 321-374, the deletion of amino acids at positions 331-364, the deletion of amino acids at positions 344-354, and the deletion of amino acids at positions 345-353;

[0242] Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0243] (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;

[0244] (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;

[0245] (c) substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, 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: 2; and

[0246] (d) After optimal global alignment with SEQ ID NO: 2, the position corresponding to SEQ ID NO:2: substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, 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;

[0247] More preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0248] (a) substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 1;

[0249] (b) after optimal global alignment with SEQ ID NO: 1, substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353, corresponding to SEQ ID NO: 1;

[0250] (c) 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; and

[0251] (d) 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.

[0252] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0253] (a) deletion of amino acids 331-364, deletion of amino acids 344-354, substitution or deletion of K47, or substitution of R354 in SEQ ID NO: 1 or SEQ ID NO: 2; and

[0254] (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, deletion of amino acids 331-364, deletion of amino acids 344-354, substitution or deletion of K47, substitution of R354 at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0255] Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0256] (a) amino acids 331-364 of SEQ ID NO: 1 or SEQ ID NO: 2 are deleted, amino acids 344-354 are deleted, amino acid 47 is substituted from lysine K to glutamine Q (K47Q), or K47 is deleted, amino acid 354 is substituted from arginine R to glutamine Q (R354Q); and

[0257] (b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, amino acid deletions at positions 331-364, amino acid deletions at positions 344-354, K47Q or K47 deletion, R354Q are located corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0258] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises the following amino acids:

[0259] (a) a deletion of K47 located at SEQ ID NO: 1 or SEQ ID NO: 2; or

[0260] (b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, the K47 deletion is located at the position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2.

[0261] In some embodiments of the present invention, the first mutation includes a mutation in which the amino acid sequence comprises the following amino acids:

[0262] (a) a mutation at I331 of SEQ ID NO: 1 or SEQ ID NO: 2; or

[0263] (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at position I331 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0264] Preferably, the mutation of I331 is selected from the group consisting of: I331E, I331W, I331M, I331L, I331Q, and I331R.

[0265] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 62-67;

[0266] Relative to SEQ ID NO: 1, SEQ ID NO: 62 comprises I331E;

[0267] Relative to SEQ ID NO: 1, SEQ ID NO: 63 comprises I331W;

[0268] Relative to SEQ ID NO: 1, SEQ ID NO: 64 comprises I331M;

[0269] Relative to SEQ ID NO: 1, SEQ ID NO: 65 comprises I331L;

[0270] Relative to SEQ ID NO: 1, SEQ ID NO: 66 comprises I331Q;

[0271] Relative to SEQ ID NO: 1, SEQ ID NO: 67 comprises I331R.

[0272] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 33 or SEQ ID NO: 34.

[0273] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 3 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of 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.

[0274] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:4 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO:4; relative to SEQ ID NO:1, SEQ ID NO:4 comprises R354Q.

[0275] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:33 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO:33; relative to SEQ ID NO:2, SEQ ID NO:33 comprises a K47 deletion.

[0276] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:34 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO:34; relative to SEQ ID NO:2, SEQ ID NO:34 comprises R354Q.

[0277] In some embodiments of the present invention, the envelope glycoprotein having any of the aforementioned first mutations still retains the ability to fuse membranes and escape from endosomal / lysosomal regions.

[0278] In some embodiments of the present invention, the envelope glycoprotein further undergoes a second mutation, which enhances the ability of the envelope glycoprotein to antagonize inactivation by complement, or prevents inactivation by complement, compared to before the second mutation.

[0279] In some embodiments of the present invention, the envelope glycoprotein having the second mutation is any of the aforementioned envelope glycoproteins.

[0280] In some embodiments of the present invention, the glycoprotein with the second mutation is the envelope glycoprotein of the Indiana strain or Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0281] In some embodiments of the present invention, the second mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids:

[0282] (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0283] (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0284] (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0285] (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0286] (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0287] (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0288] (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0289] (h) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 146 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0290] Preferably, the amino acid mutation includes at least one of amino acid deletion, insertion and substitution;

[0291] More preferably, the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids:

[0292] (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0293] (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0294] (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0295] (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0296] (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2;

[0297] (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2;

[0298] (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and

[0299] (h) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the amino acid position corresponding to 146 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0300] In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as shown in SEQ ID NO: 1, comprising at least one of the following position mutations:

[0301] (a) substitution of T214, substitution of T352, substitution of K50, or substitution of S146 in SEQ ID NO: 1; and

[0302] (b) after optimal global alignment with SEQ ID NO: 1, substitutions at T214, T352, K50, and S146 corresponding to SEQ ID NO: 1;

[0303] Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence:

[0304] (a) amino acid position 214 in SEQ ID NO: 1 is substituted from threonine T to asparagine N (T214N), amino acid position 352 is substituted from threonine T to alanine A (T352A), amino acid position 50 is substituted from lysine K to threonine T (K50T), and amino acid position 146 is substituted from serine S to threonine T (S146T); and

[0305] (b) After optimal global alignment with SEQ ID NO: 1, T214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO: 1.

[0306] In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as shown in SEQ ID NO: 1, comprising a combination of any of the following site mutations:

[0307] (a) substitution of (i) T214 and T352; or (ii) T214, T352, K50, and S146 of SEQ ID NO: 1; and

[0308] (b) after optimal global alignment with SEQ ID NO: 1, substitutions at positions corresponding to (i) T214 and T352; or (ii) T214, T352, K50, and S146 of SEQ ID NO: 1;

[0309] Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

[0310] (a) (i) T214N and T352A; or (ii) T214N, T352A, K50T, and S146T; and

[0311] (b) After optimal global alignment with SEQ ID NO: 1, located at (i) T214N and T352A; or (ii) T214N, T352A, K50T, and S146T corresponding to SEQ ID NO: 1.

[0312] In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as shown in SEQ ID NO: 2, comprising at least one of the following position mutations:

[0313] (a) substitution of K214, substitution of T352, substitution of K50, substitution of S146 in SEQ ID NO: 2; and

[0314] (b) after optimal global alignment with SEQ ID NO: 2, substitutions at positions corresponding to K214, T352, K50, and S146 of SEQ ID NO: 2;

[0315] Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence:

[0316] (a) amino acid position 214 of SEQ ID NO: 2 is replaced by lysine K to asparagine N (K214N), T352A, K50T, S146T; and

[0317] (b) After optimal global alignment with SEQ ID NO: 2, K214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO: 2.

[0318] In some embodiments of the present invention, the second mutation comprises an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as shown in SEQ ID NO: 2, comprising a combination of any of the following site mutations:

[0319] (a) substitution of (i) K214 and T352; or (ii) K214, T352, K50, and S146 of SEQ ID NO: 2; and

[0320] (b) after optimal global alignment with SEQ ID NO: 2, at positions corresponding to (i) substitutions of K214 and T352; or (ii) substitutions of K214, T352, K50, and S146 of SEQ ID NO: 2;

[0321] Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence:

[0322] (a) (i) K214N and T352A; or (ii) K214N, T352A, K50T, and S146T at SEQ ID NO: 2;

[0323] (b) After optimal global alignment with SEQ ID NO: 2, located at (i) K214N and T352A; or (ii) K214N, T352A, K50T, and S146T corresponding to SEQ ID NO: 2.

[0324] In some embodiments of the present invention, the envelope glycoprotein is VSV-G or a variant thereof;

[0325] The extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 1;

[0326] The envelope glycoprotein undergoes any of the aforementioned first mutations, so that the ability of the envelope glycoprotein to bind to LDL-R is weakened or lost relative to before the first mutation; the envelope glycoprotein may also undergo any of the aforementioned second mutations, so that the ability of the envelope glycoprotein to antagonize inactivation by complement is enhanced relative to before the second mutation or is not inactivated by complement.

[0327] In some embodiments of the present invention, the envelope glycoprotein is Cocal-G or a variant thereof;

[0328] The extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 2 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 2;

[0329] The envelope glycoprotein undergoes any of the aforementioned first mutations, so that the ability of the envelope glycoprotein to bind to LDL-R is weakened or lost relative to before the first mutation; the envelope glycoprotein may also undergo any of the aforementioned second mutations, so that the ability of the envelope glycoprotein to antagonize inactivation by complement is enhanced relative to before the second mutation or is not inactivated by complement.

[0330] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38.

[0331] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 8 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 8; relative to SEQ ID NO: 1, SEQ ID NO: 8 comprises K47 deletion, T214N (relative to before K47 deletion), and T352A (relative to before K47 deletion).

[0332] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 9 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 9; relative to SEQ ID NO: 1, SEQ ID NO: 9 comprises K47 deletion, T214N (relative to before K47 deletion), T352A (relative to before K47 deletion), K50T (relative to before K47 deletion), and S146T (relative to before K47 deletion).

[0333] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 10 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 10; relative to SEQ ID NO: 1, SEQ ID NO: 10 comprises R354Q, T214N and T352A.

[0334] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 11 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 11; relative to SEQ ID NO: 1, SEQ ID NO: 11 comprises R354Q, T214N, T352A, K50T and S146T.

[0335] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 35 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 35; relative to SEQ ID NO: 2, SEQ ID NO: 35 comprises K47 deletion, K214N (relative to before K47 deletion), T352A (relative to before K47 deletion), K50T (relative to before K47 deletion) and S146T (relative to before K47 deletion).

[0336] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 36 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 36; relative to SEQ ID NO: 2, SEQ ID NO: 36 comprises K47 deletion, K214N (relative to before K47 deletion) and T352A (relative to before K47 deletion).

[0337] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 37 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 37; relative to SEQ ID NO: 2, SEQ ID NO: 37 comprises R354Q, K214N, T352A, K50T and S146T.

[0338] In some embodiments of the present invention, the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 38 or has at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% of the amino acid sequence as shown in SEQ ID NO: 38; relative to SEQ ID NO: 2, SEQ ID NO: 38 comprises R354Q, K214N and T352A.

[0339] In some embodiments of the present invention, the glycoprotein having any of the aforementioned second mutations still retains the ability to fuse membranes and escape from endosomal / lysosomal regions.

[0340] In some embodiments of the present invention, the glycoprotein having any of the aforementioned first and second mutations still retains the ability to fuse with membranes and escape from endosomal / lysosomes.

[0341] In some embodiments of the present invention, after the LVV or RVV packaged in the packaging cell using any of the aforementioned packaging systems comprising the polynucleotide encoding the T cell activation signaling molecule (T cell activation signaling molecule gene) contacts with non-activated T cells, the efficiency of the CAR in T cell membrane expression is higher.

[0342] In some embodiments of the present invention, the improvement in the efficiency of membrane expression is relative to the LVV or RVV packaged in the packaging cell by a packaging system comprising the CAR binding molecule gene but not the T cell activation signaling molecule gene.

[0343] In some embodiments of the present invention, after the LVV or RVV packaged in the packaging cells by the packaging system containing the T cell activation signaling molecule gene contacts non-activated T cells, the killing efficiency of the prepared CAR-T cells is higher.

[0344] In some embodiments of the present invention, the improvement in the CAR-T cell killing efficiency is relative to the LVV or RVV packaged in the packaging cell by a packaging system comprising the CAR binding molecule gene but not the T cell activation signaling molecule gene.

[0345] In some embodiments of the present invention, the content of CAR available for binding to an antigen on the viral envelope of the LVV or RVV packaged in the packaging cells by the packaging system is reduced or does not contain the CAR available for binding to an antigen.

[0346] In some embodiments of the present invention, the packaging system comprises one or more nucleic acid vectors. When introduced into a packaging cell for packaging a lentiviral vector or a retroviral vector, the one or more nucleic acid vectors comprise nucleic acids necessary for producing, assembling and / or packaging the lentiviral vector or retroviral vector in the packaging cell.

[0347] In some embodiments of the present invention, the packaging system further comprises:

[0348] (a) Gag gene and Pol gene; and

[0349] (b)

[0350] (i) the LVV backbone gene and the Rev gene; or

[0351] (ii) RVV backbone gene.

[0352] In some embodiments of the present invention, the packaging system comprises:

[0353] (a) a nucleic acid vector comprising a Gag gene and a Pol gene; and

[0354] (b) a nucleic acid vector comprising the following gene:

[0355] (i) the LVV backbone gene and the Rev gene; or

[0356] (ii) RVV backbone gene.

[0357] In some embodiments of the present invention, the nucleic acid vector is a plasmid.

[0358] In some embodiments of the present invention, the packaging system comprises: at least one packaging plasmid, an envelope plasmid, a shuttle plasmid and a binding plasmid; the binding plasmid comprises a polynucleotide encoding the CAR binding molecule.

[0359] In some embodiments of the present invention,

[0360] (a) the packaging plasmid comprises the Gag gene and the Pol gene (Gag / Pol packaging plasmid);

[0361] (b) the envelope plasmid comprises (i) a polynucleotide encoding the envelope glycoprotein or a variant thereof and (ii) a polynucleotide encoding the (1) T cell targeting molecule and / or (2) a polynucleotide encoding the T cell activation signaling molecule; and

[0362] (c) The shuttle plasmid comprises (i) the shuttle gene and (ii): (1) LVV backbone gene or (2) RVV backbone gene.

[0363] In some embodiments of the present invention,

[0364] (a) the packaging plasmid comprises the Gag gene and the Pol gene (Gag / Pol packaging plasmid);

[0365] (b) the envelope plasmid comprises a polynucleotide encoding the envelope glycoprotein or a variant thereof;

[0366] (c) the shuttle plasmid comprises (i) the shuttle gene and (ii): (1) the LVV backbone gene or (2) the RVV backbone gene; and

[0367] (d) The packaging system further comprises a targeting plasmid, wherein the targeting plasmid comprises (i) a polynucleotide encoding the T cell targeting molecule and / or (ii) a polynucleotide encoding the T cell activation signaling molecule.

[0368] In some embodiments of the present invention, when the packaging system is used to package LVV, the packaging system further comprises a Rev packaging plasmid, and the Rev packaging plasmid comprises the Rev gene.

[0369] In some embodiments of the present invention, the reduced content of the CAR available for antigen binding is relative to the LVV or RVV packaged in the packaging cell by a packaging system that does not contain a polynucleotide encoding any of the aforementioned CAR binding molecules (control packaging system); the only variable between the control packaging system and the packaging system provided by the present invention is whether it contains a polynucleotide encoding any of the aforementioned CAR binding molecules.

[0370] In some embodiments of the present invention, when the LVV or RVV packaged in the packaging cells of the packaging system provided by the present invention transduces host cells, no false transduction occurs or false transduction is reduced.

[0371] In some embodiments of the present invention, the reduction in false transduction is relative to LVV or RVV packaged in packaging cells using a packaging system that does not contain a polynucleotide encoding any of the aforementioned CAR binding molecules (control packaging system); the only variable between the control packaging system and any of the aforementioned packaging systems provided herein is whether or not it contains a polynucleotide encoding any of the aforementioned CAR binding molecules.

[0372] In some embodiments of the present invention, the host cell comprises an immune cell, and the immune cell is selected from one or more of B cells, T cells, NK cells and macrophages.

[0373] In some embodiments of the present invention, the immune cells are T cells.

[0374] In some embodiments of the present invention, the LVV or RVV packaged by the packaging system in the packaging cell does not transduce disease cells expressing the antigen or has a reduced ability to transduce the disease cells; the antigen binding region of the CAR can bind to the antigen.

[0375] In some embodiments of the present invention, the reduction in transduction capacity is relative to LVV or RVV packaged in packaging cells using a packaging system that does not contain a polynucleotide encoding any of the aforementioned CAR binding molecules (control group packaging system); the only variable between the control group packaging system and the packaging system provided herein is whether or not it contains a polynucleotide encoding any of the aforementioned CAR binding molecules.

[0376] In some embodiments of the present invention, the disease cells are selected from at least one of cancer cells and autoimmune disease cells, and the cancer cells include solid cancer cells and blood cancer cells.

[0377] In another aspect, the present invention further provides an LVV or RVV, wherein the LVV or RVV is packaged in a packaging cell using any of the aforementioned packaging systems provided by the present invention.

[0378] In some embodiments of the present invention, the content of CAR available for binding to the antigen on the cell membrane of the packaging cell is reduced or the CAR is absent.

[0379] In some embodiments of the present invention, the content of the CAR available for binding to the antigen on the viral envelope of the LVV or RVV is reduced or does not contain the CAR.

[0380] In some embodiments of the present invention, the reduced content of the CAR available for antigen binding on the viral envelope of the LVV or RVV is relative to the LVV or RVV packaged in a packaging cell by any of the aforementioned packaging systems not provided by the present invention.

[0381] In another aspect, the present invention further provides a complex molecule comprising the following components (a) and (b):

[0382] (a) a CAR binding molecule comprising a CAR binding region; and

[0383] (b)CAR;

[0384] The CAR binding region of the CAR binding molecule binds to the CAR and assembles into the complex molecule.

[0385] In some embodiments of the present invention, the antigen binding region of the CAR comprises an antibody or an antigen binding fragment thereof, and the antibody or the antigen binding fragment thereof comprises an antibody epitope.

[0386] In some embodiments of the present invention, the CAR binding region comprises:

[0387] (a) antigens; and / or

[0388] (b) Primary anti-antibody.

[0389] In some embodiments of the present invention, the antigen epitope of the antigen in the CAR binding region can bind to the antibody epitope.

[0390] In some embodiments of the present invention, the antigen of the CAR binding region comprises (a) a full-length antigen or (b) a functional fragment of an antigen, wherein the functional fragment of the antigen at least comprises the antigen epitope;

[0391] Optionally, the functional fragment of the antigen comprises at least the extracellular domain of the antigen;

[0392] Optionally, the functional fragment of the antigen comprises the extracellular domain and the transmembrane region of the antigen.

[0393] In some embodiments of the present invention, the first anti-antibody can bind to at least one CDR region of the antibody epitope.

[0394] In some embodiments of the present invention, the first anti-antibody can bind to the HCDR3 region and / or LCDR3 region of the antibody epitope.

[0395] In some embodiments of the present invention, the antigen and / or the first anti-antibody of the CAR binding region can specifically bind to the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR.

[0396] In some embodiments of the present invention, the CAR binding molecule comprises, from N-terminus to C-terminus or C-terminus to N-terminus, the following sequence:

[0397] (a) the antigen of the CAR binding region or the first anti-antibody;

[0398] (b) (i) an antigen of the CAR binding region, (ii) a first anti-antibody to the CAR binding region; or

[0399] (c) (i) an antigen in the CAR binding region, (ii) a polypeptide linker, and (iii) a first anti-antibody in the CAR binding region.

[0400] In some embodiments of the present invention, (a) the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR may bind to an antigen selected from the group consisting of: and / or (b) the antigen binding region of the CAR may be selected from the group consisting of:

[0401] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5TAC, 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-3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, uPAR, GCC, EPCAM, Nectin4, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut At least one of hsp70-2, CD79α, CD79β, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, and NKG2D;

[0402] Preferably, (a) the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR can bind to and / or (b) the antigen of the CAR binding region can be selected from: at least one of CD19, CD20, CD33, MSLN, CD79β, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2, B7H3 and GUCY2C.

[0403] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof in the antigen-binding region of the CAR can bind to (a) CD19 and CD20, (b) CD19 and BCMA, (c) CD20 and BCMA, or (d) BCMA and GPRC5D.

[0404] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR comprises an anti-CD19 antibody or antigen-binding fragment thereof that can bind to CD19;

[0405] Preferably, the CD19 is human CD19.

[0406] In some embodiments of the present invention, the CAR binding region comprises the antigen CD19 and / or an anti-anti-CD19 antibody or an antigen-binding fragment thereof (first anti-antibody, anti-anti-CD19 antibody).

[0407] In some embodiments of the present invention, the antigen CD19 of the CAR binding region comprises its extracellular domain and transmembrane region.

[0408] In some embodiments of the present invention, the antigen CD19 of the CAR binding region further comprises its intracellular domain.

[0409] In some embodiments of the present invention, the anti-CD19 antibody or antigen-binding fragment thereof is the FMC63-scFv.

[0410] In some embodiments of the present invention, the amino acid sequences of the HCDR1-3 regions of the anti-anti-CD19 antibody are shown as SEQ ID NOs: 102-104, respectively, and the amino acid sequences of the LCDR1-3 regions of the anti-anti-CD19 antibody are shown as SEQ ID NOs: 105-107, respectively.

[0411] In some embodiments of the present invention, the CAR is any of the aforementioned CARs.

[0412] In some embodiments of the present invention, the CAR binding region comprises a second anti-antibody, which can bind to a region of the CAR that does not comprise the antibody epitope (non-antibody epitope region).

[0413] In some embodiments of the present invention, the complex molecule is derived from the cell membrane of the packaging cell.

[0414] In some embodiments of the present invention, the CAR binding molecule comprises a leader signal peptide, and the leader signal peptide is located at the N-terminal end of the CAR binding region;

[0415] Preferably, the leader signal peptide is selected from any one of the following signal peptides: a natural leader signal peptide of an antigen, a CD8α signal peptide, a CD28 signal peptide, an IgG signal peptide, and an HLA-A signal peptide;

[0416] More preferably, the leader signal peptide is the natural leader signal peptide of the antigen or the CD8α signal peptide.

[0417] In some embodiments of the present invention, the CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, the CAR binding region comprising at least one of (i) the antigen, (ii) the first anti-antibody and (iii) the second anti-antibody.

[0418] In some embodiments of the present invention, the CAR binding molecule further comprises an endoplasmic reticulum retention signal peptide, wherein the endoplasmic reticulum retention signal peptide is located at the C-terminus of the CAR binding region;

[0419] Preferably, the endoplasmic reticulum retention signal peptide comprises the amino acid sequence shown in SEQ ID NO: 51 (KDEL).

[0420] In some embodiments of the present invention, the CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, and (c) the endoplasmic reticulum retention signal peptide.

[0421] In some embodiments of the present invention, the CAR binding molecule further comprises a polypeptide linker, and the CAR binding region is connected to the endoplasmic reticulum retention signal peptide through the polypeptide linker; the CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, (c) the polypeptide linker, and (d) the endoplasmic reticulum retention signal peptide;

[0422] Preferably, the polypeptide linker is selected from any one of the aforementioned polypeptide linkers.

[0423] In some embodiments of the present invention, the CAR binding molecule does not comprise a leader signal peptide.

[0424] In another aspect, the present invention also provides an LVV or RVV, wherein the LVV or RVV comprises at least one complex molecule displayed on its surface, wherein the complex molecule is any of the aforementioned complex molecules provided by the present invention that does not comprise an endoplasmic reticulum retention signal peptide.

[0425] In some embodiments of the present invention, the complex molecule is any one of the aforementioned complex molecules provided by the present invention that does not contain an endoplasmic reticulum retention signal peptide and a leader signal peptide.

[0426] In some embodiments of the present invention, the LVV or RVV comprises a shuttle gene, and the shuttle gene comprises a polynucleotide encoding CAR.

[0427] In some embodiments of the present invention, the complex molecule is derived from the cell membrane of the packaging cell that packages the LVV or RVV.

[0428] In some embodiments of the present invention, the LVV or RVV further comprises a T cell targeting molecule displayed on its surface, and the T cell targeting molecule can bind to a T cell surface receptor.

[0429] In some embodiments of the present invention, the T cell targeting molecule is any of the aforementioned T cell targeting molecules provided by the present invention.

[0430] In some embodiments of the present invention, the LVV or RVV further comprises a T cell activation signaling molecule displayed on its surface.

[0431] In some embodiments of the present invention, the T cell activation signaling molecule is any one of the aforementioned T cell activation signaling molecules provided by the present invention.

[0432] In some embodiments of the present invention, the envelope glycoprotein of LVV or RVV is any one of the aforementioned envelope glycoproteins or variants thereof provided by the present invention.

[0433] In another aspect, the present invention also provides a method for transducing T cells, comprising contacting the T cells with any one of the aforementioned LVVs or RVVs provided by the present invention.

[0434] In some embodiments of the present invention, the T cells are:

[0435] (a) T cells in patients;

[0436] (b) T cells in the patient's blood; or

[0437] (c) T cells from cells isolated from a patient or other donor;

[0438] The patient is an individual who is administered the LVV or RVV and / or an individual who is administered T cells transduced by the method for transducing T cells.

[0439] In some embodiments of the present invention, the cells isolated from the patient or other donor are peripheral blood mononuclear cells (PBMCs), cells collected by lymphocyte apheresis, and / or cells collected by single blood collection from the patient or other donor.

[0440] In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implantation device administration.

[0441] In some embodiments of the present invention, the intravenous administration comprises intravenous infusion.

[0442] In some embodiments of the present invention, the transduction occurs in vivo and / or in vitro in a patient;

[0443] (a) when the transduction occurs outside the patient, the patient is an individual to whom T cells transduced by the method for transducing T cells are administered; and / or

[0444] (b) When the transduction occurs in a patient, the patient is an individual to whom the LVV or RVV is administered.

[0445] In another aspect, the present invention also provides an engineered T cell, wherein the engineered T cell expresses CAR, and the engineered T cell is prepared by transducing T cells with any of the aforementioned LVV or RVV provided by the present invention.

[0446] In another aspect, the present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier and any one of the following components: any one of the aforementioned LVV or RVV provided by the present invention and engineered T cells.

[0447] In another aspect, the present invention further provides a packaging cell, comprising any one of the aforementioned packaging systems provided by the present invention.

[0448] In some embodiments of the present invention, the CAR available for antigen binding on the cell membrane of the packaging cell is reduced.

[0449] In some embodiments of the present invention, the packaging cell is selected from any one of NS0 cells, Vero cells, HeLa cells, COS cells, CHO cells, HEK cells, BHK cells and MDCKⅡ cells;

[0450] Preferably, the packaging cells are HEK-293T cells.

[0451] In some embodiments of the present invention, the packaging cell is configured to produce a lentiviral vector or a retroviral vector.

[0452] In another aspect, the present invention also provides a method for preparing any one of the aforementioned LVV or RVV provided by the present invention, comprising culturing any one of the aforementioned packaging cells provided by the present invention until the LVV or RVV can be harvested.

[0453] In another aspect, the present invention also provides a method for reducing false transduction when LVV or RVV transduces T cells, comprising reducing the content of CAR on the viral envelope of the LVV or RVV that can bind to disease cell surface antigens.

[0454] In some embodiments of the present invention, the method comprises reducing the content of CAR available for binding to disease cell surface antigens on the cell membrane of the packaging cell packaging the LVV or RVV.

[0455] In some embodiments of the present invention, the method comprises packaging the LVV or RVV in packaging cells using any of the aforementioned packaging systems provided by the present invention.

[0456] In another aspect, the present invention also provides a method for treating a disease suffered by a patient or killing diseased cells: (a) in the patient's body; (b) in the patient's blood; or (c) in cells isolated from the patient or other donor, comprising administering to the patient, the patient's blood, or cells isolated from the patient or other donor LVV or RVV packaged in packaging cells using any of the aforementioned packaging systems provided by the present invention.

[0457] In another aspect, the present invention also provides a method for treating a disease suffered by a patient or killing: (a) diseased cells in the patient's body; (b) in the patient's blood; or (c) in cells isolated from the patient or other donor, comprising administering any of the aforementioned LVVs or RVVs provided by the present invention to the patient, the patient's blood, or cells isolated from the patient or other donor.

[0458] In another aspect, the present invention also provides the use of any of the aforementioned LVV or RVV, engineered T cells, and pharmaceutical compositions provided by the present invention in the preparation of drugs for treating diseases.

[0459] In some embodiments of the present invention, the disease cells are selected from at least one of cancer cells and autoimmune disease cells; the cancer cells include solid cancer cells and blood cancer cells.

[0460] In some embodiments of the present invention, the disease is a blood cancer selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible (Transplant At least one of the following: primary mediastinal B-cell lymphoma (MBC), diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), primary plasma cell leukemia (pPCL), peripheral T-cell lymphoma (PTCL-NHL), NK / T-cell lymphoma, anaplastic large cell lymphoma (ALCL), intestinal T-cell lymphoma, T-large granular lymphocytic leukemia (T-LGL), and germinal center T-cell lymphoma (FTCL).

[0461] In some embodiments of the present invention, the disease is a blood cancer, the blood cancer is a B-cell malignancy, and the B-cell malignancy is selected from at least one of: non-Hodgkin lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant-ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia and Burkitt lymphoma (BL).

[0462] In some embodiments of the present invention, the disease is a solid cancer, and the solid cancer is selected from at least one of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer and gastrointestinal cancer.

[0463] In some embodiments of the present invention, the disease is an autoimmune disease, and the autoimmune disease is selected from at least one of systemic lupus erythematosus, psoriasis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Behçet's disease, Sjögren's syndrome, myasthenia gravis, celiac disease, type 1 diabetes, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis and scleroderma.

[0464] In some embodiments of the present invention, the mode of administering the LVV or RVV to the patient is selected from any of the aforementioned modes of administration.

[0465] In some embodiments of the present invention, the administering comprises administering a therapeutically effective amount of LVV or RVV.

[0466] In another aspect, the present invention also provides a polynucleotide encoding a CAR binding molecule, which can bind to CAR.

[0467] In some embodiments of the present invention, the antigen binding region of the CAR comprises an antibody or an antigen binding fragment thereof, and the antibody or antigen binding fragment thereof comprises an antibody epitope, and the antibody epitope can bind to an antigen epitope.

[0468] In some embodiments of the present invention, the CAR binding molecule comprises (a) a leader signal peptide, (b) a CAR binding region, and (c) an endoplasmic reticulum retention signal peptide.

[0469] In some embodiments of the present invention, the leader signal peptide is operably linked to the N-terminus of the CAR binding region, and the endoplasmic reticulum retention signal peptide is operably linked to the C-terminus of the CAR binding region;

[0470] Preferably, the endoplasmic reticulum retention signal peptide is operably linked to the C-terminus of the CAR binding region via a polypeptide linker;

[0471] More preferably, the polypeptide linker is selected from any of the aforementioned polypeptide linkers;

[0472] Further preferably, the flexible connecting peptide is selected from (G4S) n Connector peptide, Connector peptide 1 and Connector peptide 3; wherein n=1 to 4;

[0473] Connector peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23)

[0474] Connector peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 137).

[0475] In some embodiments of the present invention, the CAR binding region is selected from at least one of (a) an antigen, (b) a first anti-antibody, and (c) a second anti-antibody.

[0476] In some embodiments of the present invention, the antigen of the CAR binding region is selected from at least one of a full-length antigen, an antigen extracellular domain, an antigen variable region, and an antigen epitope.

[0477] In some embodiments of the present invention, the antigen or first anti-antibody of the CAR binding region can bind to the antibody epitope.

[0478] In some embodiments of the present invention, the antigen or first anti-antibody of the CAR binding region can bind to at least one CDR region of the antibody epitope.

[0479] In some embodiments of the present invention, the antigen or first anti-antibody of the CAR binding region can bind to at least the CDR3 region of the antibody epitope.

[0480] In some embodiments of the present invention, the antigen or first anti-antibody of the CAR binding region can bind to at least the HCDR3 and / or LCDR3 region of the antibody epitope.

[0481] In some embodiments of the present invention, the antigen or the first anti-antibody in the CAR binding region can specifically bind to the antibody or antigen-binding fragment thereof in the antigen binding region of the CAR.

[0482] In some embodiments of the present invention, the second anti-antibody can bind to a region of the CAR that does not contain the antibody epitope (non-antibody epitope region).

[0483] In some embodiments of the present invention, the leader signal peptide is selected from the following signal peptides: natural leader signal peptide of antigen, CD8α signal peptide, CD28 signal peptide, IgG signal peptide and HLA-A signal peptide;

[0484] Preferably, the leader signal peptide is the natural leader signal peptide of the antigen or the CD8α signal peptide.

[0485] In some embodiments of the present invention, the endoplasmic reticulum retention signal peptide comprises the amino acid sequence shown in SEQ ID NO: 51 (KDEL).

[0486] In some embodiments of the present invention, the CAR binding molecule comprises a CAR binding region, and the CAR binding region can bind to CAR to assemble into a complex molecule.

[0487] In some embodiments of the present invention, the CAR binding region comprises:

[0488] (a) antigens; and / or

[0489] (b) Primary anti-antibody.

[0490] In some embodiments of the present invention, the antigen epitope of the antigen in the CAR binding region can bind to the antibody epitope.

[0491] In some embodiments of the present invention, the antigen of the CAR binding region comprises (a) a full-length antigen or (b) a functional fragment of an antigen, wherein the functional fragment of the antigen at least comprises the antigen epitope;

[0492] Optionally, the functional fragment of the antigen comprises at least the extracellular domain of the antigen;

[0493] Optionally, the functional fragment of the antigen comprises the extracellular domain and the transmembrane region of the antigen.

[0494] In some embodiments of the present invention, the first anti-antibody can bind to at least one CDR region of the antibody epitope.

[0495] In some embodiments of the present invention, the first anti-antibody can bind to at least the CDR3 region of the antibody epitope.

[0496] In some embodiments of the present invention, the antigen and / or the first anti-antibody of the CAR binding region can specifically bind to the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR.

[0497] In some embodiments of the present invention, the CAR binding region further comprises a second anti-antibody, which can bind to a region of the CAR that does not comprise the antibody epitope (non-antibody epitope region).

[0498] In some embodiments of the present invention, the CAR binding molecule comprises a leader signal peptide, and the leader signal peptide is located at the N-terminal end of the CAR binding region;

[0499] Preferably, the leader signal peptide is selected from any one of the following signal peptides: a natural leader signal peptide of an antigen, a CD8α signal peptide, a CD28 signal peptide, an IgG signal peptide, and an HLA-A signal peptide;

[0500] More preferably, the leader signal peptide is the natural leader signal peptide of the antigen or the CD8α signal peptide.

[0501] In some embodiments of the present invention, the CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, the CAR binding region comprising (i) the antigen and / or (ii) the first anti-antibody;

[0502] Optionally, the CAR binding region may further comprise a second anti-antibody.

[0503] In some embodiments of the present invention, the CAR binding molecule further comprises an endoplasmic reticulum retention signal peptide, wherein the endoplasmic reticulum retention signal peptide is located at the C-terminus of the CAR binding region;

[0504] Preferably, the endoplasmic reticulum retention signal peptide comprises the amino acid sequence shown in SEQ ID NO: 51 (KDEL).

[0505] In some embodiments of the present invention, the CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, and (c) the endoplasmic reticulum retention signal peptide.

[0506] In some embodiments of the present invention, the CAR binding molecule further comprises a polypeptide linker, and the CAR binding region is connected to the endoplasmic reticulum retention signal peptide via the polypeptide linker;

[0507] The CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, (c) a polypeptide linker, and (d) the endoplasmic reticulum retention signal peptide;

[0508] Preferably, the polypeptide linker is selected from any of the aforementioned polypeptide linkers;

[0509] More preferably, the flexible connecting peptide is selected from (G4S) n Connector peptide, Connector peptide 1 and Connector peptide 3; wherein n=1 to 4;

[0510] Connector peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23)

[0511] Connector peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 137).

[0512] In some embodiments of the present invention, the CAR binding molecule does not comprise a leader signal peptide.

[0513] In some embodiments of the present invention, the CAR to which the CAR binding molecule can bind is any of the aforementioned CARs.

[0514] In some embodiments of the present invention, the polynucleotide is isolated.

[0515] In another aspect, the present invention also provides a CAR binding molecule, which is encoded by any one of the aforementioned polynucleotides encoding the CAR binding molecule provided by the present invention.

[0516] In another aspect, the present invention also provides a packaging system, comprising any one of the aforementioned polynucleotides encoding the CAR binding molecule provided by the present invention.

[0517] In another aspect, the present invention also provides a packaging cell, comprising any one of the aforementioned polynucleotides encoding the CAR binding molecule provided by the present invention.

[0518] In some embodiments of the present invention, the packaging cell is selected from any one of NS0 cells, Vero cells, HeLa cells, COS cells, CHO cells, HEK cells, BHK cells and MDCKⅡ cells;

[0519] Preferably, the packaging cells are HEK-293T cells.

[0520] In some embodiments of the present invention, the packaging cell is configured to produce a vector;

[0521] Preferably, the vector is selected from LVV, RVV, AAV (adeno-associated virus), LNPs and VLPs.

[0522] In another aspect, the present invention further provides a packaging cell line, comprising any one of the aforementioned packaging cells provided by the present invention.

[0523] The beneficial effects of the present invention include:

[0524] The packaging system provided by the present invention significantly reduces the content of CAR available for binding to antigens contained in the viral envelope of LVV or RVV packaged in the packaging cells, which can significantly reduce false transduction and significantly weaken the ability of the LVV or RVV to transduce disease cells expressing the antigen, such as cancer cells. It significantly improves the targeting of the LVV or RVV to transduce T cells in the patient's body or in the blood of patients with blood diseases, reduces antigen escape and cancer cells escaping from the recognition and killing of CAR-T cells, thereby improving the killing efficiency of the prepared CAR-T cells in killing cancer cells and other disease cells in the patient's body or in the blood of patients with blood diseases, and significantly improving the efficacy of CAR-T cell therapy.

[0525] definition:

[0526] "Epitope": Also known as an antigenic determinant, it is the site of an antigen that is recognized by the immune system, especially by antibodies, B cells, or T cells. The site in an antibody that binds to an epitope is called an antibody epitope. Although epitopes are usually non-self proteins, sequences that can be recognized by the host (such as autoimmune diseases) are also epitopes (Mahmoudi Gomari, et al., "Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry". Biotechnology Advances. 45: 107653. (2020)).

[0527] In some embodiments of the present invention, "antigen epitope" includes a site in an antigen that can be recognized and bound by an antigen binding domain (ABD) of an engineered T cell, such as a CAR-T and / or TCR-T cell.

[0528] "Anti-antibodies" include specific regions that can recognize and bind to other antibodies, including but not limited to antibody epitopes (paratopes), CDR regions, variable regions, antigen-binding fragments (Fab fragments) or constant regions.

[0529] In some embodiments of the present invention, the anti-antibody comprises a first anti-antibody and / or a second anti-antibody.

[0530] In some embodiments of the present invention, the first anti-antibody can specifically recognize and bind to an antibody epitope, CDR region, variable region and / or antigen-binding fragment of another antibody.

[0531] In some embodiments of the present invention, the first anti-antibody can specifically recognize and bind to the antibody epitope, CDR region, variable region and / or antigen binding fragment of the antibody in the antigen binding region of the CAR.

[0532] In some embodiments of the present invention, the antibody of the antigen binding region of the CAR is scFv, and the first anti-antibody can bind to the VH region and / or VL region of the scFv.

[0533] In some embodiments of the present invention, the first anti-antibody is an anti-immunoglobulin antibody that can bind to the variable region of an immunoglobulin.

[0534] Anti-immunoglobulin antibodies are proteins that detect other antibodies in the body. They can bind to either the variable or constant regions of immunoglobulins (Anti-immunoglobulin antibodies. In: Moreland, LW (eds) Rheumatology and Immunology Therapy. Springer, Berlin, Heidelberg. (2004).)

[0535] In some embodiments of the present invention, the first anti-antibody includes but is not limited to Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) (Brand: ACRO, Cat. No. FM3-Y45P1), which can specifically bind to the antigen recognition region of the anti-CD19 antibody derived from the monoclonal antibody FMC-63.

[0536] In some embodiments of the present invention, the first anti-antibody includes but is not limited to the anti-anti-CD19 antibody.

[0537] In some embodiments of the present invention, the second anti-antibody can recognize and bind to the non-antigen binding portion of the antibody of the antigen binding region of the CAR.

[0538] In some embodiments of the present invention, the antibody of the antigen binding region of the CAR is scFv, and the second anti-antibody can recognize and bind to the connecting peptide contained in the scFv, and the connecting peptide includes but is not limited to (G4S) n Connector peptide.

[0539] "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).

[0540] In some embodiments of the present invention, the T cells are CD4 + / CD8 - 、CD4 - / CD8 + 、CD4 + / CD8 + 、CD4 - / CD8 - T cells or a combination thereof. In some embodiments of the present invention, CD4 +T cells express CAR and bind to target cells such as CD19 + Cancer cells then 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 CAR and binding to target cells.

[0541] "TCR / CD3 complex subunit or its functional fragment": TCR / CD3 complex subunit is selected from at least one of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3ζ, CD3δ and CD3ε; and its functional fragment is the minimum unit of the TCR / CD3 complex subunit, including the extracellular, transmembrane, intracellular, variable and constant domains, which are sufficient to perform their respective functions.

[0542] In some embodiments of the present invention, the TSP molecule comprises a TCR / CD3 complex subunit, or a functional fragment thereof, or a variant thereof. Cells other than T cells do not contain TCR / CD3 complex subunits such as TCRα, TCRβ, CD3γ, CD3ε, CD3γ, CD3ζ, and CD3δ; therefore, even under the action of a leader signal peptide, the TSP molecule will not or rarely be expressed in cells other than T cells, such as HEK-293T cells, which are commonly used to package LVV or RVV, and will not be transferred to the viral envelope of the packaged LVV or RVV during the budding process.

[0543] "Non-activated T cells" refers to non-activated / non-activated T cells, including T cells that are not expanded, not differentiated, in a resting state, do not recognize antigens, have not been activated by T cell activation signaling molecules such as primary and secondary T cell activation signaling molecules and / or have not been activated and stimulated in vitro with common activation magnetic beads such as anti-CD3 antibody magnetic beads and anti-CD28 antibody magnetic beads, for example, T cells in the G0 phase of the cell cycle, resting / quiescent T cells or naive T cells. T cells.

[0544] “T cell activation primary signaling molecule”: T cell activation primary signaling molecule binds to T cell surface proteins and participates in T cell receptor (TCR)-mediated T cell activation (TCR-mediated T cell activation).

[0545] In some embodiments of the present invention, the T cell activation primary signal molecule is involved in converting TCR into active PTK (protein tyrosine kinase, PTK), which can phosphorylate a series of substrates to generate a large number of downstream signals. When these signals are properly integrated (together with signals from other co-receptors), they lead to T cell activation (Smith-Garvin JE., Koretzky GA., Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0546] In some embodiments of the present invention, the T cell activation primary signal molecule binds to CD3 and participates in the activation of T cells. CD3 and TCR form a TCR / CD3 complex in T cells, participating in the activation of helper T cells (CD4 + T cells) and cytotoxic T cells (CD8 + T cell) activation.

[0547] "T cell activation secondary signal molecule (Secondary Signal)" is also called co-stimulatory signal molecule (Co-Stimulatory Signal), which binds to other T cell surface receptors to provide additional signals necessary for avoiding anergy and effective T cell activation (Smith-Garvin JE., Koretzky GA., Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0548] Although other cell surface receptors (co-stimulatory receptors) can also enhance activation signals through TCR, CD28-mediated costimulation is stronger than other co-stimulatory receptors (Smith-Garvin JE., Koretzky GA., Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0549] Unlike CD28, which is stably expressed on both non-activated and activated T cells, ICOS is inducibly expressed on activated T cells (Hutloff A, et al. ICOS is an inducible T-cell costimulator structurally and functionally related to CD28. Nature 1999; 397: 263-6. [PubMed: 9930702]) (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619). Lack of ICOS results in impaired immune responses similar to, but less severe than, the CD28 knockout model, suggesting that the two molecules may function in similar pathways (Coyle AJ., et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity 2000; 13: 95-105. [PubMed: 10933398])(Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0550] Co-stimulatory receptors outside the CD28 family, TNFR family members OX40 (CD134) and 4-1BB (CD137) provide co-stimulatory signals by binding to their ligands OX40L and 4-1BBL (Smith-Garvin JE, Koretzky GA, Jordan MS. T cell activation. Annu Rev Immunol. 2009; 27: 591-619).

[0551] "Flexible linkers": Flexible linkers are usually used when the connected domains need to move or interact to a certain extent (Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69.). Flexible linkers are usually composed of small, non-polar (such as Gly) or polar (such as Ser or Thr) amino acids (Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990; 211: 943–958.). These small amino acids provide flexibility while also allowing the movement of the connected functional domains. Commonly used flexible linker peptides are described in Chen X, Zaro JL, Shen WC., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct; 65(10): 1357-69, which is incorporated herein by reference in its entirety. In some embodiments of the present invention, the flexible linker peptide includes but is not limited to (G4S) n Connecting peptide, the connecting peptide 1 and the connecting peptide 3.

[0552] "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.

[0553] 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.

[0554] In the context of antibodies, the term "variable region" or "variable domain" refers to the domain of the antibody heavy chain or light chain that participates in the binding of the antibody to the antigen. The variable regions of the heavy and light chains of natural antibodies (VH and VL regions, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL region may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can use isolated VH or VL regions from antibodies that bind to that specific antigen to screen for libraries of complementary VL or VH regions, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0555] Synonymous terms with "hypervariable region" or "HVR": "Complementarity determining region" and "CDR" are known in the art to refer to non-contiguous sequences of amino acids within an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.

[0556] In some embodiments of the present invention, the CDR regions are identified according to the IMGT numbering scheme, the Kabat numbering scheme, the Martin numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, or the Contact numbering scheme.

[0557] In some embodiments of the present invention, the CDR regions of the scFv are determined according to the Kabat numbering scheme.

[0558] The term "antibody" herein also includes single-chain variable region fragments (scFv).

[0559] 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.

[0560] The terms "VHH domain" and "single domain antibody" (sdAb) have the same meaning and are used interchangeably herein. They refer to the construction of a single domain antibody (sdAb) consisting solely of a single heavy chain variable region by cloning the variable region of a heavy chain antibody. This is the smallest fully functional antigen-binding fragment. Typically, a heavy chain antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting solely of a single heavy chain variable region.

[0561] "Antibody" herein also includes monoclonal antibodies or antigen-binding portions thereof. Monoclonal antibodies or antigen-binding portions thereof can be non-human, chimeric, humanized or human, preferably humanized or human. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

[0562] The "antibodies" herein can be derived from any animal, including but not limited to humans and non-human animals, which can be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, monkeys (such as cynomolgus monkeys and rhesus monkeys), alpacas, sheep, rabbits, mice, rats or cartilaginous fish (such as sharks).

[0563] "Antibody" herein also includes the heavy chain variable region (VH) or light chain variable region (VL) of the antibody.

[0564] 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.

[0565] In some embodiments of the present invention, there is no particular limitation on the order in which the scFv comprises the VH region or the VL region from the N-terminus to the C-terminus, such as VH-Linker-VL or VL-Linker-VH from the N-terminus to the C-terminus; the connecting peptide can be selected from a flexible connecting peptide.

[0566] "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.

[0567] "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. Exemplarily, "receptor binding fragment" herein includes, but is not limited to, the extracellular domain or variable region of the ligand.

[0568] "Endocytosis" refers to the process by which a substance enters a cell. During endocytosis, the substance to be taken in is surrounded by a region of the plasma membrane, which then buds into the cell to form a vesicle containing the taken-in substance. Endocytosis can be divided into four categories: receptor-mediated endocytosis (also known as clathrin-mediated endocytosis), caveolae, pinocytosis, and phagocytosis (Marsh M, Endocytosis. Oxford University Press. p. vii., 2001). "Endocytic receptor" refers to a receptor that can trigger endocytosis after binding to its ligand or its binding fragment and / or antibody or its antigen-binding fragment; endocytic receptors can be markers for cells such as T cells and other lymphocytes.

[0569] "Chimeric Antigen Receptor": Chimeric Antigen Receptor (CAR) refers to an artificial cell surface receptor that has been modified to be expressed on immune effector cells such as lymphocytes and specifically binds to antigens, which at least comprises (1) an extracellular antigen binding region, such as scFv or VHH; (2) a transmembrane region that anchors the CAR molecule into the immune effector cell, and (3) an intracellular signaling domain; the extracellular structure of the CAR may further comprise a hinge region, and the intracellular structure may further comprise one or more costimulatory molecules to form a costimulatory signaling domain. The CAR molecule can use the extracellular antigen binding region to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC restricted manner.

[0570] "Fusion / Chimera": The term "fusion / chimera" refers to any nucleic acid molecule or protein / polypeptide molecule that is non-endogenous and comprises a combination of sequences joined or linked together that are not naturally joined or linked together in nature. For example, a fusion / chimera nucleic acid molecule may comprise nucleic acids encoding various domains from multiple different genes. As another example, a fusion / chimera nucleic acid molecule may comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that found in nature.

[0571] "Antigen": The terms "antigen" and "Ag" refer to a molecule capable of inducing an immune response. The induced immune response may include the production of antibodies and / or the activation of specific immune competent cells. Macromolecules including proteins, glycoproteins and glycolipids can be used as antigens. Antigens can be derived from recombinant or genomic DNA. As contemplated herein, an antigen need not be (i) encoded solely by the full-length nucleotide sequence of a gene or (ii) fully encoded by a gene. Antigens can be generated or synthesized, or the antigen can be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or biological fluids.

[0572] "CD19": CD19 is the most widely used target in CAR-T therapy and has been proven to be effective and safe in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), and B-cell lymphoma. CD19 is widely and specifically expressed throughout the development of B cells until terminal differentiation into plasma cells. Therefore, CD19 has perfect coverage for B-cell malignancies, which has led to a very high complete remission rate (CRR) in CAR-T therapy (Wei, J., Han, X., Bo, J. et al. Target selection for CAR-T therapy. J Hematol Oncol 12, 62 (2019)).

[0573] "CD33": CD33 is a sialoadhesive protein composed of a membrane-proximal immunoglobulin variable region (IgV) and a membrane-proximal immunoglobulin constant region (IgC) domain. It has become a viable target due to its almost universal expression on acute myeloid leukemia (AML) cells. Notably, CD33 is also present on precursor / mature myeloid cells and hematopoietic stem cells (HSCs) but is not essential for the development and function of human myeloid cells. This provides the possibility of adopting strategies to eliminate all CD33-positive malignant and normal hematopoietic cell populations, followed by hematopoietic restoration by using engineered CD33-negative HSCs (Freeman R, Shahid S, Khan AG, et al. Developing a membrane-proximal CD33-targeting CAR T cell. J Immunother Cancer. 2024 May 20; 12(5): e009013.).

[0574] "BCMA": B-cell maturation antigen (BCMA) is selectively expressed on normal and malignant plasma cells and has become a target for multiple immunotherapy modalities. Chimeric antigen receptor (CAR) T cells are a breakthrough in cancer immunotherapy, revolutionizing the treatment of B-cell malignancies and significantly improving the prognosis of relapsed / refractory (RR) multiple myeloma (MM). BCMA-targeted CAR-T cell therapy is the most established CAR-T cell therapy for MM, and the US Food and Drug Administration has approved idecabtagene vicleucel (Ide-cel) and ciltacabtagene autoleucel (Cilta-cel) for MM. MM remains an incurable hematologic malignancy despite the development of numerous innovative therapies over the past two decades. Immunotherapy is transforming the treatment paradigm for MM and has improved overall responses and survival in patients with RRMM. (Yang J, Zhou W, Li D, Niu T, Wang W. BCMA-targeting chimeric antigen receptor T-cell therapy for multiple myeloma. Cancer Lett. 2023Jan28;553:215949.)

[0575] Claudin18.2, also known as CLDN18.2, is highly expressed in various malignancies, particularly gastrointestinal cancers, and is emerging as a new target for cancer treatment. Satricabtagene autoleucel (satri-cel) / CT041 is an autologous chimeric antigen receptor (CAR) T cell targeting CLDN18.2. Interim results from the CT041-CG4006 trial were reported in June 2022. Satri-cel has shown therapeutic potential and a manageable safety profile in patients with CLDN18.2-positive advanced gastrointestinal cancers. (Qi, C., Liu, C., Gong, J. et al. Claudin18.2-specific CAR T cells in gastrointestinal cancers: phase 1 trial final results. Nat Med 30, 2224–2234 (2024).)

[0576] "Reduction": When referring to the "reduction" of (a) the ability of the viral envelope glycoprotein to bind to the receptor of the viral glycoprotein (binding capacity) and / or (b) the ability of the LVV or RVV to off-target transduce cancer cells (transduction capacity), the term "reduction" includes complete elimination of the binding and transduction capacities, as well as significant reduction of the binding and transduction capacities. In specific embodiments, "significant reduction" refers to a reduction of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, at least 4%, at least 3%, at least 2%, and at least 1% relative to (a) wild-type viral glycoprotein and / or (b) LVV or RVV packaged by a packaging system not provided by the present invention.

[0577] "Nucleic acid" refers to any compound and / or substance including a polymer containing nucleotides, such as a polynucleotide. As used herein, "nucleic acid," "polynucleotide," and "gene" are used synonymously. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. As used herein, the term "nucleic acid" encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. "Nucleic acid" can be linear or circular. In addition, "nucleic acid" includes both a sense strand (coding strand) and an antisense strand (template strand), as well as single-stranded and double-stranded forms. Furthermore, the "nucleic acids" described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include nucleotide bases modified with derivatized sugars, phosphate backbone linkages, or chemically modified residues.

[0578] "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.

[0579] "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).

[0580] "Shuttle gene" / "transgene": i.e. Transgene, as used herein, the term "shuttle gene" refers to a gene or polynucleotide (GOI) encoding a protein of interest (e.g., an exogenous polynucleotide of any of the aforementioned CARs or POIs), the expression of which is desired in host cells / target cells and has been transferred to cells by genetic engineering technology. Transgenes can encode therapeutically significant proteins as well as proteins that serve as reporters, tags, markers, suicide proteins, and the like. Transgenes can be derived from natural sources, modifications of natural genes, or recombinant or synthetic molecules. In certain embodiments, the transgene is a component of LVV or RVV.

[0581] "Expression cassette": As used herein, the term "expression cassette" refers to a unique component of a vector nucleic acid comprising at least one transgene and regulatory sequences (e.g., promoters, 3'UTR) that control its expression in a host cell / target cell. A tandem expression cassette refers to a component of a nucleic acid vector comprising at least two transgenes, which are under the control of a set of identical regulatory sequences for expressing the at least two transgenes in tandem. In certain embodiments, the tandem expression cassette comprises at least two transgenes under the control of the same promoter. In certain embodiments, the first transgene and the second transgene are separated by an internal ribosome entry site (IRES), a furin cleavage site, or a self-cleaving viral 2A peptide to allow co-expression of two proteins by a single mRNA. In certain embodiments, the tandem expression cassette comprises at least two transgenes under the control of different promoters.

[0582] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds.

[0583] "Encoding": refers to the inherent property of a specific polynucleotide sequence (such as DNA, cDNA and mRNA sequences) used as a template for synthesizing other polymers and macromolecules in biological processes, wherein the template has a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. Therefore, if the transcription and translation of the mRNA corresponding to the polynucleotide produces a protein in a cell or other biological system, the polynucleotide encodes the protein. Both the coding strand and the non-coding strand can be referred to as encoding proteins or other products of the polynucleotide. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.

[0584] "Self-cleaving peptide" or "self-cleaving peptide" or "2A peptide": refers to a self-cleaving peptide that is configured to generate two or more proteins from a single open reading frame, including FT2A peptide, F2A peptide, E2A peptide, T2A peptide and P2A peptide, etc. 2A peptides are 18 to 25 residues long viral oligopeptides that mediate the "cleavage" of polypeptides during translation in eukaryotic cells. "2A peptide" can refer to peptides with different amino acid sequences. In the present disclosure, it should be understood that when a viral vector contains two or more 2A peptides, the 2A peptides can be the same or different from each other. Detailed methods for designing and using 2A peptides are provided by Szymczak-Workman et al. (2012) Cold Spring Harb. Protoc. 2012: 199-204.

[0585] "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).

[0586] "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 of viruses during their life cycle as they navigate within host cells. Not all viruses have a viral envelope. Many human pathogenic viruses are encapsulated in a lipid bilayer, infecting target cells by fusing the viral envelope with the cell membrane. Viruses with viral envelopes include retroviruses and lentiviruses.

[0587] "Retrovirus": Retrovirus. Retrovirus is a double-stranded RNA enveloped virus whose main characteristic is the ability to "reverse transcribe" its genome from RNA to DNA. The virus particle diameter is measured at 100-120nm and contains a dimeric genome of the same positive RNA chain complexed with the nucleocapsid protein. The genome is encapsulated in a protein capsid that also contains enzyme proteins, namely reverse transcriptase, integrase and protease required for viral infection. The matrix protein forms a layer outside the capsid core, which interacts with the envelope derived from the lipid bilayer of the host cell membrane surrounding the viral core particle. Anchored on this bilayer are viral envelope glycoproteins that are responsible for recognizing specific receptors on the host cell and initiating the infection process. The envelope protein is formed by two subunits, the transmembrane (TM) that anchors the protein to the lipid membrane, and the surface (SU) that binds to the cell receptor.

[0588] Retroviruses contain reverse transcriptase and integrase. After entering the target cell, the retrovirus uses its reverse transcriptase to transcribe its RNA molecule into a DNA molecule. Subsequently, the integrase is used to integrate the DNA molecule into the host cell genome. After integration into the host cell genome, the sequence from the retrovirus is called a provirus (e.g., a proviral sequence or a proviral sequence).

[0589] Based on their genome structure, retroviruses are classified as either simple retroviruses, such as MLV and murine leukemia virus, or complex retroviruses, such as HIV and EIAV. Retroviruses encode four genes: gag (group-specific antigen), pro (protease), pol (polymerase), and env (envelope). The gag sequence encodes the three major structural proteins: matrix protein, nucleocapsid protein, and capsid protein. The pro sequence encodes the protease responsible for cleaving Gag and Gag-Pol during viral particle assembly, budding, and maturation. The pol sequence encodes the enzymes reverse transcriptase, which catalyzes the reverse transcription of the viral genome from RNA to DNA during infection, and integrase, which is responsible for integrating the proviral DNA into the host cell genome. The env sequence encodes the SU and TM subunits of the envelope glycoprotein. In addition, the retroviral genome presents non-coding cis-acting sequences, such as: two LTRs (long terminal repeats), which contain elements required to drive gene expression, reverse transcription, and integration into host cell chromosomes; a sequence called the packaging signal (ψ) required for specific packaging of viral RNA into newly formed virions; and a polypurine tract (PPT) that serves as a site for initiating positive-strand DNA synthesis during reverse transcription. In addition to gag, pro, pol, and env, complex retroviruses such as lentiviruses have accessory genes including vif, vpr, vpu, nef, tat, and rev, which regulate viral gene expression, assembly of infectious particles, and control viral replication in infected cells.

[0590] "Lentivirus": Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. The greater complexity allows the virus to regulate its life cycle, as it does during latent infection. Lentiviruses belong to a genus of retroviruses that can infect both dividing and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV types I and II), equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0591] “Lentiviral Vector (LVV) and Retroviral Vector (RVV)”: As used herein, the term “lentiviral vector (LVV) or retroviral vector (RVV)” is intended to mean a self-inactivating viral particle that includes a viral envelope, has one or more characteristics of a lentivirus or a retrovirus, is capable of invading target cells and delivering a gene of interest, and does not have the ability to replicate itself.

[0592] LVV and RVV can stably integrate exogenous cargo genes, such as CAR genes, into the chromosomes of target cells, allowing them to express the delivered transgenes 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 relatively large cloning capacities, sufficient for most anticipated clinical applications.

[0593] "Packaging system" as used herein refers to a vector system comprising one or more nucleic acid vectors that, when introduced into a packaging cell / packaging cell line for packaging a lentiviral vector or a retroviral vector, contain the nucleic acids necessary for the production, assembly and / or packaging of the lentiviral vector or retroviral vector in the packaging cell / packaging cell line.

[0594] In some embodiments of the present invention, the packaging system comprises (a) nucleic acids encoding viral proteins necessary for the production, assembly and / or packaging of lentiviral or retroviral vectors in a packaging cell line and (b) signals required for key functions such as viral replication, packaging, reverse transcription and integration.

[0595] Commonly used lentiviral vector packaging systems include so-called third-generation lentiviral vector packaging systems. Third-generation lentiviral vector packaging systems typically include four plasmids, typically a transfer plasmid and three packaging plasmids: a transfer plasmid / master plasmid encoding a gene of interest (GOI), such as a CAR gene, a transgenic / shuttle gene, a GagPol plasmid, a Rev plasmid, and an envelope plasmid (containing viral glycoprotein genes such as VSV-G or its variants or Cocal-G or its variants).

[0596] Generally speaking, the "shuttle / transfer plasmid" (Transfer Vector) contains the lentiviral backbone gene, transgene and signals required for key functions such as viral replication, packaging, reverse transcription and integration. The transfer plasmid usually has one or more transgenes flanked by long terminal repeat (LTRs) sequences, which helps to integrate the transgene contained in the transfer plasmid, such as the CAR gene, into the host genome. LTRs are responsible for the reverse transcription and integration process of the viral genome. Through these sequences, the lentivirus can integrate the transgene into the genome of the host cell. For safety reasons, the transfer plasmid is usually designed to make the resulting viral vector unable to replicate itself, for example, the transfer plasmid lacks the genetic elements necessary to produce an infectious lentiviral vector in the host cell. In addition, the transfer plasmid can be designed to lack the 3'LTR, thereby making the virus "self-inactivating". Compared with the traditional second-generation pseudotype lentiviral vector packaging system (usually a single packaging plasmid containing nucleic acids encoding Gag, Pol, Rev and Tat and a separate envelope plasmid), the TAT gene is eliminated from the third-generation pseudotype lentiviral vector packaging system by adding a chimeric 5'LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) to the transfer plasmid. The transfer plasmid usually contains a Ψ sequence (Psi sequence, also known as Ψ packaging signal) located downstream of the 5'LTR. The Ψ sequence is responsible for packaging the transgenic RNA into the viral vector. The Ψ sequence ensures that only RNA containing the transgene is packaged into the viral vector. The transfer plasmid may also optionally contain an internal ribosome entry site (IRENS). In some embodiments, the present invention provides an intracellular recombinant protein (IRES) site ("IRES") to allow simultaneous translation of two or more open reading frames (ORFs) on one mRNA, thereby achieving multi-gene expression. Some transfer plasmids, such as the master plasmid / transfer plasmid used in some embodiments of the present invention, may also contain a selection marker gene, such as an antibiotic resistance gene (e.g., PuroR, encoding puromycin resistance) or a fluorescent protein gene (e.g., GFP), for screening or tracking transduced cells.

[0597] Transfer plasmids for packaging systems of lentiviral vectors are known in the art, see Naldini et al. (1996) Science 272: 263-7; Zufferey et al. (1998) J. Virol. 72: 9873-9880; Dull et al. (1998) J. Virol. 72: 8463-8471; U.S. Pat. No. 6,013,516; and U.S. Pat. No. 5,994,136, each of which is incorporated herein by reference in its entirety. Generally speaking, a transfer plasmid comprises a basic nucleic acid sequence configured to carry cells for selecting vector-containing cells, for incorporating foreign nucleic acids into lentiviral particles, and for transferring nucleic acids into target cells.

[0598] The lentiviral backbone gene generally refers to the most basic and essential cis-acting elements that make up the lentiviral vector transfer plasmid. These sequences do not encode viral proteins, but rather provide the signals required for key functions such as viral replication, packaging, reverse transcription, and integration. Specifically, the lentiviral backbone gene typically includes: long terminal repeats (LTRs): located at both ends of the genome, containing promoter, enhancer, and terminator functions, regulating viral gene transcription and integration; packaging signal (Ψ): determines which RNA molecules can be recognized and packaged into viral particles; central polypurine tract (cPPT) and central termination signal (CTS): help improve reverse transcription efficiency and nuclear transport; Rev response element (RRE): binds to the Rev protein and regulates the transport of viral RNA from the cell nucleus to the cytoplasm.

[0599] When constructing a transfer plasmid, to ensure safety and efficiency, only the necessary backbone sequences are typically retained in the transfer plasmid, while sequences encoding viral structural proteins and enzymes (such as gag, pol, and env) are removed and provided by the packaging system in a helper plasmid. This design not only ensures vector functionality but also reduces the risk of generating replicative viruses.

[0600] In some embodiments of the present invention, "backbone gene" is intended to include nucleic acids encoding slow virus or retroviral cis nucleic acid sequences required for genome packaging. Backbone gene can also encode other cis nucleic acid sequences that are beneficial to gene delivery, including, for example, cis sequences required for reverse transcription, proviral integration or genomic transcription. Therefore, the exact composition of the backbone gene will depend on the genetic material that is desired to be imported into the target cell. Therefore, the backbone gene can encode, for example, other polypeptides or functions other than those required for packaging, reverse transcription, integration or transcription. Such functions generally include cis elements required for encoding expression of target nucleic acid / shuttle gene.

[0601] Transfer plasmids for lentiviral and retroviral vectors are known in the art, see Naldini, et al., (1996) Science 272:263-7; Zufferey et al., (1998) J. Virol. 72:9873-9880; Dull et al., (1998) J. Virol. 72:8463-8471, U.S. Pat. No. 6,013,516, and U.S. Pat. No. 5,994,136, each of which is incorporated herein by reference in its entirety.

[0602] For a comparison and discussion of the packaging systems of lentiviral vectors and retroviral vectors and the transfer plasmids they contain, see: Stripecke, R., Kasahara, N. (2007). Lentiviral and Retroviral Vector Systems. In: Hunt, KK, Vorburger, SA, Swisher, SG (eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

[0603] The packaging system of third-generation lentiviral vectors typically includes three packaging plasmids: the GagPol plasmid, the Rev plasmid, and the envelope plasmid. The envelope plasmid typically carries the gene for a viral envelope glycoprotein, such as wild-type VSV-G or Cocal-G, which are commonly used viral glycoproteins. The viral glycoprotein gene is operably linked to a promoter, typically the CMV promoter, to initiate transcription of the viral glycoprotein gene. In some embodiments of the present invention, the envelope plasmid comprises a polynucleotide encoding any of the aforementioned targeting molecules and / or activation signaling molecules displayed on the surface of LVV or RVV.

[0604] The packaging system of third-generation lentiviral vectors also includes two packaging plasmids: one containing genes encoding Gag and Pol proteins (GagPol packaging plasmid), and the other containing the gene encoding Rev protein (Rev plasmid) as a further safety feature, an improvement over the single packaging plasmid of so-called second-generation packaging systems. The Gag gene encodes the Gag polyprotein precursor, which contains the lentiviral structural proteins, including the matrix, capsid, and nucleocapsid; the Pol gene encodes the Pol polyprotein precursor, which provides the lentiviral enzyme functions necessary for replication, including protease, reverse transcriptase, and integrase; and the Rev gene encodes the Rev protein, which binds to the Rev response element (RRE) to allow nuclear export of unspliced ​​and singly spliced ​​HIV RNA during viral replication. The Gag and Pol polyprotein precursors are cleaved during viral vector production. The Rev protein binds to the Rev response element (RRE) sequence on the viral RNA and, by interacting with the host cell's nuclear export machinery, facilitates the transport of incompletely spliced ​​viral RNA from the nucleus to the cytoplasm. In the cytoplasm, this unspliced ​​RNA can be translated into viral structural proteins and enzymes or assembled into new viral vectors.

[0605] Exemplarily, the packaging plasmid includes but is not limited to pMD2.G, pRSV-rev, pMDLG-pRRE and pRRL-GOI.

[0606] Compared to the LVV packaging system, the RVV packaging system typically does not include a Rev plasmid. This is because the genomic RNA derived from retroviruses such as Moloney Murine Leukemia Virus (MMLV) is naturally transported from the nucleus to the cytoplasm for translation and assembly, thus eliminating the need for specialized nuclear export mechanisms such as the Rev protein. The RVV packaging system typically comprises a transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transgene sequence contained in the transfer plasmid is flanked by long terminal repeats (LTRs), which facilitate integration of the transfer plasmid sequence into the host genome. Typically, during viral transduction, sequences between and including the LTRs are integrated into the host genome. The genome of MMLV or murine stem cell virus (MSCV), including its respective LTRs, is typically used to construct the transfer plasmid in the RVV packaging system. The GagPol packaging plasmid contains the Gag gene and the Pol gene; the envelope plasmid generally contains a polynucleotide encoding a viral glycoprotein, such as VSV-G or Cocal-G. In some embodiments of the present invention, the envelope plasmid may further contain a polynucleotide encoding any of the aforementioned targeting molecules and / or activation molecules.

[0607] In some embodiments of the present invention, the packaging system further comprises a binding nucleic acid vector comprising a polynucleotide encoding any one of the CAR binding molecules provided by the present invention.

[0608] In some embodiments of the present invention, the packaging system further comprises at least one binding plasmid, wherein the binding plasmid comprises a polynucleotide encoding any one of the CAR binding molecules provided by the present invention.

[0609] In some embodiments, the production cells are transfected with a defined ratio of transfer plasmid, GagPol plasmid, envelope plasmid, Rev plasmid, and binding plasmid. In some embodiments, the ratio of each plasmid is determined by mass, and there is no particular limitation as long as it can package a non-integrating lentiviral vector or retroviral vector with biological activity. In some embodiments, the mass of each of the transfer plasmid and GagPol plasmid that package the lentiviral vector is higher than the mass of each of the envelope plasmid, Rev plasmid, and binding plasmid. In some embodiments, the defined ratio of transfer plasmid, GagPol plasmid, Rev plasmid, envelope plasmid, and binding plasmid is about 1:1:1:1:0.1 to about 10:5:4:4:5. In some embodiments, the defined ratio of transfer plasmid, GagPol plasmid, envelope plasmid, Rev plasmid, and binding plasmid is about 9:4:2:2:0.1 to about 9:4:2:2:5. In some embodiments of the present invention, the envelope plasmid may contain a nucleic acid encoding the targeting molecule and / or activation signal molecule displayed on the surface of LVV or RVV.

[0610] In some embodiments, the envelope plasmid includes a tandem expression cassette encoding any of the aforementioned targeting molecules and / or activation signal molecules displayed on the surface of LVV or RVV, such as T cell activation primary signal molecules and / or T cell activation secondary signals / co-stimulatory molecules. In certain embodiments, the tandem expression cassette contained in the envelope plasmid includes a polynucleotide encoding a first signal peptide, a polynucleotide encoding the targeting molecule and / or activation molecule, a polynucleotide encoding an internal ribosome entry site (IRES), a furin cleavage site or a viral 2A peptide, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding any of the aforementioned viral glycoproteins or their variants. In certain embodiments, the polynucleotide encoding any of the aforementioned viral glycoproteins or their variants is located at the 5' end of the polynucleotide encoding the targeting molecule and / or activation molecule. In other embodiments, the polynucleotide encoding any of the aforementioned viral glycoproteins or their variants is located at the 3' end of the polynucleotide encoding the targeting molecule and / or activation molecule. The polynucleotide encoding the targeting molecule and / or activating molecule and the polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof are separated in a tandem box by a polynucleotide encoding an IRES, a furin cleavage site or a viral 2A peptide, which allows the co-expression of the two proteins by a single mRNA. In certain embodiments, the viral 2A peptide is porcine Teschovirus-1 (P2A), Thosea asigna virus (T2A), Equine rhinovirus (E2A), foot-and-mouth disease virus (F2A) or variants thereof. In certain embodiments, the viral 2A peptide includes derivatives thereof, such as derivatives of the T2A peptide, a T2A cleavage site with a furin cleavage site and a GSG linker, and an FT2A peptide. In certain embodiments, at least two different promoters independently drive the expression of the polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof and the polynucleotide encoding the targeting molecule and / or activating molecule.

[0611] "Packaging cell line": The use of a packaging system for packaging lentiviral / retroviral vectors relies on the terms "packaging cells" and "packaging cell line." Generally speaking, a packaging cell line is a cell line that, when a packaging system, for example, including a transfer plasmid and one or more packaging plasmids, is introduced into the cells, capable of producing a non-replication-competent lentiviral or retroviral vector capable of infecting / transducing target cells. An overview of available packaging lines is provided in J.M. Coffin, S.M. Hughes, et al., Cold Spring Harbor Laboratory Press, 1997, p. 447, which is incorporated herein by reference in its entirety.

[0612] Exemplarily, various plasmids can be introduced into the packaging cell line using transfection methods including chemical-mediated transfection methods, physical-mediated transfection methods or biological-mediated transfection methods. For example, chemical-mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-dextran or PEI (Polyethylenimine, polyethyleneimine transfection reagent), and physical-mediated transfection methods include transfection methods such as electroporation.

[0613] The production / host / packaging cells that can be used to prepare the LVV or RVV provided by the present invention include but are not limited to human embryonic kidney (HEK) 293 cells and their derivatives. The production cells can be adherent cell lines such as HEK-293T production cells, or suspension cell lines such as HEK-293T / 17SF production cells.

[0614] Exemplarily, the packaging cell / production cell is selected from CHO cells, BHK cells, MDCK cells, C3H-10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells and 211 cells;

[0615] Preferably, the packaging cells / producer cells are HEK-293T cells.

[0616] The packaging cells can be genetically engineered to improve the immune properties of the LVV and RVV provided by the present invention and / or increase the efficiency of LVV and RVV transduction of target cells in other ways; such other ways include but are not limited to adding genes, deleting genes, and introducing point mutations into genes.

[0617] "Promoter": As used herein, the term "promoter" is defined as a DNA sequence that is recognized by the cellular synthetic machinery or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence. As used herein, the term "promoter / regulatory sequence" means a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

[0618] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0619] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0620] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is of the tissue type corresponding to the promoter.

[0621] "Envelope (viral) 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 the host cell, pathogenicity, downregulation of the expression of host surface proteins, and increase in viral packaging and budding.

[0622] "VSV-G and Cocal-G": The envelope glycoproteins of Vesicular Stomatitis Virus strains, such as the envelope glycoprotein (VSV-G) of the Indiana strain and the envelope glycoprotein (Cocal-G) of the Cocal strain, can bind to the low-density lipoprotein receptor (LDL-R) that is widely present on the surface of various cells and have a wide range of infectivity.

[0623] In some embodiments of the present invention, the extracellular domain of VSV-G comprises the amino acid sequence shown in SEQ ID NO: 1; and the extracellular domain of Cocal-G comprises the amino acid sequence shown in SEQ ID NO: 2.

[0624] In some embodiments of the present invention, the amino acid sequence of the full-length protein of the wild-type VSV-G excluding its signal peptide is shown in SEQ ID NO: 109.

[0625] Activated T cells express LDL-R; therefore, artificially synthesized, biosafe LVV or RVV usually uses wild-type VSV-G or Cocal-G to construct its envelope glycoprotein (VSV-G type LVV or RVV) to transduce activated T cells.

[0626] In some embodiments of the present invention:

[0627] The full-length protein of wild-type VSV-G (including its signal peptide, which is underlined):

[0628] The full-length protein of wild-type Cocal-G (including its signal peptide, underlined):

[0629] "Complement": The complement system is composed of a series of proteins and is part of the innate immune system. Complement (complement, C) exists in the serum, tissue fluid and cell membrane surface of normal humans and animals. After activation, it has enzymatic activity and can undergo complex cascade reactions. The complement system is activated by a series of enzymes (enzymes) cutting each other, and eventually forms a membrane attack complex similar to 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, conditioning, phagocytosis and mediating inflammatory reactions, showing 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.

[0630] Regulatory complement components exist in soluble or membrane-bound forms, including properdin (P factor), C1 inhibitor (C1INH), factor I, factor H, C4 binding protein (C4BP), S protein, SP40 / 40, membrane cofactor protein (MCP), decay accelerating factor (DAF), homologous restriction factor (HRF), and membrane inhibitor of reactive lysis (MIRL).

[0631] After entering the serum, VSV-G type LVV or RVV may be recognized and inactivated by complement, making it difficult to efficiently reach the target cells and exert its effect; therefore, when used to prepare engineered T cells such as CAR-T in the patient's body or blood, the efficiency of VSV-G type LVV or RVV in transducing T cells is low.

[0632] By causing VSV-G to undergo the second mutation, the ability of the mutated VSV-G to antagonize complement inactivation is improved, thereby making the mutated VSV-G type LVV or RVV more suitable for use in the in vivo preparation of engineered T cells such as CAR-T cells.

[0633] "Variant" refers to a mutant that has at least about 50% identity with the amino acid sequence of a non-mutant (wild type), and "at least about 50% identity" refers to 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, about 99.5% or about 99.9% identical to the amino acid sequence of the non-mutant (wild type).

[0634] In some embodiments of the present invention, variants include mutants comprising conservative substitutions relative to non-mutants." Conservative substitutions" are considered in the art to substitute one amino acid for another amino acid with similar properties. For example, conservative substitutions are well known in the art (see, for example, WO 97 / 09433, page 10, published on March 13, 1997; Lehninger, Biochemistry, 2nd edition; Worth Publishers, Inc. NY: NY (1975), pages 71-77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), page 8).

[0635] "Pharmaceutically acceptable excipient or carrier": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, preservatives, and / or adjuvants. Excipients are preferably nontoxic or substantially nontoxic to the recipient at the dosages and concentrations employed. Such excipients include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, pharmaceutical compositions may contain substances for improving, maintaining, or preserving, for example, the pH, osmotic properties, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition. The optimal pharmaceutical composition can be determined based on the intended route of administration, mode of delivery, and desired dosage.

[0636] Pharmaceutical compositions for in vivo administration are typically provided as sterile formulations. Sterilization is achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization, reconstitution, or dilution. The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, they can be prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposomal carriers, bioerodible microparticles or porous beads, and depot injection) are also known to those skilled in the art.

[0637] Once formulated, the pharmaceutical composition is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) before administration.

[0638] "Subject": As used herein, "subject," "patient," and "individual" are used synonymously and include, but are not limited to, mammals, such as humans or non-human mammals, such as domestic, agricultural, or wild animals, as well as birds and aquatic animals.

[0639] "Patient" is a subject who suffers from a disease, disorder or condition, is at risk of developing a disease, disorder or condition, or is otherwise in need of any of the LVVs or RVVs, engineered immune cells such as CAR-T cells, compositions or treatment methods provided herein.

[0640] A "disease" is a state of health in a subject in which the subject is unable to maintain homeostasis and in which the subject's health continues to deteriorate if the disease does not improve. Conversely, a "disorder" or "adverse condition" in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's health is less favorable than it would be in the absence of the disorder or adverse condition. Without treatment, the disorder or adverse condition does not necessarily result in a further deterioration in the subject's health. "Disease" includes, but is not limited to, cancer and autoimmune diseases.

[0641] In some embodiments of the present invention, disease-associated antigens include, but are not limited to, antigens that trigger, cause, or worsen a disease.

[0642] "Cancer": As used herein, the term "cancer" is defined as a disease characterized by the rapid, uncontrolled growth of abnormal cells. Abnormal cells may form solid tumors or constitute hematological malignancies. Cancer cells may spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, hematological cancers, such as B-lymphocyte malignancies and multiple myeloma, and solid cancers, such as breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, and lymphoma lung cancer.

[0643] "Treatment" refers to the use of a treatment method described herein to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor size, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth) in a subject. The treatment method that effectively treats a patient may vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the treatment to elicit an anti-cancer response in the subject.

[0644] As used herein, "treatment" includes any beneficial or desired effect associated with treatment."Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or its associated symptoms.

[0645] The therapeutically effective amount of any one of the LVV or RVV or pharmaceutical compositions provided herein will be used and will depend on, for example, the extent of treatment and the goal. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will vary depending in part on the molecule being delivered, the indication, the route of administration, and the patient's condition (body weight, body surface or organ size) and / or condition (age and general health). In certain embodiments, the clinician can titrate the dosage and change the route of administration to obtain the best therapeutic effect.

[0646] The frequency of administration will depend on the pharmacokinetic parameters of the LVV or RVV in the formulation used. The clinician typically administers the pharmaceutical composition until a dose is achieved that achieves the desired effect. The pharmaceutical composition can thus be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantable device or catheter.

[0647] “And / or”: should be understood to mean one or two alternatives.

[0648] "About" / "approximately": As used herein, the term "about" refers to the usual error range for the corresponding value that is readily known to those skilled in the art, including, by way of example, but not limited to, reference to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. Reference herein to "about" a value or parameter includes (and describes) embodiments for the value or parameter itself, e.g., a description referring to "about X" includes a description of "X."

[0649] "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.

[0650] "Embodiments": Reference throughout this specification to "some embodiments," "some embodiments," "embodiments," "specific embodiments," "related embodiments," "an embodiment," "another embodiment," or "other embodiments" or combinations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Therefore, the various appearances of the foregoing phrases throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0651] "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.

[0652] "Stable integration": also known as "stable transfection, refers to the integration of exogenous polynucleotides into the host cell genome after introduction into the host cell, and their long-term stable expression in the host cell (Stable Gene Expression); in contrast to transient transfection and transient expression (Transient Expression).

[0653] "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). When the interaction of two species produces a non-covalently bound complex, the binding that occurs is typically the result of electrostatic, hydrogen bonding, or lipophilic interactions. In various embodiments, the specific binding between one or more species is direct. In some embodiments of the invention, the affinity of the specific binding is about 2 times greater than background binding (non-specific binding), about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.

[0654] "Sequence identity": Generally speaking, "sequence identity" or "sequence homology" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity." Whether it is a nucleic acid or amino acid sequence, the percent identity of two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. For example, the advanced BLAST computer program available from the National Institutes of Health can also be used to compare sequence information to determine the percent identity. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and discussed in Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997). Briefly, the BLAST program defines identity as the number of aligned symbols (usually nucleotides or amino acids) that are identical divided by the total number of shorter symbols in the two sequences. The program can be used to determine percent identity over the entire length of the compared proteins.

[0655] "Signal peptide": Signal peptide, sometimes also called signal sequence, targeting signal, localization signal, localization sequence, transit peptide or leader signal peptide, is a short peptide (usually 16-30 amino acids long) (Kapp, Katja; Schrempf, Sabrina; Lemberg, Marius K.; Dobberstein, Bernhard (2013-01-01).), present at the N-terminus of most newly synthesized proteins that enter the secretory pathway (occasionally non-classically present at the C-terminus or internally) (Owji, et al., A comprehensive review of signal peptides: Structure, roles, and applications, European Journal of Cell Biology. 97(6): 422-441. (2018)) (Blobel G, Dobberstein B, et al., Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma, The Journal of Cell Biology, 67(3):835-51.(1975)).

[0656] The effect of signal peptide is to promote cell transfer protein, usually transferred to the cell membrane. In prokaryotes, the newly synthesized protein is guided to the SecYEG protein conduction channel present in the plasma membrane by signal peptide. There is a homologous system in eukaryotes, in which the newly synthesized protein is guided to the Sec6L channel by signal peptide, which has structural and sequence homology with SecYEG, but is present in the endoplasmic reticulum (Rapoport TA, Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes, Nature.450(7170):663-9(2007).). SecYEG and Sec6L channels are commonly referred to as transporters, and the transport by the channel is called translocation. When secreted protein passes through the channel, the transmembrane region may diffuse through the side gate in the translocon to be distributed in the surrounding membrane.

[0657] Some antigens naturally carry a leader signal peptide sequence. For example, the amino acid sequence at positions 1 to 19 of the amino acid sequence of the full-length protein of the human CD19 antigen is the amino acid sequence of the natural leader signal peptide of the CD19 antigen: MPPPRLLFFLLFLTPMEVR (SEQ ID NO: 68).

[0658] The amino acid sequence at positions 1 to 17 of the amino acid sequence of the full-length protein of the human CD33 antigen is the amino acid sequence of the natural leader signal peptide of the CD33 antigen: MPLLLLLPLLWAGALAM (SEQ ID NO: 69).

[0659] "Built-in retention signal peptide": (Endoplasmic reticulum) retention signals are recognized by specific receptors in the ER and cis-GA (Golgi Apparatus, "GA"), which retrieve proteins from post-ER compartments and return them to the ER via retrograde translocation in COPI-coated vesicles. Examples of such sequences in animals include the C-terminal sequence moti Lys-Asp-Glu-Leu (Lys(Lysine)-Asp(Aspartic Acid)-Glu(Glutamic Acid)-Leu(Leucine), KDEL), which is recognized by the KDEL receptor (KDELR) and present on soluble secretory proteins, and the Lys-Lys-XX sequence (KKXX, where X represents any amino acid), which interacts with the circulating COPI protein complex and is often present on the cytoplasmic side of transmembrane proteins. In principle, labeling a POI (Protein of Interest, POI, target protein) with an N-terminal signal sequence and a C-terminal retention signal allows the POI to be loaded and localized within the ER (Praznik, A., Fink, T., Franko, N., et al., Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage. Nat Commun 13, 1323 (2022)).

[0660] "MOI": Multiplicity of Infection (MOI) refers to the number of viral particles added to each cell during infection. For example, when one million viral particles are added to one million cells, MOI = 1.

[0661] "Operably": A polynucleotide is "operably linked" when it is in a functional relationship with another polynucleotide. For example, if the DNA for a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned so as to promote translation, the ribosome binding site is operably linked to a coding sequence. In general, "operably linked" means that the polynucleotides being linked are contiguous, and in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is achieved by ligation at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.

[0662] "Transduction": As used herein, the terms "transfection," "transformation," and "transduction" are used synonymously to refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell, packaging cell, or the like. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0663] Methods for introducing vectors such as viral particles or isolated polynucleotides into mammalian cells are known in the art. The vectors described can be transferred to immune effector cells by physical, chemical or biological methods.

[0664] Physical methods for introducing vectors or isolated polynucleotides into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, EF and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments of the present invention, vectors are introduced into cells by electroporation. In some embodiments of the present invention, vectors are introduced into cells by PEI transfection reagent.

[0665] Biological methods for introducing vectors or isolated polynucleotides into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.

[0666] Chemical methods for introducing vectors or isolated polynucleotides into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome.

[0667] Autoimmune diseases: Autoimmune diseases include a series of diseases that cause tissue or organ damage and inflammation and are characterized by immune tolerance disorders (Vivas AJ, Boumediene S, Tobón GJ. Predicting autoimmune diseases: A comprehensive review of classic biomarkers and advances in artificial intelligence. Autoimmun Rev. 2024 Sep; 23(9): 103611.).

[0668] All publications, documents, and patents mentioned herein are hereby incorporated by reference in their entirety. In the event of a conflict, the present application (including any definitions herein) will control. However, any reference, article, publication, patent, patent publication, and patent application cited herein is not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0669] 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

[0670] Figure 1 shows the flow cytometry results of packaging LVV in HEK-293T cells using packaging systems containing different amounts of CD19-binding plasmids in Example 1. Day 3 shows the expression of FMC-63 / CAR-19 and CD19 in each group of HEK-293T cells.

[0671] Figure 2 shows the flow cytometry results of FMC-63 / CAR-19 expression in Jurkat cells of each group on Day 2, after Jurkat cells were transduced with VG1A7-CAR19-0 / 0.5 / 1 / 2, respectively, in Example 2;

[0672] FIG3 is a flow cytometry result showing the expression of FMC-63 / CAR-19 on the surface of Jurkat cells of each group on Day 6 after Jurkat cells were transduced with VG1A7-CAR19-0 / 0.5 / 1 / 2 in Example 2;

[0673] FIG4 is a flow cytometry result showing the expression of CD19 on the surface of Nalm-6 cells in each group on Day 2 after Nalm-6 cells were transduced with VG1A7-CAR19-0 / 0.5 / 1 / 2 in Example 2;

[0674] Figure 5 shows the flow cytometry results of FMC-63 / CAR-19 and CD19 expression in each group of HEK-293T cells on Day 3, using the packaging system containing different amounts of CD19 binding plasmids to package the VG1A3X28-CAR19-0 / 0.1 / 0.5 / 1 / 2 / 5 in HEK-293T cells in Example 3;

[0675] Figure 6 shows the flow cytometry results of Jurkat cells transduced with VG1A3X28-CAR19-0 / 0.1 / 0.5 / 1 / 2 / 5 in Example 3, and the expression of FMC-63 / CAR-19 and CD19 in Jurkat cells of each group was detected on Day 5;

[0676] FIG7 is a flow cytometry result showing the expression of CD19 in Nalm-6 cells of each group on Day 5 after transduction of Nalm-6 cells with VG1A3X28-CAR19-0 / 0.1 / 0.5 / 1 / 2 / 5 in Example 3;

[0677] Figure 8 shows the flow cytometry results of packaging VG1A3X28-CAR19TSP-0 / 0.1 / 0.2 / 0.3 / 0.5 / 1 in HEK-293T cells using a packaging system containing different amounts of CD19 binding plasmid in Example 4. The expression of FMC-63 / CAR-19 and CD19 in each group of HEK-293T cells was detected on Day 3.

[0678] Figure 9 shows the flow cytometry results of Jurkat cells transduced with VG1A3X28-CAR19TSP-0 / 0.1 / 0.2 / 0.3 / 0.5 / 1 in Example 4, and the expression of FMC-63 / CAR-19 and CD19 in each group of Jurkat cells was detected on Day 5;

[0679] Figure 10 shows the flow cytometry results of transducing Nalm-6 cells with VG1A3X28-CAR19TSP-0 / 0.1 / 0.2 / 0.3 / 0.5 / 1, respectively, using a packaging system containing different amounts of CD19-binding plasmids in Example 4, and detecting the expression of CD19 in Nalm-6 cells in each group on Day 5;

[0680] FIG11 is a graph showing the flow cytometry results of packaging VG1A3X28-CAR33-0 / 0.1 / 1 / 5 in HEK-293T cells using a packaging system containing different amounts of CD33-binding plasmids in Example 5, and detecting the expression of CAR-33 molecules in each group of HEK-293T cells on Day 3;

[0681] FIG12 is a graph showing the flow cytometry results of CD33 expression in HEK-293T cells of each group on Day 3, using a packaging system containing different amounts of CD33-binding plasmids to package the VG1A3X28-CAR33-0 / 0.1 / 1 / 5 in Example 5;

[0682] FIG13 is a flow cytometry result showing the expression of CAR-33 molecules in each group of Jurkat cells detected on Day 5 after transducing each group of Jurkat cells with VG1A3X28-CAR33-0 / 0.1 / 1 / 5 in Example 5;

[0683] FIG14 is a flow cytometry result showing the expression of CD33 in each group of Nalm-6 cells overexpressing CD33 detected on Day 5 using the VG1A3X28-CAR33-0 / 0.1 / 1 / 5 transduction reagents in Example 5;

[0684] FIG15 shows the flow cytometry results of packaging VG1A3X28-CARBCMA-0 / 0.1 / 1 / 5 in HEK-293T cells using a packaging system containing different amounts of BCMA-binding plasmids in Example 6, and detecting the expression of CAR-BCMA molecules in each group of HEK-293T cells on Day 3;

[0685] FIG16 shows the flow cytometry results of packaging VG1A3X28-CARBCMA-0 / 0.1 / 1 / 5 in HEK-293T cells using a packaging system containing different amounts of BCMA-binding plasmids in Example 6, and detecting BCMA expression in each group of HEK-293T cells on Day 3;

[0686] FIG17 is a flow cytometry result showing the expression of CAR-BCMA molecules in Jurkat cells of each group detected on Day 5 after transducing Jurkat cells of each group with VG1A3X28-CARBCMA-0 / 0.1 / 1 / 5 in Example 6;

[0687] FIG18 is a flow cytometry result showing the expression of BCMA in each group of Nalm-6 cells overexpressing BCMA, detected on Day 5 using the VG1A3X28-CARBCMA-0 / 0.1 / 1 / 5 transduction reagents in Example 6;

[0688] Figure 19 shows the flow cytometry results of packaging VG1A3X28-CAR18.2-0 / 0.1 / 1 / 5 in HEK-293T cells using a packaging system containing different amounts of CLDN18.2 binding plasmid in Example 7. The expression of CAR-CLDN18.2 molecules in each group of HEK-293T cells was detected on Day 3.

[0689] FIG20 shows the flow cytometry results of packaging VG1A3X28-CAR18.2-0 / 0.1 / 1 / 5 in HEK-293T cells using a packaging system containing different amounts of CLDN18.2-binding plasmid in Example 7. The expression of CLDN18.2 in each group of HEK-293T cells was detected on Day 3.

[0690] FIG21 is a flow cytometry result showing the expression of CAR-CLDN18.2 molecules in each group of Jurkat cells detected on Day 5 after transduction of each group of Jurkat cells with VG1A3X28-CAR18.2-0 / 0.1 / 1 / 5 in Example 7;

[0691] FIG22 is a flow cytometry result showing the expression of CLDN18.2 in each group of Nalm-6 cells overexpressing CLDN18.2 detected on Day 5 using VG1A3X28-CAR18.2-0 / 0.1 / 1 / 5 in Example 7;

[0692] FIG23 shows the flow cytometry results of packaging LVV in HEK-293T cells using a packaging system containing or not containing the CD19 ECD + TM-R binding plasmid in Example 8, and detecting the expression of the FMC-63 / CAR-19 molecule capable of binding to CD19 on the cell membrane of HEK-293T cells on Day 3;

[0693] Figure 24 shows the flow cytometry results of Jurkat cells transduced with VG2A3X28-19RCAR19-0 / 1 packaged with or without the CD19ECD+TM-R binding plasmid packaging system in Example 8 to deliver and express the CAR-19 gene;

[0694] Figure 25 shows the flow cytometry results of the transduction efficiency of Nalm-6 cells by VG2A3X28-19RCAR19-0 / 1 packaged with or without the CD19ECD+TM-R binding plasmid packaging system in Example 8;

[0695] FIG26 shows the flow cytometry results of packaging LVV in HEK-293T cells using the packaging system without or with different amounts of the anti-FMC63-R binding plasmid in Example 9. The results show that on Day 3, the expression of the FMC-63 / CAR-19 molecule capable of binding to CD19 on the cell membrane of HEK-293T cells was detected.

[0696] Figure 27 is a flow cytometry result showing the efficiency of delivering and expressing the CAR-19 gene by transducing Jurkat cells with the LVV, VG2A3X28-A63RCAR19-0 / 1 / 2 / 5 packaged in HEK-293T cells using the packaging system containing no or different amounts of the anti-FMC63-R binding plasmid in Example 9;

[0697] Figure 28 is a flow cytometry result showing the efficiency of VG2A3X28-A63RCAR19-0 / 1 / 2 / 5 in transducing Nalm-6 cells in Example 9;

[0698] Figure 29 shows the flow cytometry results of the in vitro killing efficiency of CD19-CAR-T cells prepared by transducing healthy human non-activated PBMCs with VG2A3X28-CAR19-2 in Example 10 against Nalm-6 cells;

[0699] Figure 30: In vivo imaging images of Nalm-6 cell model mice injected with the VG2A3X28-CAR19-2 via tail vein in Example 10 on Days 1, 3, 7, 14 and 21. DETAILED DESCRIPTION

[0700] The following is a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments, so that the purpose, features and effects of the present invention are fully understood. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without inventive effort are all within the scope of protection of the present invention.

[0701] In the following examples, the experimental methods without specific conditions are based on conventional methods and conditions known in the art, or are selected according to the product specifications. Reagents and raw materials not specified in the present invention are all commercially available.

[0702] Example 1

[0703] The lentiviral vector was packaged using a five-plasmid lentiviral packaging system containing a conjugated plasmid containing a polynucleotide encoding a CAR binding molecule (human CD19).

[0704] 1. Preparation of 5-plasmid LVV Packaging System

[0705] The following five plasmids were prepared: an envelope plasmid carrying a polynucleotide encoding a mutant VSV-G1 and a polynucleotide encoding a membrane-expressed anti-CD7 antibody (envelope plasmid 1), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, a plasmid containing a polynucleotide encoding a CAFR binding molecule, i.e., human CD19 (CD19 binding plasmid), a main plasmid (shuttle plasmid / transfer plasmid) carrying a polynucleotide encoding a CAR-19 molecule (CAR-19 gene), and a main plasmid CAR19; the envelope plasmid 1, CD19 binding plasmid, and main plasmid CAR19 were synthesized by conventional molecular cloning methods.

[0706] A. Mutant VSV-G1

[0707] The extracellular domain of the mutant VSV-G1 comprises the amino acid sequence shown in SEQ ID NO: 8; relative to the extracellular domain of wild-type VSV-G (SEQ ID NO: 1), the extracellular domain of the mutant VSV-G1 (SEQ ID NO: 8) comprises a K47 deletion, T214N (relative to before K47 deletion), and T352A (relative to before K47 deletion); the ability of the mutant VSV-G1 to specifically bind to LDL-R is reduced or inhibited, and the ability to antagonize complement inactivation is enhanced;

[0708] The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence contained in the extracellular domain of the wild-type envelope glycoprotein VSV-G (wild-type VSV-G) of the Indiana strain of the vesicular stomatitis virus genus; the amino acid sequence of the full-length protein of the wild-type VSV-G including its signal peptide is shown in SEQ ID NO: 24.

[0709] B. Membrane-expressed anti-CD7 antibodies

[0710] Constructing a membrane-expressed anti-CD7 antibody, wherein the polynucleotide encoding the membrane-expressed anti-CD7 antibody sequentially encodes from the 5' end to the 3' end: a human CD8α signal peptide, a scFv specifically binding to CD7 (scFv derived from the monoclonal antibody TH-69, TH69-scFv), a human CD8α hinge region, and a human CD8α transmembrane region;

[0711] The heavy chain variable region (VH region) of the TH69-scFv is connected to the light chain variable region (VL region) of the TH69-scFv via the connecting peptide 2 ((G4S)3 connecting peptide);

[0712] (1) The amino acid sequence of the human CD8α signal peptide is shown in SEQ ID NO: 12;

[0713] (2) The amino acid sequence of the VH region of the TH69-scFv is shown in SEQ ID NO: 25;

[0714] (3) The amino acid sequence of the connecting peptide 2 is shown in SEQ ID NO: 20;

[0715] (4) The amino acid sequence of the VL region of the TH69-scFv is shown in SEQ ID NO: 26;

[0716] (5) The amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 87-89, respectively; the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 90-92, respectively; and the amino acid sequence of the TH69-scFv is shown in SEQ ID NO: 86.

[0717] (6) The amino acid sequence of the hinge region of human CD8α is shown in SEQ ID NO: 14;

[0718] (7) The amino acid sequence of the transmembrane region of human CD8α is shown in SEQ ID NO: 15.

[0719] C. Construction of CAR-19 molecules

[0720] A CAR molecule (CAR-19 molecule) that can specifically bind to human CD19 is constructed, wherein the polynucleotide encoding the CAR-19 molecule sequentially encodes from the 5' end to the 3' end: the human CD8α signal peptide, the anti-CD19 antigen binding region, the human CD8α hinge region, the human CD8α transmembrane region, the human 4-1BB co-stimulatory signaling domain, and the human CD3ζ intracellular signaling domain; the anti-CD19 antigen binding region is a scFv that can specifically bind to human CD19, and is derived from the scFv of the monoclonal antibody FMC-63 (FMC63-scFv);

[0721] (1) The amino acid sequence of the VH region of the FMC63-scFv is shown in SEQ ID NO: 18, and the amino acid sequence of the VL region of the FMC63-scFv is shown in SEQ ID NO: 19; the VH region and the VL region are connected by the connecting peptide 2; the amino acid sequences of the HCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 94-96, respectively, and the amino acid sequences of the LCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 97-99, respectively; the amino acid sequence of the FMC63-scFv is shown in SEQ ID NO: 93;

[0722] (2) the amino acid sequence of the human 4-1BB costimulatory signaling domain is shown in SEQ ID NO: 49;

[0723] (3) the amino acid sequence of the intracellular signaling domain of human CD3ζ is shown in SEQ ID NO: 50;

[0724] (4) The amino acid sequence of the CAR-19 is shown in SEQ ID NO: 110.

[0725] D. CD19 binding plasmid

[0726] A CD19 binding plasmid comprising a polynucleotide encoding a CAR binding molecule, human CD19, is constructed, wherein the amino acid sequence of human CD19 is shown in SEQ ID NO: 52; the CAR binding molecule can specifically bind to an anti-human CD19 antibody, FMC-63 or an antigen-binding fragment thereof.

[0727] Human CD19, UniProtKB No.: P15391; CD19 is an effective target for the treatment of B-cell malignancies, especially B-cell lymphomas and acute lymphoblastic leukemia.

[0728] 2. Packaging LVV

[0729] A. mixing the five plasmids, transfecting the five plasmids into packaging cells HEK-293T cells using PEI reagent to package LVV;

[0730] The specific steps are:

[0731] (1) Operating environment: The laboratory should maintain a constant temperature and humidity: temperature 22°C ± 3°C; humidity 50% ± 10%, and the biosafety cabinet should operate normally.

[0732] (2) Preparation before operation:

[0733] (1) Equipment preparation: Open the biosafety cabinet in advance, irradiate with UV light for 30 minutes (min), and ventilate.

[0734] (2) Reagent preparation:

[0735] ① Plant 4.5×10 in a 10 cm dish. 6 HEK-293T cells were cultured in a 5% CO2, 37°C incubator for 36 hours (h).

[0736] ② Remove the PEI tube and return it to room temperature, shake it well, and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0737] ③ Extract sufficient amount of endotoxin-free packaging plasmids and envelope plasmids, shake well before use, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0738] ④ Prepare complete HEK-293T cell culture medium: Take 56 mL of FBS, filter it into 500 mL of DMEM / high glucose medium, add 4 mL of P / S (double antibiotics; penicillin, streptomycin), shake well, (place it in a CO2 incubator to preheat and neutralize it for transfection).

[0739] ⑤ Preparation of consumables: Wash the ultracentrifuge tubes with pure water and place them in an aluminum lunch box for high-pressure sterilization.

[0740] (3) Operation steps

[0741] Five plasmid packaging systems containing 0 μg, 0.5 μg, 1.0 μg or 2.0 μg of CD19 binding plasmid were prepared, respectively packaging VG1A7-CAR19-0, VG1A7-CAR19-0.5, VG1A7-CAR19-1 and VG1A7-CAR19-2 containing the CAR-19 gene and whose viral envelope contains the membrane-expressed anti-CD7 antibody and the mutant VSV-G1.

[0742] ① Prepare solution A, the ingredients are shown in the following table 1.

[0743] Table 1 (Liquid A)

[0744] ② Prepare Solution B, the ingredients are shown in Table 2 below.

[0745] Table 2 (Liquid B)

[0746] Transfer solution B into solution A with a pipette, blow down, shake vigorously for 2 minutes, and let it stand for 10 minutes.

[0747] ③ On Day 0, remove HEK-293T cells from the incubator and remove the existing culture medium using an electric aspirator. Add 9.5-10 mL of the culture medium containing the A and B solutions, then return the cells to the 5% CO2, 37°C incubator for incubation. Record the start time of the culture.

[0748] ④ After 6 hours, fresh HEK-293T cell culture medium (DMEM+10% FBS) was replaced and the cells were returned to the 5% CO2 37°C incubator for further culture.

[0749] ⑤ 48 hours after transfection, the virus solution was collected for the first time and completely fresh HEK-293T cell culture medium was added. The cells were returned to the 5% CO2 37℃ incubator for further culture. The collected lentiviral vectors were labeled and stored in a 4℃ refrigerator.

[0750] ⑥ 72 hours after transfection, the virus solution was collected for the second time.

[0751] ⑦ Take out the virus liquid collected for the first time, together with the virus liquid collected for the second time, centrifuge at 500×g for 3 minutes, take the supernatant, filter the virus liquid using a 0.45μm filter membrane and a 50mL syringe, and filter it into a sterilized ultracentrifuge tube.

[0752] ⑧After strictly balancing the virus solution, centrifuge it at 50,000×g at 4°C for 150 min.

[0753] ⑨ After centrifugation, a small amount of white precipitate can be seen at the bottom of the centrifuge tube. Mark it and discard the supernatant. Be careful not to suck up the white precipitate when discarding the supernatant with an aspirator. Use 200μL F12 culture medium for each tube of virus and repeatedly pipette and resuspend it at the marked position. Try not to create too many bubbles.

[0754] ⑩ Pool all resuspended viruses into one tube, shake well, centrifuge briefly to remove water droplets on the inner wall of the tube cap, divide into smaller pieces as needed, and store in a -80°C refrigerator.

[0755] On Day 3, HEK-293T cells from each group were collected, and the expression of FMC-63 / CAR-19 and CD19 in HEK-293T cells from each group was detected by flow cytometry. The results are shown in Figure 1.

[0756] As can be seen from Figure 1, during the packaging of LVV, as the amount of CD19 binding plasmid increased, the content of FMC-63 / CAR-19 available for binding to CD19 on the cell membrane of HEK-293T cells decreased significantly. It can be seen that the CAR binding molecule, i.e., the antigen CD19, can effectively bind to the antigen binding region contained in the CAR-19 molecule, i.e., the FMC63-scFv, in HEK-293T cells and / or on the cell membrane, thereby effectively reducing the CAR-19 molecules available for binding to antigens expressed on the cell membrane of HEK-293T cells.

[0757] Flow cytometry antibody for detecting FMC-63: Trade name: PE-Labeled Monoclonal Anti-FMC63 Antibody, Mouse IgG1 (Y45) (Site-specific conjugation) (0.03% Proclin) DMF Filed, Brand: ACRO, Product Number: #FM3-PY54A2-200 tests.

[0758] HEK-293T cell culture medium: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog Number: #C12430500BT; FBS: Brand: EXCELL, Catalog Number: #FSP500;

[0759] F12 culture medium: Brand: GIBCO, catalog number: #C11330500BT;

[0760] Syringe filter: Brand: SORFA, item number: #622120;

[0761] Opti-MEM alpha Reduced Serum Medium: Brand: GIBCO, Catalog Number: #SP0272.

[0762] Example 2

[0763] Transduction of Jurkat and Nalm-6 cells.

[0764] Each group of LVV packaged in Example 1, VG1A7-CAR19-0 / 0.5 / 1 / 2 were used to transduce LDL-R + CD7 + CD19 - Jurkat cells (human T lymphoid leukemia cells) and LDL-R + CD7 - CD19 + Nalm-6 cells (human B lymphoid leukemia cells).

[0765] The specific steps are:

[0766] Day 0, take 4 groups of 1×10 5 Each group of Jurkat cells and four groups of Nalm-6 cells were resuspended in 200 μL of Jurkat cell culture medium and Nalm-6 cell culture medium, respectively. The Jurkat cell culture medium and the Nalm-6 cell culture medium included 1640 (brand: Elgbio, product number: EH80809) culture medium and 10% FBS.

[0767] After the VG1A7-CAR19-0 / 0.5 / 1 / 2 virus stock solutions were concentrated 200 times, 10 μL of the virus supernatant of each group was added to 4 groups of Jurkat cell culture medium and 4 groups of Nalm-6 cell culture medium, mixed, and placed in a 5% CO2, 37°C incubator for cell culture.

[0768] On Day 2 and Day 6, flow cytometry was used to detect the expression of FMC-63 / CAR-19 in the four groups of Jurkat cells. The results are shown in Figures 2 and 3, respectively.

[0769] The positive rates of FMC-63 / CAR-19 detected in Jurkat cells transduced with VG1A7-CAR19-0 / 0.5 / 1 / 2 on Day 2 were approximately 93.41%, 66.11%, 52.54% or 31.05%, respectively ( FIG. 2 ); the positive rates of FMC-63 / CAR-19 detected on Day 6 were approximately 36.23%, 37.31%, 36.85% or 38.97%, respectively ( FIG. 3 ).

[0770] As shown in Figures 2 and 3, since the viral envelope of the VG1A7-CAR19-0 / 0.5 / 1 / 2 contains the membrane-expressed anti-CD7 antibody, when the ability of the mutant VSV-G1 to bind to LDL-R is weakened, the VG1A7-CAR19-0 / 0.5 / 1 / 2 can bind to the endocytic receptor CD7 through the membrane-expressed anti-CD7 antibody, thereby effectively transducing CD7. + CD19 - Jurkat cells;

[0771] Moreover, the VG1A7-CAR19-0.5 / 1 / 2 packaged with the CD19 binding plasmid packaging system can still effectively transduce CD7 + CD19 - Jurkat cells;

[0772] At the same time, the positive rates of FMC-63 / CAR-19 expressed by Jurkat cells transduced by the VG1A7-CAR19-0 were detected on Day 2 and Day 6, respectively, and significant differences were found. That is, approximately 93.41% of the FMC-63 / CAR-19 positive rate was detected on Day 2, while only 36.23% of the FMC-63 / CAR-19 positive rate was detected on Day 6.

[0773] This is because when the VG1A7-CAR19-0 was packaged, the CD19 binding plasmid was not added to the packaging system, and the CAR-19 molecules containing FMC-63 for binding to CD19 were expressed in large quantities on the cell membrane of HEK-293T cells. As the LVV budded, the viral envelope of the VG1A7-CAR19-0 contained a large amount of FMC-63 / CAR-19 derived from the cell membrane of HEK-293T cells. When transducing Jurkat cells, the viral envelope of the VG1A7-CAR19-0 fused with the cell membrane of the Jurkat cells, thereby transferring the CAR-19 molecules on the viral envelope of the VG1A7-CAR19-0 to the cell membrane of the Jurkat cells. As a result, when the positive rate of FMC-63 / CAR-19 on the surface of Jurkat cells was detected by flow cytometry on Day 2, the detection result was inaccurate due to severe false transduction. On Day 2, the positive rate of FMC-63 / CAR-19 on the surface of Jurkat cells was detected by flow cytometry. 6. The CAR-19 molecules and the FMC-63 contained in them transferred to the cell membrane of Jurkat cells through membrane fusion are metabolized and degraded, resulting in a significant decrease in the positive rate of FMC-63 / CAR-19 detected at this time compared with the positive rate detected on Day 2;

[0774] After the VG1A7-CAR19-0.5 / 1 / 2 were transduced into Jurkat cells, Day 2 and Day 6 The differences in the positive rates of FMC-63 / CAR-19 on the surface of Jurkat cells in each group were significantly lower than those of the Jurkat cells transduced by VG1A7-CAR19-0, especially the Jurkat cells transduced by VG1A7-CAR19-2 packaged by the packaging system containing 2 μg of the CD19 binding plasmid; this is because the packaging systems for packaging the VG1A7-CAR19-0.5 / 1 / 2 all contain the CD19 binding plasmid. Therefore, during the packaging process, CD19 can bind to the FMC-63 contained in the antigen binding region of the CAR-19 molecule in the cytoplasm and / or on the cell membrane of HEK-293T cells, thereby reducing the FMC-63 / CAR-19 available for binding to CD19 on the cell membrane of HEK-293T cells and on the viral envelope transferred to LVV as budding, thereby reducing false transduction.

[0775] On Day 2, flow cytometry was used to detect the expression of CD19 in the four groups of Nalm-6 cells. The results are shown in Figure 4.

[0776] As shown in Figure 4, compared with the control group Nalm-6 cells (CD19 positive rate is about 97.03%), the CD19 positive rate of Nalm-6 cells transduced by the VG1A7-CAR19-0 is significantly reduced (about 64.78%); it can be seen that the VG1A7-CAR19-0 can bind to the antigen CD19 on the surface of Nalm-6 cells through the FMC-63 contained in its viral envelope, thereby effectively transducing CD19 + Nalm-6 cells, thereby resulting in a decrease in the antigen CD19 on the surface of the transduced Nalm-6 cells available for binding to FMC-63; in addition, the transduced Nalm-6 cells will also express the CAR-19 molecule containing FMC-63, which can further bind to the antigen CD19 on the surface of the remaining Nalm-6 cells. In an environment containing tumor cells, such as in the body of a cancer patient or in the blood of a blood cancer patient, the antigen CD19 on the surface of the remaining Nalm-6 cells can be further reduced, ultimately causing the antigen to escape and allowing the Nalm-6 cells to evade recognition and killing by engineered immune cells such as CAR-T cells;

[0777] Compared with the control group Nalm-6 cells, the CD19 positivity rates of the three groups of Nalm-6 cells transduced by the VG1A7-CAR19-0.5 / 1 / 2 were effectively maintained (about 98.04%, 97.91%, and 98.19%, respectively); it can be seen that the content of FMC-63 / CAR-19 available for binding to the antigen CD19 contained in the viral envelope of the VG1A7-CAR19-0.5 / 1 / 2 was significantly reduced compared with that of the VG1A7-CAR19-0; this proves that in Adding a CD19 binding plasmid, such as 0.5 μg, 1.0 μg or 2.0 μg, to the packaging system of LVV can effectively reduce the content of FMC-63 / CVAR-19 available for binding to CD19 on the viral envelope of the packaged LVV; thereby significantly weakening the ability of the LVV to transduce Nalm-6 cells by binding to the Nalm-6 cell surface antigen CD19, effectively reducing antigen escape and reducing the ability of Nalm-6 cells to evade recognition and killing by engineered immune cells such as CAR-T cells.

[0778] Example 3

[0779] The viral envelope of the LVV contains anti-CD3 and anti-CD28 antibodies.

[0780] 1. Construction of membrane-expressed anti-CD3 antibody × anti-CD28 antibody structure

[0781] In this embodiment, the polynucleotide encoding the membrane-expressed anti-CD3 antibody × anti-CD28 antibody (membrane-expressed anti-CD3 × CD28 dual antibody) sequentially encodes from the 5' end to the 3' end: the human CD8α signal peptide, the anti-CD3 antibody (scFv), the human CD8α hinge region, the human CD8α transmembrane region, the FT2A peptide, the human CD8α signal peptide, the anti-CD28 antibody (scFv), the human CD8α hinge region, and the human CD8α transmembrane region;

[0782] (1) The anti-CD3 antibody (scFv) is a scFv derived from the anti-CD3ε monoclonal antibody UCHT1 (UCHT1-scFv), the amino acid sequence of which is shown in SEQ ID NO: 13; the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NO: 72-74, respectively, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NO: 75-77, respectively; the amino acid sequence of the VH region of the UCHT1-scFv is shown in SEQ ID NO: 70, and the amino acid sequence of the VL region of the UCHT1-scFv is shown in SEQ ID NO: 71; the VH region is connected to the VL region via the connecting peptide 2;

[0783] (2) The amino acid sequence of the FT2A peptide is shown in SEQ ID NO: 16;

[0784] (3) The anti-CD28 antibody (scFv) is a scFv (15E8-scFv) derived from the monoclonal antibody 15E8, the amino acid sequence of which is shown in SEQ ID NO: 17; the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 80-82, respectively; the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 83-85, respectively; the amino acid sequence of the VH region of the 15E8-scFv is shown in SEQ ID NO: 78, and the amino acid sequence of the VL region of the 15E8-scFv is shown in SEQ ID NO: 79; the VH region is connected to the VL region via the connecting peptide 2.

[0785] 2. Prepare the LVV Packaging System

[0786] Prepare the following five plasmids: an envelope plasmid carrying a polynucleotide encoding the mutant VSV-G1 and a polynucleotide encoding the membrane-expressed anti-CD3×CD28 dual antibody (envelope plasmid 2), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, the CD19 binding plasmid, and the main plasmid CAR19.

[0787] 3. Packaging LVV

[0788] Referring to the method for packaging LVV described in Example 1, the usage amounts of the CD19 binding plasmid are: 0 μg, 0.1 μg, 0.5 μg, 1 μg, 2 μg, and 5 μg, respectively; on Day 0, LVV containing the CAR-19 gene, whose viral envelope contains the membrane-expressed anti-CD3×CD28 dual antibody and the mutant VSV-G1, VG1A3X28-CAR19-0 / 0.1 / 0.5 / 1 / 2 / 5, are packaged respectively.

[0789] On Day 3, HEK-293T cells from each group were collected, and the expression of FMC-63 / CAR-19 and CD19 in HEK-293T cells from each group was detected by flow cytometry. The results are shown in Figure 5.

[0790] As shown in Figure 5, during the packaging of the VG1A3X28-CAR19-0 / 0.1 / 0.5 / 1 / 2 / 5, as the amount of CD19 binding plasmid increased, the content of FMC-63 / CAR-19 available for binding to CD19 on the cell membrane of each group of HEK-293T cells was significantly reduced.

[0791] 4. Transduction of Jurkat and Nalm-6 Cells

[0792] Referring to the method for transducing Jurkat cells and Nalm-6 cells described in Example 2, on Day 0, Jurkat cells and Nalm-6 cells were transduced using the VG1A3X28-CAR19-0 / 0.1 / 0.5 / 1 / 2 / 5, respectively. On Day 5, the expression of FMC-63 / CAR-19 and CD19 in each group of Jurkat cells, as well as the expression of CD19 in Nalm-6 cells, were detected. The results are shown in Figures 6 and 7, respectively.

[0793] As shown in Figure 6, the VG1A3X28-CAR19-0.1 / 0.5 / 1 / 2 / 5 packaged by the 5-plasmid packaging system with the addition of CD19 binding plasmid can still express the anti-CD3×CD28 dual antibody through the membrane to bind to the endocytic receptors CD3 and CD28, effectively transducing CD3 + CD28 + Jurkat cells, delivering and expressing the CAR-19 gene.

[0794] As shown in Figure 7, compared with the control group Nalm-6 cells (CD19 positivity of approximately 93.82%), the CD19 positivity of Nalm-6 cells transduced by the VG1A3X28-CAR19-0 was significantly decreased (approximately 65.65%); while the CD19 positivity of the five groups of Nalm-6 cells transduced by the VG1A3X28-CAR19-0.1 / 0.5 / 1 / 2 / 5 were effectively maintained (approximately 93.99%, 93.40%, 95.07%, 94.28%, and 95.31%, respectively);

[0795] This demonstrates that the addition of the CD19 binding plasmid to the packaging system can effectively attenuate the ability of VG1A3X28-CAR19-0.1 / 0.5 / 1 / 2 / 5 to transduce Nalm-6 cells.

[0796] Example 4

[0797] The LVV is packaged, wherein the viral envelope of the LVV comprises the membrane expressing anti-CD3×CD28 dual antibodies and a shuttle gene encoding CAR, wherein the CAR comprises TSP (CAR-TSP).

[0798] 1. Build a CAR containing TSP

[0799] In this example, a CAR that can specifically bind to human CD19 was constructed, wherein the CAR comprises a TSP (CAR19-TSP), and the polynucleotide encoding the CAR19-TSP sequentially encodes from the 5' end to the 3' end: the human CD8α signal peptide, the FMC63-scFv, the connecting peptide 1, and CD3γ;

[0800] (1) The amino acid sequence of CD3γ is as shown in SEQ ID NO: 22; the CD3γ does not contain its signal peptide;

[0801] (2) The C-terminus of the FMC63-scFv is connected to the N-terminus of the CD3γ via a connecting peptide 1; the amino acid sequence of the connecting peptide 1 is shown in SEQ ID NO: 23;

[0802] (3) The amino acid sequence of the CAR19-TSP is shown in SEQ ID NO: 111.

[0803] 2. Prepare the LVV Packaging System

[0804] Prepare the following five plasmids: the envelope plasmid 2, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, the CD19 binding plasmid, and the main plasmid carrying the polynucleotide encoding the CAR19-TSP (CAR19-TSP gene).

[0805] 3. Packaging Lentiviral Vectors

[0806] Referring to the method for packaging LVV described in Example 1, the usage amounts of the CD19 binding plasmid are: 0 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.5 μg, and 1 μg, respectively; on Day 0, LVV containing the CAR19-TSP gene and the viral envelope containing the membrane-expressed anti-CD3×CD28 dual antibody and the mutant VSV-G1, VG1A3X28-CAR19TSP-0 / 0.1 / 0.2 / 0.3 / 0.5 / 1, respectively, are packaged.

[0807] On Day 3, HEK-293T cells from each group were collected, and the expression of FMC-63 / CAR-19 and CD19 in HEK-293T cells from each group was detected by flow cytometry. The results are shown in Figure 8.

[0808] As shown in Figure 8, compared with the groups of HEK-293T cells packaged with VG1A7-CAR19-0 and VG1A3X28-CAR19-0 described in Examples 1 and 3, the content of FMC-63 on the cell membrane of the HEK-293T cells of VG1A3X28-CAR19TSP-0 packaged by the packaging system containing the CAR19-TSP gene is relatively reduced; this may be because the CAR19-TSP can be effectively expressed in T cells along with the endogenous TCR / CD3 complex or its subunits, while cells other than T cells, such as HEK-293T cells, do not contain endogenous TCR / CD3 complex or its subunits, and therefore the CAR19-TSP is difficult to be effectively expressed on the cell membrane of HEK-293T cells;

[0809] Moreover, as shown in FIG8 , as the amount of CD19 binding plasmid increased, the content of FMC-63 on the cell membrane of HEK-293T cells in each group also decreased significantly.

[0810] 4. Transduction of Jurkat and Nalm-6 Cells

[0811] On Day 0, referring to the transduction method described in Example 2, Jurkat cells and Nalm-6 cells were transduced using the VG1A3X28-CAR19TSP-0 / 0.1 / 0.2 / 0.3 / 0.5 / 1. On Day 5, the expression of FMC-63 / CAR19-TSP in each group of Jurkat cells and the expression of CD19 in Nalm-6 cells were detected, and the results are shown in Figures 9 and 10, respectively.

[0812] As shown in Figure 9, the VG1A3X28-CAR19TSP-0.1 / 0.2 / 0.3 / 0.5 / 1 packaged by the packaging system with different amounts of CD19 binding plasmid can still express the anti-CD3×CD28 dual antibody through the membrane to bind to the endocytic receptors CD3 and CD28, thereby effectively transducing CD3 + CD28 + Jurkat cells were used to deliver and express the CAR19-TSP (the positive rates were 30.37%, 26.97%, 17.29%, 14.12% and 15.15% respectively).

[0813] As shown in Figure 10, compared with the control group Nalm-6 cells (CD19 positivity of approximately 97.18%), the CD19 positivity of Nalm-6 cells transduced by the VG1A3X28-CAR19TSP-0 was significantly decreased (approximately 84.30%); while the CD19 positivity of the five groups of Nalm-6 cells transduced by the VG1A3X28-CAR19TSP-0.1 / 0.2 / 0.3 / 0.5 / 1 were effectively maintained (approximately 98.07%, 98.78%, 98.82%, 98.72%, and 98.49%, respectively);

[0814] This demonstrates that the addition of the CD19 binding plasmid to the packaging system can effectively attenuate the ability of VG1A3X28-CAR19TSP-0.1 / 0.2 / 0.3 / 0.5 / 1 to transduce Nalm-6 cells.

[0815] Example 5

[0816] A binding plasmid containing a polynucleotide encoding human CD33 (CD33 binding plasmid) was constructed, and LVV was packaged using an LVV packaging system containing the CD33 binding plasmid.

[0817] 1. Construction of CAR-33 molecules

[0818] Referring to the CAR-19 molecule described in Example 1, a CAR molecule (CAR-33 molecule) was constructed whose antigen binding region could specifically bind to human CD33;

[0819] The polynucleotide encoding the CAR-33 molecule (CAR-33 gene) sequentially encodes from the 5' end to the 3' end: the human CD8α signal peptide, the antigen binding region that can specifically bind to human CD33, the human CD8α hinge region, the human CD8α transmembrane region, the human 4-1BB costimulatory signaling domain, and the human CD3ζ intracellular signaling domain;

[0820] The antigen-binding region that can specifically bind to human CD33 is a scFv derived from the anti-CD33 monoclonal antibody Gemtuzumab (Gemtuzumab-scFv); the amino acid sequence of the Gemtuzumab-scFv is set forth in SEQ ID NO: 112, and the amino acid sequences of the HCDR1-3 regions of the Gemtuzumab-scFv are set forth in SEQ ID NOs: 113-115, respectively; the amino acid sequences of the LCDR1-3 regions of the Gemtuzumab-scFv are set forth in SEQ ID NOs: 116-118, respectively; the amino acid sequence of the VL region of the Gemtuzumab-scFv is set forth in SEQ ID NO: 53; and the amino acid sequence of the VH region of the Gemtuzumab-scFv is set forth in SEQ ID NO: 54; the VL region is connected to the VH region via the connecting peptide 2.

[0821] 2. Prepare the LVV Packaging System

[0822] Prepare the following five plasmids: the envelope plasmid 2, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, the CD33 binding plasmid, and the main plasmid carrying the CAR-33 gene;

[0823] The CD33 binding plasmid contains a polynucleotide encoding a CAR binding molecule, human CD33, the amino acid sequence of which is shown in SEQ ID NO: 55; the CAR binding molecule, antigen CD33 can specifically bind to a CD33 antibody, Gemtuzumab, or an antigen-binding fragment thereof.

[0824] Human CD33, UniProtKB No.: P20138. CD33 is an effective target for the treatment of blood cancers such as acute myeloid leukemia.

[0825] 3. Packaging LVV

[0826] Referring to the method for packaging LVV as described in Example 1, the amount of the CD33 binding plasmid in the solution A contained in the packaging system is: 0 μg, 0.1 μg, 1 μg or 5 μg, respectively, to package LVV, VG1A3X28-CAR33-0 / 0.1 / 1 / 5.

[0827] On Day 3, HEK-293T cells were collected from each group, and the expression of CD33 in each group of HEK-293T cells was detected using anti-CD33 flow cytometry antibodies. The expression of the CAR-33 molecule in each group of HEK-293T cells was detected using fluorescently labeled and His Tag-tagged CD33 protein (detection CD33). The results are shown in Figures 11 and 12, respectively.

[0828] As can be seen from Figures 11 and 12, during the packaging of each group of LVV, as the amount of CD33 binding plasmid increased, the expression levels of the CAR-33 molecules in the remaining groups of HEK-293T cells packaging the VG1A3X28-CAR33-0.1 / 1 / 5 were significantly reduced (the positive rates were approximately 40.11%, 2.51%, and 1.98%, respectively). Adding the CD33 binding plasmid to the packaging system can effectively reduce the content of the CAR-33 molecules available for binding to CD33 on the cell membrane of HEK-293T cells.

[0829] 4. Transduction of Jurkat cells and CD33-overexpressing Nalm-6 cell lines

[0830] The Nalm-6 cell line overexpressing CD33 was constructed. The construction method is well known to those skilled in the art.

[0831] Referring to the transduction method described in Example 2, on Day 0, the VG1A3X28-CAR33-0 / 0.1 / 1 / 5 were used to transduce Jurkat cells and Nalm-6 cells overexpressing CD33 (CD33 + Nalm-6 cells); Day 5, the expression of the CAR-33 molecule in each group of Jurkat cells was detected using the detection CD33; and the expression of CD33 in each group of Nalm-6 cells was detected by flow cytometry. The results are shown in Figures 13 and 14, respectively.

[0832] As shown in Figure 13, VG1A3X28-CAR33-0.1 / 1 / 5 packaged by the packaging system with different amounts of CD33 binding plasmid can still express anti-CD3×CD28 dual antibodies through the membrane to bind to endocytic receptors CD3 and CD28, thereby effectively transducing CD3 + CD28 + The positive rates of CAR-33 molecules in each group were 14.49%, 13.87% and 9.77% respectively.

[0833] As shown in Figure 14, compared with the control group CD33 + Nalm-6 cells (CD33 positive rate is about 98.86%), CD33 transduced by VG1A3X28-CAR33-0 + The CD33 positive rate of Nalm-6 cells decreased significantly (to about 82.71%); while the CD33 positive rate of the three groups of cells transduced by VG1A3X28-CAR33-0.1 / 1 / 5 was significantly decreased (to about 82.71%).+ The CD33 positivity rates of Nalm-6 cells were effectively maintained (approximately 86.62%, 99.15%, and 99.06%, respectively);

[0834] This demonstrates that the addition of the CD33 binding plasmid to the packaging system can effectively attenuate the ability of VG1A3X28-CAR33-0.1 / 1 / 5 to transduce CD33-overexpressing Nalm-6 cells.

[0835] CD33 detection: Brand: Acro; Reagent name: PE-Labeled Siglec-3 / CD33 Protein, His Tag, Catalog Number: #CD3-HP2H7.

[0836] Antibody for CD33 flow cytometry detection: APC anti-human CD33 Antibody, Brand: Biolegend, Catalog Number: #366606.

[0837] Example 6

[0838] A binding plasmid containing a polynucleotide encoding human BCMA (BCMA binding plasmid) was constructed, and LVV was packaged using an LVV packaging system containing the BCMA binding plasmid.

[0839] 1. Construction of CAR-BCMA molecules

[0840] Referring to the CAR-19 molecule described in Example 1, a CAR molecule (CAR-BCMA molecule) was constructed whose antigen binding region could specifically bind to human BCMA.

[0841] The polynucleotide encoding the CAR-BCMA molecule (CAR-BCMA gene) sequentially encodes from the 5' end to the 3' end: the human CD8α signal peptide, the antigen binding region that can specifically bind to human BCMA, the human CD8α hinge region, the human CD8α transmembrane region, the human 4-1BB costimulatory signaling domain, and the human CD3ζ intracellular signaling domain;

[0842] The antigen-binding region specific for human BCMA is an anti-BCMA scFv (anti-BCMA-scFv), the amino acid sequence of which is shown in SEQ ID NO: 119; the amino acid sequences of the HCDR1-3 regions of the anti-BCMA-scFv are shown in SEQ ID NOs: 120-122, respectively; the amino acid sequences of the LCDR1-3 regions of the anti-BCMA-scFv are shown in SEQ ID NOs: 123-125, respectively; the amino acid sequence of the VL region of the anti-BCMA-scFv is shown in SEQ ID NO: 56; and the amino acid sequence of the VH region of the anti-BCMA-scFv is shown in SEQ ID NO: 57; the VL region is connected to the VH region via the connecting peptide 2.

[0843] 2. Prepare the LVV Packaging System

[0844] Prepare the following five plasmids: the envelope plasmid 2, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, the BCMA binding plasmid, and the main plasmid carrying the CAR-BCMA gene.

[0845] The BCMA-binding plasmid contains a polynucleotide encoding a CAR-binding molecule, human BCMA, the amino acid sequence of which is shown in SEQ ID NO: 58; the CAR-binding molecule can specifically bind to the anti-BCMA-scFv.

[0846] Human BCMA: Uniprot ID: Q02223. BCMA is a promising target for the treatment of multiple myeloma.

[0847] 3. Packaging LVV

[0848] Referring to the method for packaging LVV described in Example 1, the amounts of BCMA-binding plasmid in solution A contained in the packaging system are: 0 μg, 0.1 μg, 1 μg, and 5 μg, respectively, for packaging LVV, VG1A3X28-CARBCMA-0 / 0.1 / 1 / 5, respectively.

[0849] On Day 3, HEK-293T cells were collected from each group, and the expression of the CAR-BCMA molecule in each group of HEK-293T cells was detected using a fluorescently labeled and His Tag-tagged BCMA protein (BCMA for detection); and the expression of BCMA in each group of HEK-293T cells was detected using flow cytometry. The results are shown in Figures 15 and 16, respectively.

[0850] As can be seen from Figures 15 and 16, during the packaging of each group of LVV, as the amount of BCMA-binding plasmid increased, the expression levels of the CAR-BCMA molecules on the surface of the HEK-293T cells of the remaining groups packaging the VG1A3X28-CARBCMA-0.1 / 1 / 5 were significantly reduced (the positive rates were approximately 31.07%, 0.44%, and 0.48%, respectively).

[0851] 4. Transduction of Jurkat cells and Nalm-6 cell lines overexpressing BCMA

[0852] Construction of Nalm-6 cell line overexpressing BCMA (BCMA + Nalm-6 cells), and the construction method is well known to those skilled in the art.

[0853] Referring to the method for transducing Jurkat cells and Nalm-6 cells described in Example 2, on Day 0, the VG1A3X28-CARBCMA-0 / 0.1 / 1 / 5 was used to transduce Jurkat cells and BCMA, respectively. + Nalm-6 cells; Day 5, using the detection BCMA to detect the expression of the CAR-BCMA molecule in each group of Jurkat cells; and using flow cytometry to detect the expression of the BCMA in each group + The expression of BCMA in Nalm-6 cells is shown in Figures 17 and 18, respectively.

[0854] As shown in Figure 17, VG1A3X28-CARBCMA-0.1 / 1 / 5 packaged by the packaging system with different amounts of BCMA binding plasmid can still express anti-CD3×CD28 dual antibodies through the membrane to bind to endocytic receptors CD3 and CD28, thereby effectively transducing CD3 + CD28 + The positive rates of CAR-BCMA molecules in each group of Jurkat cells were approximately 28.53%, 25.24%, and 23.59%, respectively.

[0855] As shown in Figure 18, compared with the control group BCMA + Nalm-6 cells (BCMA positive rate is about 97.18%), BCMA transduced by VG1A3X28-CARBCMA-0 + The BCMA positive rate of Nalm-6 cells decreased significantly (about 55.50%); while the BCMA positive rate of the three groups of cells transduced with VG1A3X28-CARBCMA-0.1 / 1 / 5 was significantly decreased (about 55.50%).+ The BCMA positivity rates of Nalm-6 cells were effectively maintained (approximately 98.11%, 98.70%, and 98.92%, respectively);

[0856] Adding a BCMA-binding plasmid to the packaging system effectively attenuated the ability of VG1A3X28-CARBCMA-0.1 / 1 / 5 to transduce BCMA-overexpressing Nalm-6 cells.

[0857] BCMA protein used for detection: Brand: ACRO, Reagent Name: PE-Labele Human BCMA / TNFRSF17 Protein, His Tag (Site-specific conjugation), Catalog Number: #BCA-HP2H2.

[0858] Flow cytometry antibody for detecting BCMA: Brand: Suzhou Si'anchi Biopharmaceutical Technology Co., Ltd., Product No.: S0237.

[0859] Example 7

[0860] A binding plasmid comprising a polynucleotide encoding human CLDN18.2 (CLDN18.2 binding plasmid) was constructed, and LVV was packaged using an LVV packaging system comprising the CLDN18.2 binding plasmid.

[0861] 1. Construction of CAR-CLDN18.2 molecules

[0862] Referring to the CAR-19 molecule described in Example 1, a CAR molecule (CAR-CLDN18.2 molecule) was constructed whose antigen binding region could specifically bind to CLDN18.2;

[0863] The polynucleotide encoding the CAR-CLDN18.2 molecule (CAR-CLDN18.2 gene) sequentially encodes from the 5' end to the 3' end: the human CD8α signal peptide, the antigen binding region that can specifically bind to human CLDN18.2, the human CD8α hinge region, the human CD8α transmembrane region, the human 4-1BB costimulatory signaling domain, and the human CD3ζ intracellular signaling domain;

[0864] The antigen binding region that can specifically bind to CLDN18.2 is an anti-CLDN18.2 scFv (anti-CLDN18.2-scFv), and the amino acid sequence of the anti-CLDN18.2-scFv is shown in SEQ ID NO: 126; the amino acid sequences of the HCDR1-3 regions of the anti-CLDN18.2-scFv are shown in SEQ ID NOs: 127-129, respectively; the amino acid sequences of the LCDR1-3 regions of the anti-CLDN18.2-scFv are shown in SEQ ID NOs: 130-132, respectively; the amino acid sequence of the VL region of the anti-CLDN18.2-scFv is shown in SEQ ID NO: 59; the amino acid sequence of the VH region of the anti-CLDN18.2-scFv is shown in SEQ ID NO: 60; the VL region is connected to the VH region via the connecting peptide 2.

[0865] 2. Prepare the LVV Packaging System

[0866] Prepare the following five plasmids: the envelope plasmid 2, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, the CLDN18.2 binding plasmid, and the main plasmid carrying the CAR-CLDN18.2 gene;

[0867] The CLDN18.2 binding plasmid contains a polynucleotide encoding a CAR binding molecule, CLDN18.2, the amino acid sequence of which is shown in SEQ ID NO: 61; the CAR binding molecule can specifically bind to the anti-CLDN18.2-scFv.

[0868] In this example, the CLDN18.2 is a peptide having GenBank accession number NP001002026 (RNA: NM 001002026).

[0869] 3. Packaging LVV

[0870] Day 0: Refer to the method for packaging LVV as described in Example 1, wherein the amounts of the CLDN18.2 binding plasmid in Solution A contained in the packaging system are: 0 μg, 0.1 μg, 1 μg, and 5 μg, respectively, for packaging LVV, VG1A3X28-CAR18.2-0 / 0.1 / 1 / 5, respectively.

[0871] On Day 3, HEK-293T packaging cells were collected from each group and incubated with His-Tag CLDN18.2 for 0.5 h. After washing once, the cells were co-incubated with a fluorescently labeled anti-His Tag secondary antibody for 0.5 h (detection CLDN18.2). The expression of the CAR-CLDN18.2 molecule in each group of HEK-293T cells was then detected using the detection CLDN18.2. Flow cytometry was also used to detect the expression of CLDN18.2 in each group of HEK-293T cells. The results are shown in Figures 19 and 20, respectively.

[0872] As can be seen from Figures 19 and 20, during the packaging of each group of LVV, as the amount of CLDN18.2 binding plasmid increased, the expression level of the CAR-CLDN18.2 molecule on the surface of the remaining groups of HEK-293T cells packaging the VG1A3X28-CAR18.2-0.1 / 1 / 5 was significantly reduced (the positive rates were approximately 14.65%, 0.33%, and 0.22%, respectively).

[0873] 4. Transduction of Jurkat cells and Nalm-6 cell lines overexpressing CLDN18.2

[0874] Construction of Nalm-6 cell line overexpressing CLDN18.2 + Nalm-6 cells), and the construction method is well known to those skilled in the art.

[0875] Referring to the method for transducing Jurkat cells and Nalm-6 cells described in Example 2, on Day 0, the VG1A3X28-CAR18.2-0 / 0.1 / 1 / 5 was used to transduce Jurkat cells and CLDN18.2 + Nalm-6 cells; Day 5, using the detection CLDN18.2 to detect the expression of the CAR-CLDN18.2 molecule in each group of Jurkat cells; and using flow cytometry to detect the expression of the CLDN18.2 molecule in each group + The expression results of CLDN18.2 in Nalm-6 cells are shown in Figures 21 and 22, respectively.

[0876] As shown in Figure 21, VG1A3X28-CAR18.2-0.1 / 1 / 5 packaged by the packaging system with different amounts of CLDN18.2 binding plasmid can still express anti-CD3×CD28 dual antibodies through the membrane to bind to endocytic receptors CD3 and CD28, thereby effectively transducing CD3 + CD28 + The positive rates of CAR-CLDN18.2 molecules in each group were 25.24%, 23.59% and 23.00%, respectively.

[0877] As shown in Figure 22, compared with the control group, CLDN18.2 + Nalm-6 cells (CLDN18.2 positive rate is about 94.98%), CLDN18.2 transduced by VG1A3X28-CAR18.2-0 + The CLDN18.2 positive rate of Nalm-6 cells decreased significantly (to about 81.82%); while the CLDN18.2 positive rate of the three groups transduced with VG1A3X28-CAR18.2-0.1 / 1 / 5 was significantly decreased (to about 81.82%). + The CLDN18.2 positivity rates of Nalm-6 cells were effectively maintained (approximately 97.97%, 96.92%, and 97.69%, respectively);

[0878] Adding a CLDN18.2-binding plasmid to the packaging system effectively attenuated the ability of VG1A3X28-CAR18.2-0.1 / 1 / 5 to transduce CLDN18.2-overexpressing Nalm-6 cells.

[0879] Detection of CLDN18.2 in the CAR-CLDN18.2 molecule: Brand: ACRO, Reagent Name: Biotinylated Human / Cynomolgus Claudin-18.2 Protein, Avitag TM ,His Tag (Nanodisc), catalog number: #CL2-H85Q7;

[0880] Anti-His Tag secondary antibody: His-Tag (D3I1O) Rabbit mAb (Alexa 647Conjugate), item number: #14931;

[0881] Flow cytometry antibody for detecting CLDN18.2: Trade name: Anti-Claudin-18 Isoform 2 Antibody, PE-Labeled, Brand: BPS Bioscience, Catalog No. #101676.

[0882] Example 8

[0883] 1. Construction of CAR binding molecule CD19ECD+TM-R

[0884] Constructing a CAR binding molecule CD19ECD+TM-R, wherein the polynucleotide encoding the CD19ECD+TM-R sequentially encodes from the 5' end to the 3' end: human CD19 signal peptide, human CD19 extracellular domain (ECD), human CD19 transmembrane region (TM), (G4S)4 connecting peptide, and endoplasmic reticulum retention signal peptide (R);

[0885] (1) The amino acid sequence of the human CD19 signal peptide is shown in SEQ ID NO: 52;

[0886] (2) The amino acid sequence of the human CD19 extracellular domain is shown in SEQ ID NO: 134;

[0887] (3) The amino acid sequence of the human CD19 transmembrane region is shown in SEQ ID NO: 135;

[0888] (4) The amino acid sequence of the (G4S)4 connecting peptide is shown in SEQ ID NO: 133;

[0889] (5) The amino acid sequence of the endoplasmic reticulum retention signal peptide is shown in SEQ ID NO: 51 (KDEL);

[0890] (6) The amino acid sequence of the CD19ECD+TM-R is shown in SEQ ID NO:138.

[0891] The CD19ECD+TM-R contains a human CD19 extracellular domain that can specifically bind to the anti-CD19 antibody FMC-63, and an endoplasmic reticulum retention signal peptide (KDEL). In theory, the CD19ECD+TM-R will be retained in the endoplasmic reticulum of HEK-293T cells due to its C-terminal endoplasmic reticulum retention signal peptide, and specifically bind to the antigen-binding fragment of FMC-63 contained in the CAR-19 molecule through the CD19 extracellular domain contained in its N-terminus, and the CAR-19 molecules produced in HEK-293T cells will also be retained in the endoplasmic reticulum of HEK-293T cells, preventing the CAR-19 molecules from being expressed on the cell membrane of HEK-293T cells.

[0892] 2. Prepare 5-plasmid LVV packaging system and package LVV

[0893] Prepare the following five plasmids: an envelope plasmid carrying a polynucleotide encoding mutant VSV-G2 and a polynucleotide encoding the membrane-expressed anti-CD3×CD28 dual antibody (envelope plasmid 3), a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, a plasmid containing a polynucleotide encoding the CD19ECD+TM-R (CD19ECD+TM-R binding plasmid), and the main plasmid CAR19; the envelope plasmid 3 and the CD19ECD+TM-R binding plasmid were synthesized by conventional molecular cloning methods.

[0894] The extracellular domain of the mutant VSV-G2 comprises the amino acid sequence shown in SEQ ID NO: 10; relative to the extracellular domain of wild-type VSV-G (SEQ ID NO: 1), the extracellular domain of the mutant VSV-G2 (SEQ ID NO: 10) comprises R354Q, T214N and T352A; the ability of the mutant VSV-G2 to specifically bind to LDL-R is reduced or inhibited, and the ability to antagonize complement inactivation is enhanced.

[0895] Day 0, referring to the packaging method in Example 1, wherein the amount of the CD19ECD+TM-R binding plasmid in Solution A of the 5-plasmid packaging system is 0 μg or 1 μg, respectively, and LVV, VG2A3X28-19RCAR19-0 / 1 is packaged; the VG2A3X28-19RCAR19-0 / 1 contains the CAR-19 gene, and its viral envelope contains the mutant VSV-G2 and the membrane-expressed CD3×CD28 dual antibody;

[0896] On Day 3, HEK-293T cells from each group were collected, and the expression of FMC-63 / CAR-19 in HEK-293T cells from each group was detected by flow cytometry. The results are shown in Figure 23.

[0897] As can be seen from Figure 23, the FMC-63 / CAR-19 positivity rate (about 1.59%) of HEK-293T cells containing a packaging system without the addition of the CD19ECD+TM-R binding plasmid was significantly reduced compared to the FMC-63 / CAR-19 positivity rate (about 37.84%) of HEK-293T cells containing a packaging system with the addition of 1 μg of the CD19ECD+TM-R binding plasmid; this proves that the number of CAR-19 molecules available for binding to CD19 on the cell membrane of HEK-293T cells containing a packaging system with the addition of the CD19ECD+TM-R binding plasmid is effectively reduced.

[0898] 3. Transduction of Jurkat and Nalm-6 Cells

[0899] Day 0: Referring to the transduction method described in Example 2, 200 μL of the collected VG2A3X28-19RCAR19-0 / 1 viral supernatant (unconcentrated) was used to transduce Jurkat cells and Nalm-6 cells, respectively;

[0900] On Day 2, flow cytometry was used to detect the expression of FMC-63 / CAR-19 in Jurkat cells of each group, as well as the expression of CD19 and FMC-63 / CAR-19 in Nalm-6 cells of each group. The results are shown in Figures 24 and 25, respectively.

[0901] As shown in Figure 24, the VG2A3X28-19RCAR19-1 packaged by the packaging system containing 1 μg of the CD19ECD+TM-R combined plasmid can effectively transduce CD3 + CD28 + Jurkat cells delivered and expressed the CAR-19 gene (positive rate was approximately 16.74%).

[0902] As can be seen from Figure 25, compared with the control group Nalm-6 cells, the VG2A3X28-19RCAR19-0 packaged by the packaging system without adding the CD19ECD+TM-R binding plasmid can still effectively transduce Nalm-6 cells (the CD19 expression level of Nalm-6 cells is significantly reduced), and the effect of avoiding off-target effects is not ideal; while the VG2A3X28-19RCAR19-1 packaged by the packaging system with 1 μg of the CD19ECD+TM-R binding plasmid is difficult to effectively transduce Nalm6 cells (the CD19 expression level of Nalm-6 cells is effectively maintained), and the off-target effect is successfully controlled.

[0903] Example 9

[0904] 1. Construction of CAR binding molecule anti-FMC63-R

[0905] Constructing a CAR binding molecule anti-FMC63-R, wherein the polynucleotide encoding the anti-FMC63-R sequentially encodes from the 5' end to the 3' end: the human CD8α signal peptide, the anti-FMC-63 antibody (anti-anti-CD19 antibody), the human CD8α hinge region, and the endoplasmic reticulum retention signal peptide (KDEL);

[0906] The anti-FMC-63 antibody is a scFv (antiFMC63-scFv), and the polynucleotide encoding the antiFMC63-scFv sequentially comprises, from the 5' end to the 3' end: antiFMC63-VH, (G4S)4 connecting peptide, and antiFMC63-VL;

[0907] The amino acid sequences of the HCDR1-3 regions of the antiFMC63-scFv are shown in SEQ ID NOs: 102-104, respectively, and the amino acid sequences of the LCDR1-3 regions of the antiFMC63-scFv are shown in SEQ ID NOs: 105-107, respectively; the amino acid sequence of the antiFMC63-scFv is shown in SEQ ID NO: 108; the amino acid sequence of the antiFMC63-VH is shown in SEQ ID NO: 100, and the amino acid sequence of the antiFMC63-VL is shown in SEQ ID NO: 101. The VH region is connected to the VL region via a (G4S)4 linker peptide, and the amino acid sequence of the (G4S)4 linker peptide is shown in SEQ ID NO: 133.

[0908] antiFMC63-scFv-HCDR1:

[0909] GFDFSRYW (SEQ ID NO: 102)

[0910] antiFMC63-scFv-HCDR2:

[0911] INLDSSTI (SEQ ID NO: 103)

[0912] antiFMC63-scFv-HCDR3:

[0913] ARRYDAMDY (SEQ ID NO: 104)

[0914] antiFMC63-scFv-LCDR1:

[0915] ESVDDYGISF (SEQ ID NO: 105)

[0916] antiFMC63-scFv-LCDR2:

[0917] AAP (SEQ ID NO: 106)

[0918] antiFMC63-scFv-LCDR3:

[0919] QQSKD (SEQ ID NO: 107)

[0920] The amino acid sequence of the anti-FMC63-R is shown in SEQ ID NO: 139.

[0921] 2. Prepare 5-plasmid LVV packaging system and package LVV

[0922] Prepare the following five plasmids: the envelope plasmid 3, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, a plasmid containing a polynucleotide encoding the anti-FMC63-R (anti-FMC63-R binding plasmid), and the main plasmid CAR19; the anti-FMC63-R binding plasmid is synthesized by conventional molecular cloning methods.

[0923] Day 0, referring to the packaging method in Example 1, wherein the amount of the anti-FMC63-R binding plasmid in Solution A of the 5-plasmid packaging system is 0 μg, 1 μg, 2 μg or 5 μg, respectively, to package LVV, VG2A3X28-A63RCAR19-0 / 1 / 2 / 5, respectively; the VG2A3X28-A63RCAR19-0 / 1 / 2 / 5 contains the CAR-19 gene, and its viral envelope contains the mutant VSV-G2 and the membrane-expressed CD3×CD28 dual antibody;

[0924] On Day 3, HEK-293T cells from each group were collected, and the expression of FMC-63 / CAR-19 in HEK-293T cells from each group was detected by flow cytometry. The results are shown in Figure 26.

[0925] As can be seen from Figure 26, the FMC-63 / CAR-19 positivity rate of HEK-293T cells in a packaging system that does not contain the anti-FMC63-R binding plasmid (about 44.21%) was significantly reduced (about 1.70%, 1.44%, and 1.12%, respectively) of the HEK-293T cells in a packaging system containing 1 μg, 2 μg, or 5 μg of the anti-FMC63-R binding plasmid; this proves that the number of CAR-19 molecules available for binding to CD19 on the cell membrane of HEK-293T cells containing a packaging system containing the anti-FMC63-R binding plasmid is effectively reduced.

[0926] 3. Transduction of Jurkat and Nalm-6 Cells

[0927] Day 0: Referring to the transduction method described in Example 2, 200 μL of the collected viral supernatant of VG2A3X28-A63RCAR19-0 / 1 / 2 / 5 (unconcentrated) was used to transduce Jurkat cells and Nalm-6 cells, respectively;

[0928] On Day 2, flow cytometry was used to detect the expression of FMC-63 / CAR-19 in Jurkat cells of each group, as well as the expression of CD19 and FMC-63 / CAR-19 in Nalm-6 cells of each group. The results are shown in Figures 27 and 28, respectively.

[0929] As shown in Figure 27, the VG2A3X28-A63RCAR19-1 / 2 / 5 packaged by the packaging system containing 1 μg, 2 μg or 5 μg of the anti-FMC63-R binding plasmid can effectively transduce CD3 + CD28 + Jurkat cells, delivering and expressing the CAR-19 gene (positive rates were approximately 13.34%, 6.46%, and 2.16%, respectively).

[0930] As can be seen from Figure 28, compared with the control group Nalm-6 cells, the VG2A3X28-A63RCAR19-0 packaged by the packaging system without the addition of the anti-FMC63-R binding plasmid can still effectively transduce Nalm-6 cells (CD19 expression is significantly reduced); while the VG2A3X28-A63RCAR19-1 / 2 / 5 packaged by the packaging system with the addition of 1 μg, 2 μg or 5 μg of the anti-FMC63-R binding plasmid is difficult to effectively transduce Nalm6 cells (CD19 expression is effectively maintained), successfully controlling the off-target effect.

[0931] Example 10

[0932] Detect the killing efficiency of LVV in vivo and in vitro.

[0933] 1. Detecting the killing efficiency of LVV in vitro

[0934] A. Prepare 5 plasmid packaging systems to package LVV

[0935] Prepare the following five plasmids: the envelope plasmid 3, the pMDLg / pRRE packaging plasmid, the pRSV-REV packaging plasmid, the CD19 binding plasmid (2 μg), and the main plasmid CAR19;

[0936] Day 0: Package LVV, VG2A3X28-CAR19-2, according to the packaging method in Example 1; the VG2A3X28-CAR19-2 comprises the CAR-19 gene, and its viral envelope comprises the mutant VSV-G2 and the membrane-expressed anti-CD3×CD28 dual antibody.

[0937] B. Transduced PBMCs kill Nalm-6 cells in vitro

[0938] Day 0, prepare two groups of the following mixed cells: resuscitated 2 × 10 5 Cryopreserved non-activated PBMCs of healthy donors (human) were mixed with 1×10 5 CD19 +Nalm-6 cells were mixed (the mixed cell culture medium was 1640 culture medium + 10% FBS); according to MOI = 2, the VG2A3X28-CAR19-2 was added to one group of the mixed cells and the mixture was incubated in 5% CO 2、 Cells were cultured in a 37°C incubator;

[0939] Day 5, flow cytometry was used to detect the CD3 + The expression of FMC-63 / CAR-19 and CD19 in the cells are shown in Figure 29.

[0940] As shown in Figure 29, compared with the control group without LVV, the CD3 + The FMC-63 / CAR-19 positivity rate of the cells was approximately 17.31%, demonstrating that the VG2A3X28-CAR19-2 can effectively transduce T cells in non-activated PBMCs of healthy donors, deliver and express the CAR-19 molecule, and prepare CD19-targeted CAR-T cells (CD19-CAR-T cells);

[0941] Moreover, compared with the expression level of CD19 in the control group without LVV, the expression level of CD19 in the mixed cells with LVV was significantly reduced, which proves that the CD19-CAR-T cells prepared by VG2A3X28-CAR19-2 transduced non-activated PBMCs can effectively kill CD19 + Nalm-6 cells.

[0942] 2. Detecting the Killing Efficiency of LVV in Vivo

[0943] On Day 1, three groups of mice were selected, with three mice in each group, and Nalm-6 tumor cell models were established in each group of mice. Specifically, 1×10 6 Nalm-6-Luci (luciferase) cells, and in vivo imaging showed that the model was successfully established;

[0944] On Day 0, 1×10 7 Human non-activated PBMCs from healthy donors were obtained; subsequently, 1E6 TU of the VG2A3X28-CAR19-2 virus solution was injected into the tail vein of one of the mice injected with the PBMCs;

[0945] On Days 3, 7, 14, and 21, live imaging was performed on each group of mice to detect the growth of Nalm-6 tumor cells in each group of mice. The results are shown in FIG30 .

[0946] As shown in Figure 30, the VG2A3X28-CAR19-2 can effectively transduce non-activated T cells in human non-activated PBMCs in mice, prepare CAR-T cells and can efficiently kill Nalm-6 tumor cells in mice.

Claims

1. A packaging system for packaging a lentiviral vector (LVV) or a retroviral vector (RVV), characterized in that: (a) the shuttle gene (Transgene) contained in the packaging system comprises a polynucleotide encoding a chimeric antigen receptor (CAR); and (b) The packaging system comprises a polynucleotide encoding a CAR-binding molecule (CAR-Binding Molecule), and the CAR-binding molecule comprises a CAR-binding domain (CAR-Binding Domain), and the CAR-binding domain can bind to the CAR.

2. The packaging system according to claim 1, characterized in that The CAR binding region can bind to the CAR to reduce the availability of the antigen-binding CAR for expression on the cell membrane of the packaging cell when the packaging system packages LVV or RVV in the packaging cell.

3. The packaging system according to claim 1 or 2, characterized in that: In the cytoplasm and / or on the cell membrane of the packaging cell containing the packaging system, the CAR binding region of the CAR binding molecule can bind to the CAR and assemble into a complex molecule.

4. The packaging system according to claim 1, wherein: The antigen-binding region of the CAR comprises an antibody or an antigen-binding fragment thereof, and the antibody or the antigen-binding fragment thereof comprises an antibody epitope.

5. The packaging system according to claim 4, characterized in that The CAR binding region comprises an antigen, and the antigenic epitope of the antigen can bind to the antibody epitope.

6. The packaging system according to claim 5, characterized in that The antigen is selected from at least one of a full-length antigen, an extracellular domain of an antigen, a variable region of an antigen, and an antigen epitope.

7. The packaging system according to claim 5, characterized in that The antigen comprises (a) a full-length antigen or (b) a functional fragment of the antigen, wherein the functional fragment of the antigen at least comprises the antigen epitope; Optionally, the functional fragment of the antigen comprises at least the extracellular domain of the antigen; Optionally, the functional fragment of the antigen comprises the extracellular domain and the transmembrane region of the antigen.

8. The packaging system according to claim 4, characterized in that The CAR binding region comprises a first anti-antibody, which can bind to at least one CDR region (Complementarity-Determining Region) of the antibody epitope.

9. The packaging system according to any one of claims 4 to 8, characterized in that: The CAR binding region comprises (a) the antigen and (b) the first anti-antibody.

10. The packaging system according to any one of claims 5 to 9, characterized in that: The CAR binding molecule does not comprise a leader signal peptide.

11. The packaging system according to any one of claims 5 to 9, characterized in that: The CAR binding molecule further comprises a leader signal peptide, which is located at the N-terminal end of the CAR binding region; Preferably, the leader signal peptide is selected from any one of the following signal peptides: a natural leader signal peptide of an antigen, a CD8α signal peptide, a CD28 signal peptide, an IgG signal peptide, and an HLA-A signal peptide; More preferably, the leader signal peptide is the natural leader signal peptide of the antigen or the CD8α signal peptide.

12. The packaging system according to claim 11, characterized in that The CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, and the CAR binding region comprises (i) the antigen and / or (ii) the first anti-antibody.

13. The packaging system according to claim 11 or 12, characterized in that: The CAR binding molecule further comprises an endoplasmic reticulum retention signal peptide, wherein the endoplasmic reticulum retention signal peptide is located at the C-terminal end of the CAR binding region; Preferably, the N-terminus of the endoplasmic reticulum retention signal peptide is operably linked to the C-terminus of the CAR binding region.

14. The packaging system according to claim 13, characterized in that The endoplasmic reticulum retention signal peptide comprises the amino acid sequence (KDEL) shown in SEQ ID NO:

51.

15. The packaging system according to claim 13 or 14, characterized in that The CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, and (c) the endoplasmic reticulum retention signal peptide.

16. The packaging system according to claim 13 or 14, characterized in that The CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, (c) a polypeptide linker, and (d) the endoplasmic reticulum retention signal peptide; Preferably, the polypeptide linker is selected from: (a) an immunoglobulin hinge region selected from the group consisting of wild-type and modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (b) a hinge region selected from the wild-type and 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; (c) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; (d) a 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; and (e) flexible linker peptide; More preferably, the flexible connecting peptide is selected from (G4S) n Connector peptide, Connector peptide 1 and Connector peptide 3; wherein n=1 to 4; Connector peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23) Connector peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 137).

17. The packaging system according to claim 4, wherein: The CAR binding molecule further comprises (a) a leader signal peptide, (b) the CAR binding region and (c) an endoplasmic reticulum retention signal peptide; The CAR binding region comprises a second anti-antibody, which can bind to a region of the CAR that does not comprise the antibody epitope (non-antibody epitope region); Preferably, the endoplasmic reticulum retention signal peptide comprises the amino acid sequence (KDEL) as shown in SEQ ID NO:

51.

18. The packaging system according to any one of claims 4 to 17, characterized in that The antibody or antigen-binding fragment thereof is selected from the group consisting of: an immunoglobulin (full-length antibody), a half antibody, Fab, Fab', F(ab')2, an Fv fragment, a single-chain variable region fragment / single-chain antibody (scFv), a disulfide bond-stabilized antibody (dsFv), an antibody heavy chain variable region (VH) or light chain variable region (VL), an Fd fragment consisting of a VH and a CH1 domain, a linear antibody, and a single-domain antibody (nanoantibody VHH).

19. The packaging system according to any one of claims 1 to 18, characterized in that The antigen binding region of the CAR binds to a member selected from the group consisting of: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38, CD40, CD40 L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5TAC, TEM1 / CD 248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, Erb B4, MUC16, MAGE-3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, uPAR, GCC (guanylate cyclase C), EPCAM, Nectin4, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.

2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut At least one of hsp70-2, CD79α, CD79β, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, and NKG2D; Preferably, the antigen binding region of the CAR binds to at least one selected from the group consisting of: CD19, CD20, CD33, MSLN, CD79β, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2, B7H3 and GUCY2C.

20. The packaging system according to any one of claims 1 to 19, characterized in that The antibody or antigen-binding fragment thereof in the antigen-binding region of the CAR comprises an anti-CD19 antibody or antigen-binding fragment thereof that can bind to CD19; Preferably, the CD19 is human CD19 (UniProtKB No.: P15391).

21. The packaging system according to claim 20, characterized in that The CAR binding region comprises the antigen CD19 and / or an anti-anti-CD19 antibody or an antigen-binding fragment thereof (first anti-antibody, anti-anti-CD19 antibody).

22. The packaging system according to claim 21, characterized in that The antigen CD19 of the CAR binding region comprises its extracellular domain and transmembrane region.

23. The packaging system according to claim 22, characterized in that The antigen CD19 of the CAR binding region also includes its intracellular domain.

24. The packaging system according to any one of claims 20 to 23, characterized in that The anti-CD19 antibody or antigen-binding fragment thereof is a scFv derived from the monoclonal antibody FMC-63 (FMC63-scFv), the amino acid sequences of the HCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 94-96, respectively, and the amino acid sequences of the LCDR1-3 regions of the anti-CD19 antibody or antigen-binding fragment thereof are shown in SEQ ID NOs: 97-99, respectively.

25. The packaging system according to claim 24, characterized in that The amino acid sequences of the HCDR1-3 regions of the anti-anti-CD19 antibody are shown in SEQ ID NOs: 102-104, respectively, and the amino acid sequences of the LCDR1-3 regions of the anti-anti-CD19 antibody are shown in SEQ ID NOs: 105-107, respectively.

26. The packaging system according to any one of claims 1 to 25, characterized in that The transmembrane region of the CAR is derived from the transmembrane region of at least one of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; Preferably, the transmembrane region of the CAR is derived from the transmembrane region of CD8α or CD28; More preferably, the transmembrane region of the CAR is derived from the transmembrane region of CD8α.

27. The packaging system according to any one of claims 1 to 26, characterized in that The intracellular signaling domain of the CAR is derived from the intracellular signaling domain of at least one of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79α, CD79β, FcεRlγ, FcεRβ, FcγRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12, and other intracellular signaling domains of proteins containing at least one ITAM; Preferably, the intracellular signaling domain of the CAR is derived from the intracellular signaling domain of CD3ζ.

28. The packaging system according to any one of claims 1 to 27, characterized in that The CAR further comprises a costimulatory signaling domain; Preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of at least one of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRlγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-l, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88; More preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of 4-1BB and / or CD28.

29. The packaging system according to any one of claims 1 to 28, characterized in that The CAR further comprises a hinge region, which sequentially connects the antigen binding region and the transmembrane region of the CAR; Preferably, the hinge region is derived from the hinge region of at least one of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154; More preferably, the hinge region is derived from the hinge region of CD8α or CD28.

30. The packaging system according to any one of claims 1 to 25, characterized in that The region of the CAR that does not contain an antigen binding region (non-antigen binding region) contains a TCR / CD3 complex subunit-related polypeptide (TCR / CD3 Complex Subunit Related Peptide, "TSP") derived from a TCR / CD3 complex subunit, wherein the TSP contains at least one of a TCR / CD3 complex subunit, a TCR / CD3 complex subunit functional fragment, a TCR / CD3 complex subunit variant, and a variant of a TCR / CD3 complex subunit functional fragment; the antigen binding region is located in the N-terminal direction of the TSP.

31. The packaging system according to claim 30, wherein: The TCR / CD3 complex subunit is selected from at least one of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ζ and CD3ε.

32. The packaging system according to claim 31, wherein: The functional fragment of the TCR / CD3 complex subunit comprises at least one of the full-length protein, extracellular region, transmembrane region, intracellular region, variable region and constant region of the TCR / CD3 complex subunit.

33. The packaging system according to claim 32, wherein: The TSP comprises (a) CD3γ or a functional fragment thereof or (b) CD3ε or a functional fragment thereof.

34. The packaging system according to any one of claims 30 to 33, characterized in that The antigen binding region is connected to the N-terminus of the TSP via a connecting peptide; Preferably, the connecting peptide is selected from a flexible connecting peptide and a hinge region of a protein; More preferably, the flexible connecting peptide is selected from (G4S) n A connecting peptide and a connecting peptide 1 comprising the amino acid sequence shown in SEQ ID NO: 23; wherein n=1 to 4.

35. The packaging system according to claim 34, wherein: The flexible connecting peptide is the connecting peptide 1.

36. The packaging system according to any one of claims 30 to 35, characterized in that The CAR does not comprise a leader signal peptide.

37. The packaging system according to any one of claims 1 to 35, characterized in that The CAR comprises a leader signal peptide; Preferably, the leader signal peptide is selected from any one of the following signal peptides: CD8α signal peptide, CD28 signal peptide, IgG signal peptide, HLA-A signal peptide, CD3γ signal peptide, CD3δ signal peptide, CD3ζ signal peptide and CD3ε signal peptide; More preferably, the leader signal peptide is CD8α signal peptide.

38. The packaging system according to any one of claims 1 to 37, characterized in that The packaging system further comprises a polynucleotide encoding a T cell targeting molecule that can bind to a T cell surface receptor.

39. The packaging system according to claim 38, wherein: The T cell targeting molecule binds to CD3, CD5, CD7, CD28, TCRγ, TCRδ, TCRα or TCRβ.

40. The packaging system according to claim 39, wherein: The T cell targeting molecule binds to CD5 or CD7.

41. The packaging system according to claim 40, wherein: The T cell targeting molecule binds to CD7, and the T cell targeting molecule comprises at least one selected from (a) an anti-CD7 antibody or an antigen-binding fragment thereof and (b) a CD7 ligand or a binding fragment thereof; Preferably, the CD7 is human CD7 (UniProtKB No.: P09564); More preferably, the T cell targeting molecule comprises anti-CD7 scFv and / or VHH; Further preferably, the T cell targeting molecule includes an anti-CD7 scFv, which is derived from the monoclonal antibody TH-69 (TH69-scFv), and the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 87-89, respectively; the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 90-92, respectively.

42. The packaging system according to any one of claims 1 to 41, characterized in that The packaging system further comprises a polynucleotide encoding a T cell activation signaling molecule.

43. The packaging system according to claim 42, wherein: The T cell activation signal molecule includes a T cell activation primary signal molecule.

44. The packaging system according to claim 43, wherein: The T cell activation primary signal molecule can bind to at least one selected from TCR / CD3 complex subunits and TCR / CD3 complex subunit functional fragments; the TCR / CD3 complex subunits are selected from at least one of CD3ε, CD3γ, CD3δ, CD3ζ, TCRγ, TCRδ, TCRα and TCRβ.

45. The packaging system according to claim 43 or 44, characterized in that The T cell activation primary signal molecule can bind to CD3; Preferably, the T cell activation primary signal molecule can bind to human CD3, and the human CD3 is selected from human CD3ε / CD3E (UniProtKB No.: P07766), CD3γ / CD3G (UniProtKB No.: P09693), CD3δ / CD3D (UniProtKB No.: P04234) and CD3ζ / CD3Z (UniProtKB No.: P20963).

46. ​​The packaging system according to claim 45, wherein: The T cell activation primary signal molecule includes an anti-CD3 antibody or an antigen-binding fragment thereof; Preferably, the anti-CD3 antibody or antigen-binding fragment thereof is an anti-CD3 scFv and / or VHH.

47. The packaging system according to claim 46, wherein: The anti-CD3 antibody is selected from UCHT1 or a variant or derivative thereof, OKT3 or a variant or derivative thereof, SP34 or a variant or derivative thereof, HuM291 or a variant or derivative thereof, and TR66 or a variant or derivative thereof; Preferably, the anti-CD3 antibody is an anti-CD3ε scFv, and the anti-CD3ε scFv is derived from the monoclonal antibody UCHT1 (UCHT1-scFv); the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NOs: 72-74, respectively, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NOs: 75-77, respectively.

48. The packaging system according to any one of claims 42 to 47, characterized in that The T cell activation signaling molecules include T cell activation secondary signaling molecules.

49. The packaging system according to claim 48, characterized in that The T cell activation secondary signaling molecule can bind to CD28; Preferably, the T cell activation secondary signaling molecule can bind to human CD28 (UniProtKB No.: P10747).

50. The packaging system of claim 49, wherein: The T cell activation secondary signal molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof; Preferably, the CD28 ligand or its receptor binding fragment comprises CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.

51. The packaging system according to claim 50, characterized in that The T cell activation secondary signal molecule includes an anti-CD28 antibody or an antigen-binding fragment thereof; Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is an anti-CD28 scFv; the anti-CD28 scFv is derived from the monoclonal antibody 15E8 (15E8-scFv); the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 80-82, respectively; the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 83-85, respectively.

52. The packaging system according to any one of claims 38 to 51, characterized in that The T cell targeting molecule further comprises an adhesion molecule; preferably, the adhesion molecule can bind to CD2; more preferably, the adhesion molecule comprises CD58 or its extracellular domain or a functional fragment thereof.

53. The packaging system according to any one of claims 38 to 52, characterized in that The T cell targeting molecule and / or T cell activation signaling molecule further comprises a transmembrane domain, and the transmembrane domain is anchored on the surface of the LVV or RVV packaged in the packaging cell by the packaging system.

54. The packaging system according to claim 53, wherein: The transmembrane domain is selected from the transmembrane regions of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; Preferably, the transmembrane domain is the CD8α transmembrane region; More preferably, the CD8α transmembrane region is a human CD8α transmembrane region, and the amino acid sequence of the human CD8α transmembrane region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:

15.

55. The packaging system according to claim 53 or 54, characterized in that The T cell targeting molecule and / or T cell activation signaling molecule further comprises a linker domain, which connects the extracellular domain of the T cell targeting molecule and / or T cell activation signaling molecule and the transmembrane domain.

56. The packaging system of claim 55, wherein: The connecting domain is selected from the group consisting of: (a) immunoglobulin hinge region, (b) the immunoglobulin hinge region is selected from wild-type and modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions; (c) a hinge region selected from the wild-type and 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; (d) all or a portion of an Fc domain, wherein the Fc domain is selected from at least one of a CH1 domain, a CH2 domain, and a CH3 domain; (e) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and (f) flexible linker peptide; Preferably, the connecting domain is the CD8α hinge region; More preferably, the CD8α hinge region is a human CD8α hinge region, and the amino acid sequence of the human CD8α hinge region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:

14.

57. The packaging system according to any one of claims 1 to 56, characterized in that The polynucleotide encoding the envelope glycoprotein contained in the packaging system encodes any one of the following envelope glycoproteins: the envelope glycoprotein of the vesicular stomatitis virus strain and its variants, the envelope glycoprotein of the baboon endogenous retrovirus BaEV and its variants, the envelope glycoprotein RD114 of the feline endogenous retrovirus and its variants, and the envelope glycoprotein GALV of the gibbon ape leukemia virus and its variants.

58. The packaging system of claim 57, wherein: The envelope glycoprotein is selected from any one of the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof; Preferably, the envelope glycoprotein of the vesicular stomatitis virus strain and its variants include the following envelope glycoproteins and their variants: envelope glycoprotein of the vesicular stomatitis virus Indiana strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Cocal strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Maraba strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Morreton strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Alagoas strain and its variants, envelope glycoprotein of the vesicular stomatitis virus New The envelope glycoprotein of Jersey strain and its variants, the envelope glycoprotein of Carajas strain and its variants, the envelope glycoprotein of Chandipura strain and its variants, the envelope glycoprotein of Eptesicus strain and its variants, the envelope glycoprotein of Isfahan strain and its variants, the envelope glycoprotein of Jurona strain and its variants, the envelope glycoprotein of Malpais strain and its variants, the envelope glycoprotein of Perinet strain and its variants, the envelope glycoprotein of Piry strain and its variants, the envelope glycoprotein of Radi strain and its variants, the envelope glycoprotein of Rhinolopus strain and its variants, and the envelope glycoprotein of Yug Bogdanovac strain and its variants.

59. The packaging system according to claim 58, characterized in that The envelope glycoprotein is the envelope glycoprotein of the Indiana strain of the vesicular stomatitis virus genus (VSV-G) or its variant, or the envelope glycoprotein of the Cocal strain (Cocal-G) or its variant, and the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

60. The packaging system according to any one of claims 57 to 59, characterized in that The envelope glycoprotein undergoes a first mutation, which reduces or loses the ability of the envelope glycoprotein to bind to the envelope glycoprotein receptor compared to before the first mutation occurs.

61. The packaging system according to claim 60, characterized in that The envelope glycoprotein is selected from the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof.

62. The packaging system according to claim 61, characterized in that The envelope glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the envelope glycoprotein receptor is the low-density lipoprotein receptor LDL-R; the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

63. The packaging system according to claim 62, characterized in that The first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 209, substitution or deletion of amino acid at position 347 in SEQ ID NO: 1 or SEQ ID NO:

2. the substitution or deletion of the amino acid at position 350, the substitution or deletion of the amino acid at position 352, the substitution or deletion of the amino acid at position 353, the substitution of the amino acid at position 354, the deletion of amino acids at positions 1-18, the deletion of amino acids at positions 19-36, the deletion of amino acids at positions 37-51, the deletion of amino acids at positions 314-384, the deletion of amino acids at positions 321-374, the deletion of amino acids at positions 331-364, the deletion of amino acids at positions 344-354, and the deletion of amino acids at positions 345-353; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, substitution or deletion of amino acid 8, substitution or deletion of amino acid 9, substitution or deletion of amino acid 10, substitution or deletion of amino acid 47, substitution or deletion of amino acid 50, substitution or deletion of amino acid 51, substitution or deletion of amino acid 183, substitution or deletion of amino acid 179, substitution or deletion of amino acid 180, substitution or deletion of amino acid 182, substitution or deletion of amino acid 184, substitution or deletion of amino acid 209, substitution or deletion of amino acid 347, substitution or deletion of amino acid 358, substitution or deletion of amino acid 360, substitution or deletion of amino acid 361, substitution or deletion of amino acid 362, substitution or deletion of amino acid 364, substitution or deletion of amino acid 365, substitution or deletion of amino acid 366, substitution or deletion of amino acid 367, substitution or deletion of amino acid 368, substitution or deletion of amino acid 369, substitution or deletion of amino acid 370, substitution or deletion of amino acid 371, substitution or deletion of amino acid 372, substitution or deletion of amino acid 373, substitution or deletion of amino acid 374, substitution or deletion of amino acid 375, substitution or deletion of amino acid 376, substitution or deletion of amino acid 377, substitution or deletion of amino acid 378, substitution or deletion of amino acid 379, substitution or deletion of amino acid 371, substitution or deletion of amino acid 377 the substitution or deletion of the amino acid at position 350, the substitution or deletion of the amino acid at position 352, the substitution or deletion of the amino acid at position 353, the substitution of the amino acid at position 354, the deletion of amino acids at positions 1-18, the deletion of amino acids at positions 19-36, the deletion of amino acids at positions 37-51, the deletion of amino acids at positions 314-384, the deletion of amino acids at positions 321-374, the deletion of amino acids at positions 331-364, the deletion of amino acids at positions 344-354, and the deletion of amino acids at positions 345-353; Preferably, the first mutation comprises 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, 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; (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; (c) substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, 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: 2; and (d) After optimal global alignment with SEQ ID NO: 2, the position corresponding to SEQ ID NO:2: substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, 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 comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 1; (b) after optimal global alignment with SEQ ID NO: 1, substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353, corresponding to SEQ ID NO: 1; (c) 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; and (d) 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.

64. The packaging system according to claim 63, characterized in that The first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) deletion of amino acids 331-364, deletion of amino acids 344-354, substitution or deletion of K47, or substitution of R354 in SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, deletion of amino acids 331-364, deletion of amino acids 344-354, substitution or deletion of K47, substitution of R354 at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acids 331-364 of SEQ ID NO: 1 or SEQ ID NO: 2 are deleted, amino acids 344-354 are deleted, amino acid 47 is substituted from lysine K to glutamine Q (K47Q), or K47 is deleted, and amino acid 354 is substituted from arginine R to glutamine Q (R354Q); (b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, amino acid deletions at positions 331-364, amino acid deletions at positions 344-354, K47Q or K47 deletion, R354Q are located corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

65. The packaging system of claim 64, wherein: The first mutation includes a mutation in which the amino acid sequence comprises the following amino acids: (a) a deletion of K47 located at SEQ ID NO: 1 or SEQ ID NO: 2; or (b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, the K47 deletion is located at the position corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

66. The packaging system of claim 62, wherein: The first mutation includes a mutation in which the amino acid sequence comprises the following amino acids: (a) a mutation at I331 of SEQ ID NO: 1 or SEQ ID NO: 2; or (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at position I331 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the mutation of I331 is selected from the group consisting of: I331E, I331W, I331M, I331L, I331Q, and I331R.

67. The packaging system of claim 63, wherein: The extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 33 or SEQ ID NO:

34.

68. The packaging system according to any one of claims 57 to 67, characterized in that The envelope glycoprotein undergoes a second mutation, which enhances the ability of the envelope glycoprotein to antagonize inactivation by complement, or prevents the envelope glycoprotein from being inactivated by complement, compared with before the second mutation occurs.

69. The packaging system according to claim 68, characterized in that The envelope glycoprotein is an envelope glycoprotein of the Indiana strain or Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

70. The packaging system of claim 69, wherein: The second mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 146 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the amino acid mutation includes at least one of amino acid deletion, insertion and substitution; More preferably, the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the amino acid position corresponding to 146 of SEQ ID NO: 1 or SEQ ID NO:

2.

71. The packaging system of claim 70, wherein: The second mutation includes an amino acid sequence as shown in SEQ ID NO: 1 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1, comprising at least one of the following position mutations: (a) substitution of T214, substitution of T352, substitution of K50, or substitution of S146 in SEQ ID NO: 1; and (b) after optimal global alignment with SEQ ID NO: 1, substitutions at T214, T352, K50, and S146 corresponding to SEQ ID NO: 1; Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence: (a) amino acid position 214 in SEQ ID NO: 1 is substituted from threonine T to asparagine N (T214N), amino acid position 352 is substituted from threonine T to alanine A (T352A), amino acid position 50 is substituted from lysine K to threonine T (K50T), and amino acid position 146 is substituted from serine S to threonine T (S146T); and (b) After optimal global alignment with SEQ ID NO: 1, T214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO:

1.

72. The packaging system of claim 71, wherein: The second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) substitution of (i) T214 and T352; or (ii) T214, T352, K50, and S146 of SEQ ID NO: 1; and (b) after optimal global alignment with SEQ ID NO: 1, substitutions at positions corresponding to (i) T214 and T352; or (ii) T214, T352, K50, and S146 of SEQ ID NO: 1; Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) (i) K214N and T352A; or (ii) K214N, T352A, K50T, and S146T at SEQ ID NO: 2; (b) After optimal global alignment with SEQ ID NO: 2, located at (i) K214N and T352A; or (ii) K214N, T352A, K50T, and S146T corresponding to SEQ ID NO:

2. Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) (i) T214N and T352A; or (ii) T214N, T352A, K50T, and S146T at SEQ ID NO: 1; (b) After optimal global alignment with SEQ ID NO: 1, located at (i) T214N and T352A; or (ii) T214N, T352A, K50T, and S146T corresponding to SEQ ID NO:

1.

73. The packaging system of claim 70, wherein: The second mutation includes an amino acid sequence as shown in SEQ ID NO: 2 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 2, comprising at least one of the following position mutations: (a) substitution of K214, substitution of T352, substitution of K50, substitution of S146 in SEQ ID NO: 2; and (b) after optimal global alignment with SEQ ID NO: 2, substitutions at positions corresponding to K214, T352, K50, and S146 of SEQ ID NO: 2; Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence: (a) amino acid position 214 of SEQ ID NO: 2 is replaced by lysine K to asparagine N (K214N), T352A, K50T, S146T; and (b) After optimal global alignment with SEQ ID NO: 2, K214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO:

2.

74. [Corrected 09.05.2025 according to Rule 26] The packaging system according to claim 73, characterized in that The second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) substitution of (i) K214 and T352; or (ii) K214, T352, K50, and S146 of SEQ ID NO: 2; and (b) After optimal global alignment with SEQ ID NO: 2, the substitutions are located at (i) K214 and T352; or (ii) K214, T352, K50, and S146, which are equivalent to SEQ ID NO:

2.

75. The packaging system of claim 70, wherein: The extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:

38.

76. The packaging system according to any one of claims 1 to 75, characterized in that The content of CAR available for binding to an antigen on the viral envelope of the LVV or RVV packaged in the packaging cells by the packaging system is reduced or does not contain the CAR available for binding to an antigen.

77. The packaging system according to any one of claims 1 to 76, characterized in that The packaging system comprises one or more nucleic acid vectors, which, when introduced into a packaging cell for packaging a lentiviral vector or a retroviral vector, contain nucleic acids necessary for producing, assembling and / or packaging the lentiviral vector or the retroviral vector in the packaging cell.

78. The packaging system according to any one of claims 1 to 77, characterized in that The packaging system further comprises: (a) Gag gene and Pol gene; and (b) (i) the LVV backbone gene and the Rev gene; or (ii) RVV backbone gene.

79. The packaging system according to any one of claims 1 to 78, characterized in that The packaging system comprises: at least one packaging plasmid, an envelope plasmid, a shuttle plasmid and a binding plasmid; the binding plasmid comprises a polynucleotide encoding the CAR binding molecule.

80. The packaging system of claim 79, wherein: (a) the packaging plasmid comprises the Gag gene and the Pol gene (Gag / Pol packaging plasmid); (b) the envelope plasmid comprises (i) a polynucleotide encoding the envelope glycoprotein or a variant thereof and (ii) a polynucleotide encoding the (1) T cell targeting molecule and / or (2) a polynucleotide encoding the T cell activation signaling molecule; and (c) The shuttle plasmid comprises (i) the shuttle gene and (ii): (1) LVV backbone gene or (2) RVV backbone gene.

81. The packaging system according to claim 79 or 80, characterized in that When the packaging system is used to package LVV, the packaging system further comprises a Rev packaging plasmid, and the Rev packaging plasmid comprises the Rev gene.

82. An LVV or RVV, characterized in that The LVV or RVV is packaged in a packaging cell by the packaging system of any one of claims 1-81.

83. The LVV or RVV according to claim 82, wherein: The content of CAR available for binding to the antigen on the cell membrane of the packaging cell is reduced or the CAR is absent.

84. The LVV or RVV according to claim 82 or 83, wherein: The viral envelope of the LVV or RVV has a reduced content of the CAR available for binding to the antigen or does not contain the CAR.

85. A complex molecule, characterized in that The complex molecule comprises the following components (a) and (b): (a) a CAR binding molecule comprising a CAR binding region; and (b)CAR; The CAR binding region of the CAR binding molecule binds to the CAR and assembles into the complex molecule.

86. The complex molecule according to claim 85, characterized in that The antigen binding region of the CAR comprises an antibody or an antigen binding fragment thereof, and the antibody or the antigen binding fragment thereof comprises an antibody epitope.

87. The complex molecule according to claim 85 or 86, characterized in that The CAR binding region comprises: (a) antigens; and / or (b) Primary anti-antibody.

88. The complex molecule according to claim 87, characterized in that The antigenic epitope of the antigen in the CAR binding region can bind to the antibody epitope.

89. The complex molecule according to claim 88, characterized in that The antigen in the CAR binding region comprises (a) a full-length antigen or (b) a functional fragment of an antigen, wherein the functional fragment of the antigen at least comprises the antigen epitope; Optionally, the functional fragment of the antigen comprises at least the extracellular domain of the antigen; Optionally, the functional fragment of the antigen comprises the extracellular domain and the transmembrane region of the antigen.

90. The complex molecule according to any one of claims 87 to 89, characterized in that The first anti-antibody can bind to at least one CDR region of the antibody epitope.

91. The complex molecule according to any one of claims 87 to 90, characterized in that The antigen binding region of the CAR and / or the first anti-antibody can specifically bind to the antibody or antigen binding fragment thereof of the antigen binding region of the CAR.

92. The complex molecule according to any one of claims 87 to 91, characterized in that The CAR binding molecule comprises, from N-terminus to C-terminus or C-terminus to N-terminus: (a) the antigen or the first anti-antibody; (b) (i) the antigen, (ii) the first anti-antibody; or (c) (i) the antigen, (ii) a polypeptide linker, and (iii) the first anti-antibody.

93. The complex molecule according to any one of claims 86 to 92, characterized in that (a) the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR may bind to an antigen selected from the group consisting of: and / or (b) the antigen binding region of the CAR may be selected from the group consisting of: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5TAC, 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-3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, uPAR, GCC, EPCAM, Nectin4, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.

2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut At least one of hsp70-2, CD79α, CD79β, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, and NKG2D; Preferably, (a) the antibody or antigen-binding fragment thereof of the antigen-binding region of the CAR can bind to and / or (b) the antigen of the CAR binding region can be selected from: at least one of CD19, CD20, CD33, MSLN, CD79β, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2, B7H3 and GUCY2C.

94. The complex molecule according to any one of claims 86 to 93, characterized in that The antibody or antigen-binding fragment thereof in the antigen-binding region of the CAR comprises an anti-CD19 antibody or antigen-binding fragment thereof that can bind to CD19; Preferably, the CD19 is human CD19 (UniProtKB No.: P15391).

95. The complex molecule according to claim 94, characterized in that The CAR binding region comprises the antigen CD19 and / or an anti-anti-CD19 antibody or an antigen-binding fragment thereof (first anti-antibody, anti-anti-CD19 antibody).

96. The complex molecule according to claim 95, characterized in that The antigen CD19 of the CAR binding region comprises its extracellular domain and transmembrane region.

97. The complex molecule according to claim 96, characterized in that The antigen CD19 of the CAR binding region also includes its intracellular domain.

98. The complex molecule according to any one of claims 94 to 97, characterized in that The anti-CD19 antibody or its antigen-binding fragment is derived from the scFv of the monoclonal antibody FMC-63 (FMC63-scFv), the amino acid sequences of the HCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 94-96, respectively, and the amino acid sequences of the LCDR1-3 regions of the anti-CD19 antibody or its antigen-binding fragment are shown in SEQ ID NOs: 97-99, respectively.

99. The complex molecule according to claim 98, characterized in that The amino acid sequences of the HCDR1-3 regions of the anti-anti-CD19 antibody are shown in SEQ ID NOs: 102-104, respectively, and the amino acid sequences of the LCDR1-3 regions of the anti-anti-CD19 antibody are shown in SEQ ID NOs: 105-107, respectively.

100. The complex molecule according to any one of claims 85 to 99, characterized in that The transmembrane region of the CAR is derived from the transmembrane region of at least one of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; Preferably, the transmembrane region is derived from the transmembrane region of CD8α or CD28.

101. The complex molecule according to any one of claims 85-100, characterized in that The intracellular signaling domain of the CAR is derived from the intracellular signaling domain of at least one of the following proteins: CD3ε, CD3γ, CD3δ, CD3ζ, CD79α, CD79β, FcεRlγ, FcεRβ, FcγRⅡa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP12, and other intracellular signaling domains of proteins containing at least one ITAM; Preferably, the intracellular signaling domain is derived from the intracellular signaling domain of CD3ζ.

102. The complex molecule according to any one of claims 85 to 101, characterized in that The CAR further comprises a costimulatory signaling domain; Preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of at least one of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRlγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-l, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88; More preferably, the costimulatory signaling domain is derived from the costimulatory signaling domain of 4-1BB and / or CD28.

103. The complex molecule according to any one of claims 85 to 102, characterized in that The CAR further comprises a hinge region, which sequentially connects the antigen binding region and the transmembrane region of the CAR; Preferably, the hinge region is derived from the hinge region of at least one of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154; More preferably, the hinge region is derived from the hinge region of CD8α or CD28.

104. The complex molecule according to any one of claims 85-99, characterized in that The region of the CAR that does not contain an antigen binding region (non-antigen binding region) contains a TCR / CD3 complex subunit-associated polypeptide (TSP) derived from a TCR / CD3 complex subunit, and the TSP contains at least one of a TCR / CD3 complex subunit, a TCR / CD3 complex subunit functional fragment, a TCR / CD3 complex subunit variant, and a variant of a TCR / CD3 complex subunit functional fragment; the antigen binding region is located at the N-terminal end of the TSP.

105. The complex molecule according to claim 104, characterized in that The TCR / CD3 complex subunit is selected from at least one of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ζ and CD3ε.

106. The complex molecule according to claim 105, characterized in that The functional fragment of the TCR / CD3 complex subunit comprises at least one of the full-length protein, extracellular region, transmembrane region, intracellular region, variable region and constant region of the TCR / CD3 complex subunit.

107. The complex molecule according to claim 106, characterized in that The TSP comprises (a) CD3γ or a functional fragment thereof or (b) CD3ε or a functional fragment thereof.

108. The complex molecule according to any one of claims 104-107, characterized in that The antigen binding region is connected to the N-terminus of the TSP via a connecting peptide; Preferably, the connecting peptide is selected from a flexible connecting peptide and a hinge region of a protein; More preferably, the flexible connecting peptide is selected from (G4S) n A connecting peptide and a connecting peptide 1 comprising the amino acid sequence shown in SEQ ID NO: 23; wherein n=1 to 4; More preferably, the flexible connecting peptide is the connecting peptide 1.

109. The complex molecule according to any one of claims 104-108, characterized in that The CAR does not comprise a leader signal peptide.

110. The complex molecule according to any one of claims 85 to 109, characterized in that The CAR comprises a leader signal peptide; Preferably, the leader signal peptide is selected from any one of the following signal peptides: CD8α signal peptide, CD28 signal peptide, IgG signal peptide, HLA-A signal peptide, CD3γ signal peptide, CD3δ signal peptide, CD3ζ signal peptide and CD3ε signal peptide; More preferably, the leader signal peptide is CD8α signal peptide.

111. The complex molecule according to any one of claims 85 to 110, characterized in that The CAR binding region comprises a second anti-antibody that can bind to a region of the CAR that does not comprise the antibody epitope (non-antibody epitope region).

112. The complex molecule according to any one of claims 85 to 111, characterized in that The complex molecules are derived from the cell membrane of the packaging cells that package LVV or RVV.

113. The complex molecule according to any one of claims 85 to 112, characterized in that The CAR binding molecule comprises a leader signal peptide, which is located at the N-terminal end of the CAR binding region; Preferably, the leader signal peptide is selected from any one of the following signal peptides: a natural leader signal peptide of an antigen, a CD8α signal peptide, a CD28 signal peptide, an IgG signal peptide, and an HLA-A signal peptide; More preferably, the leader signal peptide is the natural leader signal peptide of the antigen or the CD8α signal peptide.

114. The complex molecule according to claim 113, characterized in that The CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, the CAR binding region comprising (i) the antigen, (ii) the first anti-antibody and (iii) at least one of the second anti-antibody.

115. The complex molecule according to claim 113 or 114, characterized in that The CAR binding molecule further comprises an endoplasmic reticulum retention signal peptide, wherein the endoplasmic reticulum retention signal peptide is located at the C-terminal end of the CAR binding region; Preferably, the endoplasmic reticulum retention signal peptide comprises the amino acid sequence shown in SEQ ID NO: 51 (KDEL).

116. The complex molecule according to any one of claims 115, characterized in that The CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, and (c) the endoplasmic reticulum retention signal peptide.

117. The complex molecule according to any one of claims 115, characterized in that The CAR binding molecule further comprises a polypeptide linker, and the CAR binding region is connected to the endoplasmic reticulum retention signal peptide through the polypeptide linker; the CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, (c) the polypeptide linker, and (d) the endoplasmic reticulum retention signal peptide; Preferably, the polypeptide linker is selected from: (a) an immunoglobulin hinge region selected from the group consisting of wild-type and modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (b) a hinge region selected from the wild-type and 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; (c) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; (d) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and (e) flexible linker peptide; More preferably, the flexible connecting peptide is selected from (G4S) n Connector peptide, Connector peptide 1 and Connector peptide 3; wherein n=1 to 4; Connector peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23) Connector peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 137).

118. The complex molecule according to any one of claims 85 to 112, characterized in that The CAR binding molecule does not comprise a leader signal peptide.

119. A LVV or RVV, characterized in that: The LVV or RVV comprises at least one complex molecule according to any one of claims 85 to 112 displayed on its surface.

120. The LVV or RVV according to claim 119, wherein: The LVV or RVV comprises a shuttle gene comprising a polynucleotide encoding a CAR.

121. The LVV or RVV according to claim 119 or 120, wherein: The complex molecule is derived from the cell membrane of the packaging cell that packages the LVV or RVV.

122. The LVV or RVV according to any one of claims 119 to 121, wherein: The LVV or RVV further comprises a T cell targeting molecule displayed on its surface, and the T cell targeting molecule can bind to a T cell surface receptor.

123. The LVV or RVV according to claim 122, wherein: The T cell targeting molecule binds to CD3, CD5, CD7, CD28, TCRγ, TCRδ, TCRα or TCRβ.

124. The LVV or RVV according to claim 123, wherein: The T cell targeting molecule binds to CD5 or CD7.

125. The LVV or RVV according to claim 124, wherein: The T cell targeting molecule binds to CD7, and the T cell targeting molecule comprises at least one selected from (a) an anti-CD7 antibody or an antigen-binding fragment thereof and (b) a CD7 ligand or a binding fragment thereof; Preferably, the CD7 is human CD7 (UniProtKB No.: P09564); More preferably, the T cell targeting molecule comprises anti-CD7 scFv and / or VHH; Further preferably, the T cell targeting molecule includes an anti-CD7 scFv, which is derived from the monoclonal antibody TH-69 (TH69-scFv), and the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 87-89, respectively; the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NOs: 90-92, respectively.

126. The LVV or RVV according to any one of claims 119 to 125, wherein: The LVV or RVV further comprises a T cell activation signaling molecule displayed on its surface.

127. The LVV or RVV according to claim 126, wherein: The T cell activation signal molecule includes a T cell activation primary signal molecule.

128. The LVV or RVV according to claim 127, wherein: The T cell activation primary signal molecule can bind to at least one selected from TCR / CD3 complex subunits and TCR / CD3 complex subunit functional fragments; the TCR / CD3 complex subunits are selected from at least one of CD3ε, CD3γ, CD3δ, CD3ζ, TCRγ, TCRδ, TCRα and TCRβ.

129. The LVV or RVV according to claim 127 or 128, wherein: The T cell activation primary signal molecule can bind to CD3; Preferably, the T cell activation primary signal molecule can bind to human CD3, and the human CD3 is selected from human CD3ε / CD3E (UniProtKB No.: P07766), CD3γ / CD3G (UniProtKB No.: P09693), CD3δ / CD3D (UniProtKB No.: P04234) and CD3ζ / CD3Z (UniProtKB No.: P20963).

130. The LVV or RVV according to claim 129, wherein: The T cell activation primary signal molecule includes an anti-CD3 antibody or an antigen-binding fragment thereof; Preferably, the anti-CD3 antibody or antigen-binding fragment thereof is an anti-CD3 scFv and / or VHH.

131. The LVV or RVV according to claim 130, wherein: The anti-CD3 antibody is selected from UCHT1 or a variant or derivative thereof, OKT3 or a variant or derivative thereof, SP34 or a variant or derivative thereof, HuM291 or a variant or derivative thereof, and TR66 or a variant or derivative thereof; Preferably, the anti-CD3 antibody is an anti-CD3ε scFv, and the anti-CD3ε scFv is derived from the monoclonal antibody UCHT1 (UCHT1-scFv); the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NOs: 72-74, respectively, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NOs: 75-77, respectively.

132. The LVV or RVV according to any one of claims 126-131, characterized in that The T cell activation signaling molecules include T cell activation secondary signaling molecules.

133. The LVV or RVV according to claim 132, characterized in that The T cell activation secondary signaling molecule can bind to CD28; Preferably, the T cell activation secondary signaling molecule can bind to human CD28 (UniProtKB No.: P10747).

134. The LVV or RVV according to claim 133, wherein: The T cell activation secondary signal molecule is selected from at least one of an anti-CD28 antibody or an antigen-binding fragment thereof and a CD28 ligand or a receptor-binding fragment thereof; Preferably, the CD28 ligand or its receptor binding fragment comprises CD80 or its receptor binding fragment and CD86 or its receptor binding fragment.

135. The LVV or RVV according to claim 134, wherein: The T cell activation secondary signal molecule includes an anti-CD28 antibody or an antigen-binding fragment thereof; Preferably, the anti-CD28 antibody or antigen-binding fragment thereof is an anti-CD28 scFv; the anti-CD28 scFv is derived from the monoclonal antibody 15E8 (15E8-scFv); the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 80-82, respectively; the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NOs: 83-85, respectively.

136. The LVV or RVV according to any one of claims 122-125, characterized in that The T cell targeting molecule further comprises an adhesion molecule; preferably, the adhesion molecule can bind to CD2; more preferably, the adhesion molecule comprises CD58 or its extracellular domain or a functional fragment thereof.

137. The LVV or RVV according to any one of claims 122-136, wherein: The T cell targeting molecule and / or T cell activation signaling molecule further comprises a transmembrane domain, which is anchored on the surface of the LVV or RVV.

138. The LVV or RVV according to claim 137, wherein: The transmembrane domain is selected from the transmembrane regions of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD 33. CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40, ICOS, ICA M-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, BTLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D, and CS1; Preferably, the transmembrane domain is the CD8α transmembrane region; More preferably, the CD8α transmembrane region is a human CD8α transmembrane region, and the amino acid sequence of the human CD8α transmembrane region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:

15.

139. The LVV or RVV according to claim 137 or 138, wherein: The T cell targeting molecule and / or T cell activation signaling molecule further comprises a linker domain, which connects the extracellular domain of the T cell targeting molecule and / or T cell activation signaling molecule and the transmembrane domain.

140. The LVV or RVV according to claim 139, wherein: The connecting domain is selected from the group consisting of: (a) an immunoglobulin hinge region selected from the group consisting of wild-type and modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (b) a hinge region selected from the wild-type and 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; (c) all or a portion of an Fc domain, wherein the Fc domain is selected from at least one of a CH1 domain, a CH2 domain, and a CH3 domain; (d) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and (e) flexible linker peptide; Preferably, the connecting domain is the CD8α hinge region; More preferably, the CD8α hinge region is a human CD8α hinge region, and the amino acid sequence of the human CD8α hinge region is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical to SEQ ID NO:

14.

141. The LVV or RVV according to any one of claims 119-140, wherein: The envelope glycoprotein of the LVV or RVV is selected from any one of the following envelope glycoproteins: the envelope glycoprotein of the vesicular stomatitis virus strain and its variants, the envelope glycoprotein of the baboon endogenous retrovirus BaEV and its variants, the envelope glycoprotein RD114 of the feline endogenous retrovirus and its variants, and the envelope glycoprotein GALV of the gibbon ape leukemia virus and its variants.

142. The LVV or RVV according to claim 141, wherein The envelope glycoprotein is selected from any one of the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof; Preferably, the envelope glycoprotein of the vesicular stomatitis virus strain and its variants include the following envelope glycoproteins and their variants: envelope glycoprotein of the vesicular stomatitis virus Indiana strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Cocal strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Maraba strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Morreton strain and its variants, envelope glycoprotein of the vesicular stomatitis virus Alagoas strain and its variants, envelope glycoprotein of the vesicular stomatitis virus New The envelope glycoprotein of Jersey strain and its variants, the envelope glycoprotein of Carajas strain and its variants, the envelope glycoprotein of Chandipura strain and its variants, the envelope glycoprotein of Eptesicus strain and its variants, the envelope glycoprotein of Isfahan strain and its variants, the envelope glycoprotein of Jurona strain and its variants, the envelope glycoprotein of Malpais strain and its variants, the envelope glycoprotein of Perinet strain and its variants, the envelope glycoprotein of Piry strain and its variants, the envelope glycoprotein of Radi strain and its variants, the envelope glycoprotein of Rhinolopus strain and its variants, and the envelope glycoprotein of Yug Bogdanovac strain and its variants.

143. The LVV or RVV according to claim 142, wherein: The envelope glycoprotein is the envelope glycoprotein of the Indiana strain (VSV-G) or the Cocal strain (Cocal-G) of the genus Vesicular Stomatitis Virus or a variant thereof, and the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

144. The LVV or RVV according to any one of claims 141-143, wherein The envelope glycoprotein undergoes a first mutation, which reduces or loses the ability of the envelope glycoprotein to bind to the envelope glycoprotein receptor compared to before the first mutation occurs.

145. The LVV or RVV according to claim 144, wherein: The envelope glycoprotein is selected from the envelope glycoproteins of vesicular stomatitis virus strains and variants thereof.

146. The LVV or RVV according to claim 145, characterized in that The envelope glycoprotein is the envelope glycoprotein of the Indiana strain or the Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the envelope glycoprotein receptor is the low-density lipoprotein receptor LDL-R; the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

147. The LVV or RVV according to claim 146, wherein: The first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution or deletion of amino acid at position 8, substitution or deletion of amino acid at position 9, substitution or deletion of amino acid at position 10, substitution or deletion of amino acid at position 47, substitution or deletion of amino acid at position 50, substitution or deletion of amino acid at position 51, substitution or deletion of amino acid at position 183, substitution or deletion of amino acid at position 179, substitution or deletion of amino acid at position 180, substitution or deletion of amino acid at position 182, substitution or deletion of amino acid at position 184, substitution or deletion of amino acid at position 209, substitution or deletion of amino acid at position 347 in SEQ ID NO: 1 or SEQ ID NO:

2. the substitution or deletion of the amino acid at position 350, the substitution or deletion of the amino acid at position 352, the substitution or deletion of the amino acid at position 353, the substitution of the amino acid at position 354, the deletion of amino acids at positions 1-18, the deletion of amino acids at positions 19-36, the deletion of amino acids at positions 37-51, the deletion of amino acids at positions 314-384, the deletion of amino acids at positions 321-374, the deletion of amino acids at positions 331-364, the deletion of amino acids at positions 344-354, and the deletion of amino acids at positions 345-353; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, substitution or deletion of amino acid 8, substitution or deletion of amino acid 9, substitution or deletion of amino acid 10, substitution or deletion of amino acid 47, substitution or deletion of amino acid 50, substitution or deletion of amino acid 51, substitution or deletion of amino acid 183, substitution or deletion of amino acid 179, substitution or deletion of amino acid 180, substitution or deletion of amino acid 182, substitution or deletion of amino acid 184, substitution or deletion of amino acid 209, substitution or deletion of amino acid 347, substitution or deletion of amino acid 358, substitution or deletion of amino acid 360, substitution or deletion of amino acid 361, substitution or deletion of amino acid 362, substitution or deletion of amino acid 364, substitution or deletion of amino acid 365, substitution or deletion of amino acid 366, substitution or deletion of amino acid 367, substitution or deletion of amino acid 368, substitution or deletion of amino acid 369, substitution or deletion of amino acid 370, substitution or deletion of amino acid 371, substitution or deletion of amino acid 372, substitution or deletion of amino acid 373, substitution or deletion of amino acid 374, substitution or deletion of amino acid 375, substitution or deletion of amino acid 376, substitution or deletion of amino acid 377, substitution or deletion of amino acid 378, substitution or deletion of amino acid 379, substitution or deletion of amino acid 371, substitution or deletion of amino acid 377 the substitution or deletion of the amino acid at position 350, the substitution or deletion of the amino acid at position 352, the substitution or deletion of the amino acid at position 353, the substitution of the amino acid at position 354, the deletion of amino acids at positions 1-18, the deletion of amino acids at positions 19-36, the deletion of amino acids at positions 37-51, the deletion of amino acids at positions 314-384, the deletion of amino acids at positions 321-374, the deletion of amino acids at positions 331-364, the deletion of amino acids at positions 344-354, and the deletion of amino acids at positions 345-353; Preferably, the first mutation comprises 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, 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; (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; (c) substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, 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: 2; and (d) After optimal global alignment with SEQ ID NO: 2, the position corresponding to SEQ ID NO:2: substitution or deletion of Q8, substitution or deletion of S9, substitution or deletion of Q10, substitution or deletion of K47, substitution or deletion of K50, substitution or deletion of A51, substitution or deletion of D183, substitution or deletion of A179, substitution or deletion of T180, substitution or deletion of V182, substitution or deletion of T184, substitution or deletion of Y209, substitution or deletion of I347, substitution or deletion of S350, 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 comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353 of SEQ ID NO: 1; (b) after optimal global alignment with SEQ ID NO: 1, substitution of H8, substitution of N9, substitution of Q10, substitution or deletion of K47, substitution of K50, substitution of A51, substitution of S183, substitution of S179, substitution of N180, substitution of I182, substitution of M184, substitution of Y209, substitution of I347, substitution of T350, substitution of T352, substitution of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353, corresponding to SEQ ID NO: 1; (c) 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; and (d) 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.

148. The LVV or RVV according to claim 147, characterized in that The first mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) deletion of amino acids 331-364, deletion of amino acids 344-354, substitution or deletion of K47, or substitution of R354 in SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, deletion of amino acids 331-364, deletion of amino acids 344-354, substitution or deletion of K47, substitution of R354 at positions corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the first mutation comprises a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acids 331-364 of SEQ ID NO: 1 or SEQ ID NO: 2 are deleted, amino acids 344-354 are deleted, amino acid 47 is substituted from lysine K to glutamine Q (K47Q), amino acid 354 is substituted from arginine R to glutamine Q (R354Q), and K47 is deleted; (b) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, amino acid deletions at positions 331-364, amino acid deletions at positions 344-354, K47Q, R354Q, and K47 deletion are located at positions corresponding to SEQ ID NO: 1 or SEQ ID NO:

2.

149. The LVV or RVV according to claim 148, wherein The first mutation includes a mutation in which the amino acid sequence comprises the following amino acids: (c) a deletion of K47 located at SEQ ID NO: 1 or SEQ ID NO: 2; or (d) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, the K47 position corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 is deleted.

150. The LVV or RVV of claim 146, wherein: The first mutation includes a mutation in which the amino acid sequence comprises the following amino acids: (a) a mutation at I331 of SEQ ID NO: 1 or SEQ ID NO: 2; or (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at position I331 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the mutation of I331 is selected from the group consisting of: I331E, I331W, I331M, I331L, I331Q, and I331R.

151. The LVV or RVV of claim 147, wherein: The extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 33 or SEQ ID NO:

34.

152. The LVV or RVV according to any one of claims 141-151, characterized in that The envelope glycoprotein undergoes a second mutation, which enhances the ability of the envelope glycoprotein to antagonize inactivation by complement, or prevents the envelope glycoprotein from being inactivated by complement, compared with before the second mutation occurs.

153. The LVV or RVV according to claim 152, wherein: The envelope glycoprotein is an envelope glycoprotein of the Indiana strain or Cocal strain of the vesicular stomatitis virus genus or a variant thereof, and the extracellular domain of the envelope glycoprotein comprises an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

154. The LVV or RVV according to claim 153, wherein: The second mutation includes a mutation in which the amino acid sequence comprises at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 146 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; Preferably, the amino acid mutation includes at least one of amino acid deletion, insertion and substitution; More preferably, the second mutation comprises a substitution of the amino acid sequence comprising at least one of the following amino acids: (a) amino acid position 214 of SEQ ID NO: 1 or SEQ ID NO: 2; (b) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 214 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (c) amino acid position 352 of SEQ ID NO: 1 or SEQ ID NO: 2; (d) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, located at amino acid position 352 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (e) amino acid position 50 of SEQ ID NO: 1 or SEQ ID NO: 2; (f) after optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, is located at amino acid position 50 corresponding to SEQ ID NO: 1 or SEQ ID NO: 2; (g) amino acid position 146 of SEQ ID NO: 1 or SEQ ID NO: 2; and (h) After optimal global alignment with SEQ ID NO: 1 or SEQ ID NO: 2, it is located at the amino acid position corresponding to 146 of SEQ ID NO: 1 or SEQ ID NO:

2.

155. The LVV or RVV according to claim 154, wherein: The second mutation includes an amino acid sequence as shown in SEQ ID NO: 1 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 1, comprising at least one of the following position mutations: (a) substitution of T214, substitution of T352, substitution of K50, or substitution of S146 in SEQ ID NO: 1; and (b) after optimal global alignment with SEQ ID NO: 1, substitutions at T214, T352, K50, and S146 corresponding to SEQ ID NO: 1; Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence: (a) T214N, T352A, K50T, S146T at SEQ ID NO: 1; and (b) After optimal global alignment with SEQ ID NO: 1, T214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO:

1.

156. The LVV or RVV according to claim 155, characterized in that The second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) substitution of (i) T214 and T352; or (ii) T214, T352, K50, and S146 of SEQ ID NO: 1; and (b) after optimal global alignment with SEQ ID NO: 1, substitutions at positions corresponding to (i) T214 and T352; or (ii) T214, T352, K50, and S146 of SEQ ID NO: 1; Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) (i) T214N and T352A; or (ii) T214N, T352A, K50T, and S146T at SEQ ID NO: 1; (b) After optimal global alignment with SEQ ID NO: 1, located at (i) T214N and T352A; or (ii) T214N, T352A, K50T, and S146T corresponding to SEQ ID NO:

1.

157. The LVV or RVV according to claim 154, wherein: The second mutation includes an amino acid sequence as shown in SEQ ID NO: 2 or having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 2, comprising at least one of the following position mutations: (a) substitution of K214, substitution of T352, substitution of K50, substitution of S146 in SEQ ID NO: 2; and (b) after optimal global alignment with SEQ ID NO: 2, substitutions at positions corresponding to K214, T352, K50, and S146 of SEQ ID NO: 2; Preferably, the second mutation includes at least one of the following site mutations in the amino acid sequence: (a) K214N, T352A, K50T, S146T at SEQ ID NO: 2; and (b) After optimal global alignment with SEQ ID NO: 2, K214N, T352A, K50T, and S146T are located at positions corresponding to those in SEQ ID NO:

2.

158. The LVV or RVV according to claim 157, wherein: The second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) substitution of (i) K214 and T352; or (ii) K214, T352, K50, and S146 of SEQ ID NO: 2; and (b) after optimal global alignment with SEQ ID NO: 2, at positions corresponding to (i) substitutions of K214 and T352; or (ii) substitutions of K214, T352, K50, and S146 of SEQ ID NO: 2; Preferably, the second mutation includes a combination of any one of the following site mutations in the amino acid sequence: (a) (i) K214N and T352A; or (ii) K214N, T352A, K50T, and S146T at SEQ ID NO: 2; (b) After optimal global alignment with SEQ ID NO: 2, located at (i) K214N and T352A; or (ii) K214N, T352A, K50T, and S146T corresponding to SEQ ID NO:

2.

159. The LVV or RVV according to claim 154, wherein: The extracellular domain of the envelope glycoprotein comprises the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:

38.

160. The LVV or RVV according to any one of claims 119-159, wherein The content of CAR available for binding to an antigen on the viral envelope of the LVV or RVV is reduced or does not contain the CAR available for binding to an antigen.

161. A method for transducing T cells, characterized in that Comprising contacting the T cells with the LVV or RVV of any one of claims 82-84 or 119-160.

162. The method according to claim 161, characterized in that The T cells are: (a) T cells in patients; (b) T cells in the patient's blood; or (c) T cells from cells isolated from a patient or other donor; The patient is an individual who is administered the LVV or RVV and / or an individual who is administered T cells transduced by the method for transducing T cells.

163. The method according to claim 162, characterized in that The administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system and implantation.

164. The method according to any one of claims 161 to 163, wherein: The transduction occurs in vivo and / or in vitro in the patient; (a) when the transduction occurs outside the patient, the patient is an individual to whom T cells transduced by the method for transducing T cells are administered; and / or (b) When the transduction occurs in a patient, the patient is an individual to whom the LVV or RVV is administered.

165. An engineered T cell expressing CAR, characterized in that: The engineered T cells are prepared by LVV or RVV-transduced T cells according to any one of claims 82-84 or 119-160.

166. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a pharmaceutically acceptable excipient or carrier and any one of the following ingredients: the LVV or RVV of any one of claims 82-84 or 119-160 and the engineered T cells of claim 165.

167. A packaging cell, characterized in that The packaging cell comprises the packaging system of any one of claims 1 to 81; Preferably, the CAR available for binding to antigens on the cell membrane of the packaging cell is reduced; Preferably, the packaging cell is selected from any one of NS0 cells, Vero cells, HeLa cells, COS cells, CHO cells, HEK cells, BHK cells and MDCKⅡ cells; More preferably, the packaging cells are HEK-293T cells.

168. The packaging cell according to claim 167, wherein The packaging cells are configured to produce lentiviral vectors or retroviral vectors.

169. A packaging cell line, characterized in that The packaging cell line comprises the packaging cell of claim 167 or 168.

170. A method for preparing the LVV or RVV of any one of claims 82-84 or 119-160, characterized in that The method comprises culturing the packaging cell line of claim 169 until the LVV or RVV can be harvested.

171. A method for reducing false transduction when LVV or RVV transduces T cells, characterized in that This includes reducing the content of CAR on the viral envelope of the LVV or RVV that can bind to disease cell surface antigens.

172. The method according to claim 171, characterized in that This includes reducing the content of CAR available for binding to disease cell surface antigens on the cell membrane of the packaging cell that packages the LVV or RVV.

173. The method according to claim 172, characterized in that Comprising packaging the LVV or RVV in the packaging cell using the packaging system of any one of claims 1-81.

174. A method of treating a disease in a patient or killing diseased cells: (a) in the patient; (b) in the patient's blood; or (c) in cells isolated from the patient or another donor, characterized in that: The method comprises administering the LVV or RVV packaged in packaging cells according to any one of claims 1 to 81 to the patient, the patient's blood, or cells isolated from the patient or other donor.

175. A method of treating a disease in a patient or killing diseased cells: (a) in the patient; (b) in the patient's blood; or (c) in cells isolated from the patient or another donor, characterized in that: Comprising administering the LVV or RVV of any one of claims 82-84 or 119-160 to a patient, the patient's blood, or cells isolated from a patient or other donor.

176. The method according to claim 174 or 175, characterized in that The disease cells are selected from at least one of cancer cells and autoimmune disease cells; the cancer cells include solid cancer cells and blood cancer cells.

177. The method according to claim 176, characterized in that The disease is a blood cancer selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant ineligible (Transplant At least one of the following: primary mediastinal B-cell lymphoma (MBC), diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), primary plasma cell leukemia (pPCL), peripheral T-cell lymphoma (PTCL-NHL), NK / T-cell lymphoma, anaplastic large cell lymphoma (ALCL), intestinal T-cell lymphoma, T-large granular lymphocytic leukemia (T-LGL), and germinal center T-cell lymphoma (FTCL).

178. The method according to claim 176, characterized in that The disease is a blood cancer, the blood cancer is a B-cell malignancy, and the B-cell malignancy is selected from: non-Hodgkin lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia and Burkitt lymphoma (BL) at least one.

179. The method according to claim 176, characterized in that The disease is a solid cancer, and the solid cancer is selected from at least one of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer and gastrointestinal cancer.

180. The method according to claim 176, characterized in that The disease is an autoimmune disease, and the autoimmune disease is selected from at least one of systemic lupus erythematosus, psoriasis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Behcet's disease, Sjögren's syndrome, myasthenia gravis, celiac disease, type 1 diabetes, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis and scleroderma.

181. The method according to any one of claims 174-180, characterized in that The LVV or RVV is administered to the patient by at least one route selected from the group consisting of oral, nasal, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, sustained release system, and implantable device administration.

182. The method according to any one of claims 174-181, characterized in that The administration includes administering a therapeutically effective amount of LVV or RVV.

183. Use of the LVV or RVV of any one of claims 82-84 or 119-160, the engineered T cells of claim 165, or the pharmaceutical composition of claim 166 in the preparation of a drug for treating a disease.

184. The use according to claim 183, characterized in that The disease is selected from the group consisting of autoimmune diseases and cancer; and the cancer is selected from the group consisting of solid cancer and blood cancer.

185. The use according to claim 184, characterized in that The disease is a blood cancer selected from the group consisting of non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant ineligible (Transplant At least one of the following: primary mediastinal B-cell lymphoma (MBC), diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia, Burkitt lymphoma (BL), multiple myeloma (MM), acute myeloid leukemia (AML), primary plasma cell leukemia (pPCL), peripheral T-cell lymphoma (PTCL-NHL), NK / T-cell lymphoma, anaplastic large cell lymphoma (ALCL), intestinal T-cell lymphoma, T-large granular lymphocytic leukemia (T-LGL), and germinal center T-cell lymphoma (FTCL).

186. The use according to claim 184, characterized in that The disease is a blood cancer, the blood cancer is a B-cell malignancy, and the B-cell malignancy is selected from: non-Hodgkin lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), transplant ineligible LBCL, diffuse LBCL (DLBCL), high-grade B-cell lymphoma (HGBCL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), precursor B-cell lymphoma / leukemia and Burkitt lymphoma (BL) at least one.

187. The use according to claim 184, characterized in that The disease is a solid cancer, and the solid cancer is selected from at least one of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer and gastrointestinal cancer.

188. The use according to claim 184, characterized in that The disease is an autoimmune disease, and the autoimmune disease is selected from at least one of systemic lupus erythematosus, psoriasis, psoriatic arthritis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Behcet's disease, Sjögren's syndrome, myasthenia gravis, celiac disease, type 1 diabetes, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis and scleroderma.

189. A polynucleotide encoding a CAR binding molecule, characterized in that The CAR binding molecule can bind to CAR.

190. The polynucleotide according to claim 189, characterized in that The antigen binding region of the CAR comprises an antibody or an antigen binding fragment thereof, and the antibody or the antigen binding fragment thereof comprises an antibody epitope, and the antibody epitope can bind to an antigen epitope.

191. The polynucleotide according to claim 190, characterized in that The CAR binding molecule comprises (a) a leader signal peptide, (b) a CAR binding region and (c) an endoplasmic reticulum retention signal peptide.

192. The polynucleotide according to claim 191, wherein The leader signal peptide is operably linked to the N-terminus of the CAR binding region, and the endoplasmic reticulum retention signal peptide is operably linked to the C-terminus of the CAR binding region; Preferably, the endoplasmic reticulum retention signal peptide is operably linked to the C-terminus of the CAR binding region via a polypeptide linker.

193. The polynucleotide according to claim 192, characterized in that The CAR binding region is selected from at least one of (a) an antigen, (b) a first anti-antibody, and (c) a second anti-antibody.

194. The polynucleotide according to claim 193, characterized in that The antigen is selected from at least one of a full-length antigen, an extracellular domain of an antigen, a variable region of an antigen, and an antigen epitope.

195. The polynucleotide according to claim 193 or 194, characterized in that The antigen or first anti-antibody may bind to the antibody epitope.

196. The polynucleotide according to claim 195, characterized in that The antigen or first anti-antibody may bind to at least one CDR region of the antibody epitope.

197. The polynucleotide according to claim 196, characterized in that The antigen or first anti-antibody can specifically bind to the antigen binding region of the CAR or an antigen binding fragment thereof.

198. The polynucleotide according to any one of claims 193-197, characterized in that The second anti-antibody can bind to a region of the CAR that does not contain the antibody epitope (non-antibody epitope region).

199. The polynucleotide according to any one of claims 191-198, characterized in that The leader signal peptide is selected from the following signal peptides: natural leader signal peptide of antigen, CD8α signal peptide, CD28 signal peptide, IgG signal peptide and HLA-A signal peptide; Preferably, the leader signal peptide is the natural leader signal peptide of the antigen or the CD8α signal peptide.

200. The polynucleotide according to any one of claims 191-199, characterized in that The endoplasmic reticulum retention signal peptide comprises the amino acid sequence shown in SEQ ID NO: 51 (KDEL).

201. The polynucleotide according to claim 189 or 190, characterized in that The CAR binding molecule comprises a CAR binding region, and the CAR binding region can bind to CAR to assemble into a complex molecule.

202. The polynucleotide according to claim 201, wherein The CAR binding region comprises: (a) antigens; and / or (b) Primary anti-antibody.

203. The polynucleotide according to claim 202, characterized in that The antigenic epitope of the antigen in the CAR binding region can bind to the antibody epitope.

204. The polynucleotide according to claim 203, characterized in that The antigen in the CAR binding region comprises (a) a full-length antigen or (b) a functional fragment of an antigen, wherein the functional fragment of the antigen at least comprises the antigen epitope; Optionally, the functional fragment of the antigen comprises at least the extracellular domain of the antigen; Optionally, the functional fragment of the antigen comprises the extracellular domain and the transmembrane region of the antigen.

205. The polynucleotide according to any one of claims 202-204, characterized in that The first anti-antibody can bind to at least one CDR region of the antibody epitope.

206. The polynucleotide according to any one of claims 202-205, characterized in that The antigen binding region of the CAR and / or the first anti-antibody can specifically bind to the antibody or antigen binding fragment thereof of the antigen binding region of the CAR.

207. The polynucleotide according to any one of claims 202-206, characterized in that The CAR binding region further comprises a second anti-antibody that can bind to a region of the CAR that does not comprise the antibody epitope (non-antibody epitope region).

208. The polynucleotide according to any one of claims 202-207, characterized in that The CAR binding molecule comprises a leader signal peptide, which is located at the N-terminal end of the CAR binding region; Preferably, the leader signal peptide is selected from any one of the following signal peptides: a natural leader signal peptide of an antigen, a CD8α signal peptide, a CD28 signal peptide, an IgG signal peptide, and an HLA-A signal peptide; More preferably, the leader signal peptide is the natural leader signal peptide of the antigen or the CD8α signal peptide.

209. The polynucleotide according to claim 208, characterized in that The CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, the CAR binding region comprising (i) the antigen and / or (ii) the first anti-antibody; Optionally, the CAR binding region may further comprise a second anti-antibody.

210. The polynucleotide according to claim 208 or 209, characterized in that The CAR binding molecule further comprises an endoplasmic reticulum retention signal peptide, wherein the endoplasmic reticulum retention signal peptide is located at the C-terminal end of the CAR binding region; Preferably, the endoplasmic reticulum retention signal peptide comprises the amino acid sequence shown in SEQ ID NO: 51 (KDEL).

211. The polynucleotide according to any one of claims 208-210, characterized in that The CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, and (c) the endoplasmic reticulum retention signal peptide.

212. The polynucleotide according to any one of claims 208-210, characterized in that The CAR binding molecule further comprises a polypeptide linker, and the CAR binding region is connected to the endoplasmic reticulum retention signal peptide through the polypeptide linker; The CAR binding molecule comprises, from N-terminus to C-terminus, (a) the leader signal peptide, (b) the CAR binding region, (c) a polypeptide linker, and (d) the endoplasmic reticulum retention signal peptide; Preferably, the polypeptide linker is selected from: (a) an immunoglobulin hinge region selected from the group consisting of wild-type and modified IgG1, IgG2, IgG3, IgG4, IgA, and IgD hinge regions; (b) a hinge region selected from the wild-type and 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; (c) all or a portion of an Fc domain, wherein the Fc domain is selected from one or more of a CH1 domain, a CH2 domain, and a CH3 domain; (d) a stem region of a type II C-lectin selected from the group consisting of the stem regions of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and (e) flexible linker peptide; More preferably, the flexible connecting peptide is selected from (G4S) n Connector peptide, Connector peptide 1 and Connector peptide 3; wherein n=1 to 4; Connector peptide 1: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 23) Connector peptide 3: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO: 137).

213. The polynucleotide according to claim 189, characterized in that The CAR binding molecule does not comprise a leader signal peptide.

214. The polynucleotide according to any one of claims 190-213, characterized in that The antibody epitope may bind to a selected from: TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5TAC, 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-3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, uPAR, GCC, EPCAM, Nectin4, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, CD20, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.

2. Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, bean curd protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut An antigenic epitope of at least one of hsp70-2, CD79α, CD79β, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, and NKG2D; Preferably, the antibody epitope binds to an antigenic epitope of at least one antigen selected from the group consisting of: CD19, CD20, CD33, MSLN, CD79β, CD8, ASGPR, BCMA, CEA, uPAR, DLL3, GCC, Nectin4, HER2, Claudin18.2, B7H3 and GUCY2C.

215. The polynucleotide according to any one of claims 190 to 214, characterized in that The antibody or antigen-binding fragment thereof in the antigen-binding region of the CAR comprises an anti-CD19 antibody or antigen-binding fragment thereof that can bind to CD19; Preferably, the CD19 is human CD19 (UniProtKB No.: P15391).

216. The polynucleotide according to claim 215, characterized in that The CAR binding region comprises an antigen CD19 and / or a first anti-antibody (anti-anti-CD19 antibody) of an anti-anti-CD19 antibody or an antigen-binding fragment thereof.

217. The polynucleotide according to claim 216, characterized in that The antigen CD19 of the CAR binding region comprises its extracellular domain and transmembrane region.

218. The polynucleotide according to claim 217, characterized in that The antigen CD19 of the CAR binding region also includes its intracellular domain.

219. The polynucleotide according to any one of claims 215 to 218, characterized in that The anti-CD19 antibody or its antigen-binding fragment is derived from the scFv of the monoclonal antibody FMC-63 (FMC63-scFv), the amino acid sequences of the HCDR1-3 regions of the FMC63-scFv are shown in SEQ ID NOs: 94-96, respectively, and the amino acid sequences of the LCDR1-3 regions of the anti-CD19 antibody or its antigen-binding fragment are shown in SEQ ID NOs: 97-99, respectively.

220. The polynucleotide according to claim 219, characterized in that The amino acid sequences of the HCDR1-3 regions of the anti-anti-CD19 antibody are shown in SEQ ID NOs: 102-104, respectively, and the amino acid sequences of the LCDR1-3 regions of the anti-anti-CD19 antibody are shown in SEQ ID NOs: 105-107, respectively.

221. The polynucleotide according to any one of claims 189-220, characterized in that The polynucleotide is isolated.

222. A CAR binding molecule, characterized in that The CAR binding molecule is encoded by the polynucleotide of any one of claims 189-221.

223. A packaging system, characterized in that The packaging system comprises the polynucleotide of any one of claims 189-221.

224. A packaging cell, characterized in that The packaging cell comprises the polynucleotide of any one of claims 189-221; Preferably, the packaging cell is selected from any one of NS0 cells, Vero cells, HeLa cells, COS cells, CHO cells, HEK cells, BHK cells and MDCKⅡ cells; More preferably, the packaging cells are HEK-293T cells.

225. The packaging cell according to claim 224, wherein The packaging cells are configured to produce lentiviral vectors or retroviral vectors.

226. A packaging cell line, characterized in that The packaging cell line comprises the packaging cell of claim 224 or 225.

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