Modified virus vector as well as preparation method and application thereof

Abstract

The invention relates to the field of viral vectors, in particular to a modified viral vector as well as a preparation method and application thereof. The invention discloses a modified viral vector, the surface of the modified viral vector contains viral glycoprotein, and the ability of the viral glycoprotein to bind to a ligand or receptor of the viral glycoprotein on the surface of a cell is weakened.

Description

Technical Field

[0001] This invention relates to the field of viral vectors, and more specifically to a modified viral vector, its preparation method, and its application. Background Technology

[0002] Pseudotyped lentiviral vectors (LVVs) or retroviral vectors (RVVs) are among the most commonly used vectors in gene editing. Viral glycoproteins such as VSV-G and Cocal-G are widely used to package pseudotyped LVVs or RVVs by binding to their receptors, LDL-R, which are widely expressed on the surfaces of various cells.

[0003] However, due to the broad infectivity of VSV-G and Cocal-G, in order to prepare pseudo-LVV or RVV that can target specific target cells, existing technologies usually mutate VSV-G or Cocal-G to obtain VSV-G / Cocal-G mutants with reduced ability to bind LDL-R.

[0004] However, mutations in viral glycoproteins may cause partial misfolding of proteins or peptides, leading to a decrease in viral vector yield or titer or the need for additional process screening. In clinical applications, viral vectors containing viral glycoprotein variants are generally considered to pose a greater risk and are not conducive to the large-scale clinical preparation of pseudotyped LVV or RVV. Summary of the Invention

[0005] In view of this, in order to solve at least one of the above-mentioned technical problems, the first aspect of the present invention provides a modified viral vector, the surface of which contains a viral glycoprotein, the ability of which to bind to receptors on the cell surface is weakened.

[0006] In some embodiments of the present invention, the reduced ability of the viral glycoprotein contained on the surface of the modified viral vector to bind to receptors of the viral glycoprotein on the cell surface is relative to the viral glycoprotein contained on the surface of the unmodified viral vector.

[0007] In some embodiments of the present invention, the number of binding sites for receptors on the cell surface of the modified viral vector containing viral glycoproteins is reduced.

[0008] In some embodiments of the present invention, the number of binding sites of the viral glycoprotein contained on the surface of the modified viral vector for binding to receptors of the viral glycoprotein on the cell surface is reduced relative to the viral glycoprotein contained on the surface of the unmodified viral vector.

[0009] In some embodiments of the present invention, the viral glycoprotein contained on the surface of the modified viral vector has bound to at least one of its receptor and antibody, thereby weakening the ability of the viral glycoprotein to bind to the receptor of the viral glycoprotein on the cell surface.

[0010] Preferably, the modified viral vector is an enveloped viral vector, and the receptors and / or antibodies bound to the viral glycoproteins on the surface of the modified viral vector are free, expressed on the viral membrane, or displayed on the viral membrane as receptors and / or antibodies for the viral glycoproteins.

[0011] In some embodiments of the present invention, the modified viral vector is a pseudotyped lentiviral vector (LVV) or a retroviral vector (RVV);

[0012] Preferably, the viral glycoprotein binds to receptors and / or antibodies on the packaging cells and / or cell membranes of the modified pseudo-LVV or RVV.

[0013] In some embodiments of the present invention, the modified pseudo-LVV or RVV viral glycoprotein binds to receptors and / or antibodies of the free viral glycoprotein in packaging cells that package the modified pseudo-LVV or RVV.

[0014] In some embodiments of the present invention, the modified pseudo-LVV or RVV viral glycoprotein binds to receptors and / or antibodies of the viral glycoprotein on the cell membrane of the packaging cell that packages the modified pseudo-LVV or RVV.

[0015] In some embodiments of the present invention, the viral glycoprotein contained on the surface of the modified viral vector is selected from vesicular stomatitis virus strains glycoprotein, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibberish leukemia virus glycoprotein (GaLV), ditropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein;

[0016] The vesicular stomatitis virus (VSV) glycoproteins include: VSV Indiana strain glycoprotein, VSV Cocal strain glycoprotein, VSV Maraba strain glycoprotein, VSV Morreton strain glycoprotein, VSV Alagoas strain glycoprotein, VSV New Jersey strain glycoprotein, VSV Carajas strain glycoprotein, VSV Chandipura strain glycoprotein, and vesicular stomatitis virus (VSV) glycoprotein. The glycoproteins of the following strains of vesicular stomatitis virus (VSV) are: Eptesicus, Isfahan, Jurona, Malpais, Perinet, Piry, Radi, Rhinolopus, and YugBogdanovac.

[0017] In some embodiments of the present invention, the viral glycoprotein contained on the surface of the modified viral vector is vesicular stomatitis virus strain Indiana glycoprotein (VSV-G) or vesicular stomatitis virus strain Cocal glycoprotein (Cocal-G), the receptor includes LDL-R, and the antibody includes VSV-G antibody or Cocal-G antibody.

[0018] Preferably, the amino acid sequences of the HCDR1-3 regions of the VH region of the anti-VSV-G antibody are shown in SEQ ID NO:30-32, respectively; and the amino acid sequences of the LCDR1-3 regions of the VL region of the anti-VSV-G antibody are shown in SEQ ID NO:33-35, respectively.

[0019] In some embodiments of the present invention, the amino acid sequence of the VH region of the anti-VSV-G antibody is shown in SEQ ID NO:36; and the amino acid sequence of the VL region of the anti-VSV-G antibody is shown in SEQ ID NO:37.

[0020] In some embodiments of the present invention, the viral glycoprotein contained on the surface of the modified viral vector contains a mutation that enhances or prevents the viral glycoprotein from being inactivated by complement (“complement inactivation mutation”).

[0021] In some embodiments of the present invention, the enhanced or non-inactivation of the viral glycoprotein by complement antagonism is relative to viral glycoproteins that do not contain the complement-inactivating mutation.

[0022] In some embodiments of the present invention, the viral glycoprotein is VSV-G or Cocal-G, and the mutation includes mutations at at least one of the following sites:

[0023] (a) The 214th amino acid located in SEQ ID NO:1 or SEQ ID NO:2;

[0024] (b) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at the 214th amino acid position corresponding to SEQ ID NO:1 or SEQ ID NO:2;

[0025] (c) The 352nd amino acid located in SEQ ID NO:1 or SEQ ID NO:2;

[0026] (d) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at position 352, which corresponds to SEQ ID NO:1 or SEQ ID NO:2;

[0027] (e) The 50th amino acid located in SEQ ID NO:1 or SEQ ID NO:2;

[0028] (f) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at the 50th amino acid corresponding to SEQ ID NO:1 or SEQ ID NO:2;

[0029] (g) the amino acid located at position 146 of SEQ ID NO:1 or SEQ ID NO:2; and

[0030] (h) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, it is located at the 146th amino acid position, which corresponds to SEQ ID NO:1 or SEQ ID NO:2;

[0031] Preferably, the mutation at the site is selected from amino acid substitutions, deletions, and insertions;

[0032] More preferably, the mutation at the site is an amino acid substitution.

[0033] In some embodiments of the present invention, the mutation includes a mutation at at least one of the following sites:

[0034] (a) The amino acid at position 214 of SEQ ID NO:1 is replaced by threonine T with asparagine N (T214N), the amino acid at position 352 is replaced by threonine T with alanine A (T352A), the amino acid at position 50 is replaced by lysine K with threonine T (K50T), and the amino acid at position 146 is replaced by serine S with threonine T (S146T);

[0035] (b) After best global alignment with SEQ ID NO:1, T214N, T352A, K50T, and S146T are located at the equivalent of SEQ ID NO:1;

[0036] (c) The amino acid at position 214 of SEQ ID NO:2 is replaced by lysine K with asparagine N (K214N), T352A, K50T, or S146T; and

[0037] (d) After best global alignment with SEQ ID NO:2, it is located at K214N, T352A, K50T, and S146T, which are equivalent to SEQ ID NO:2.

[0038] In some embodiments of the present invention, the mutation includes a combination of any of the following site mutations:

[0039] (a) Substitution of (1) T214 and T352 located in SEQ ID NO:1; or (2) Substitution of T214, T352, K50 and S146;

[0040] (b) After best global alignment with SEQ ID NO:1, the substitutions located at (1) T214 and T352 of SEQ ID NO:1; or (2) substitutions of T214, T352, K50 and S146;

[0041] (c) Substitution of (1) K214 and T352 located in SEQ ID NO:2; or (2) Substitution of K214, T352, K50 and S146; and

[0042] (d) After best global alignment with SEQ ID NO:2, the substitutions located at (1) K214 and T352 in SEQ ID NO:2; or (2) K214, T352, K50 and S146;

[0043] Preferably, the mutation includes a combination of any of the following site mutations:

[0044] (a) (1) T214N and T352A located in SEQ ID NO:1; or (2) T214N, T352A, K50T and S146T;

[0045] (b) After best global alignment with SEQ ID NO:1, it is located at (1) T214N and T352A, or (2) T214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:1;

[0046] (c) K214N and T352A located in SEQ ID NO:2; or (2) K214N, T352A, K50T and S146T; and

[0047] (d) After best global alignment with SEQ ID NO:2, it is located at (1) K214N and T352A, or (2) K214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:2.

[0048] In some embodiments of the present invention, the amino acid sequence of the viral glycoprotein containing the complement inactivation mutation is shown in any of SEQ ID NO:3-6.

[0049] In some embodiments of the present invention, the amino acid sequence of the viral glycoprotein containing the complement inactivation mutation is shown in SEQ ID NO:3; relative to SEQ ID NO:1, SEQ ID NO:3 contains T214N and T352A.

[0050] In some embodiments of the present invention, the amino acid sequence of the viral glycoprotein containing the complement inactivation mutation is shown in SEQ ID NO:4; relative to SEQ ID NO:1, SEQ ID NO:4 contains T214N, T352A, K50T and S146T.

[0051] In some embodiments of the present invention, the amino acid sequence of the viral glycoprotein containing the complement inactivation mutation is shown in SEQ ID NO:5; relative to SEQ ID NO:2, SEQ ID NO:5 contains K214N and T352A.

[0052] In some embodiments of the present invention, the amino acid sequence of the viral glycoprotein containing the complement inactivation mutation is shown in SEQ ID NO:6; relative to SEQ ID NO:2, SEQ ID NO:6 contains K214N, T352A, K50T and S146T.

[0053] In some embodiments of the present invention, the surface of any of the aforementioned modified viral vectors further comprises a targeting molecule, said targeting molecule binding to at least one selected from the following antigens:

[0054] 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, soybean protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, spermin 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate-specific protein, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, Cyclin B1, 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, muthsp70-2, CD79α, CD79β, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, NKG2D, TCRα, TCRβ, TCRγ, and TCRδ;

[0055] Preferably, the targeting molecule binds to at least one of CD3, CD5, CD7, CD28, TCRα, TCRβ, TCRγ, and TCRδ.

[0056] In some embodiments of the present invention, the targeting molecule binds to CD3;

[0057] Preferably, the CD3 is a human CD3.

[0058] In some embodiments of the present invention, the targeting molecule binds to human CD3, wherein the human CD3 is selected from the group consisting of human CD3ε / CD3E (UniProtKB No.: P07766), CD3γ / CD3G (UniProtKB No.: P09693), CD3δ / CD3D (UniProtKB No.: P04234) and CD3ζ / CD3Z (UniProtKB No.: P20963), or any combination thereof.

[0059] In some embodiments of the present invention, the targeting molecule binds to CD7;

[0060] Preferably, the CD7 is a human CD7 (UniProtKB number: P09564).

[0061] In some embodiments of the present invention, the targeting molecule includes an antigen-binding region that can bind the antigen, and the antigen-binding region includes at least one of (a) an antibody or an antigen-binding fragment thereof and (b) a ligand or a receptor-binding fragment thereof.

[0062] In some embodiments of the present invention, the targeting molecule further comprises a transmembrane domain, and the antigen-binding region is directly or indirectly connected to the transmembrane domain;

[0063] Preferably, the transmembrane domain is selected from the transmembrane domains of the following proteins:

[0064] CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD 95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM), CD272( BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, K IR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70;

[0065] More preferably, the transmembrane domain includes the CD8α transmembrane domain.

[0066] In some embodiments of the present invention, the transmembrane domain of the target molecule is the human CD8α transmembrane domain, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:13.

[0067] In some embodiments of the present invention, the targeting molecule further includes a linker domain, and the antigen-binding region is indirectly connected to the transmembrane domain through the linker domain;

[0068] Preferably, the connection structure domain is selected from:

[0069] (a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions;

[0070] (b) Hinge region, wherein the hinge region is selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154;

[0071] (c) All or a portion of the Fc domains, wherein the Fc domains are selected from one or more of the CH1, CH2, and CH3 domains; and

[0072] (d) Stem regions of type II C-lectins, wherein the type II C-lectins are selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A and NKG2D;

[0073] More preferably, the connection structure domain includes the hinge region of CD8α.

[0074] In some embodiments of the present invention, the hinge region of the target molecule is the human CD8α hinge region, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:12.

[0075] In some embodiments of the present invention, the antibody (membrane-expressed antibody) that expresses or displays the viral glycoprotein on the viral membrane includes a transmembrane domain;

[0076] Preferably, the membrane-expressing antibody further includes a linker domain.

[0077] In some embodiments of the present invention, the transmembrane domain of the membrane-expressing antibody is any of the aforementioned transmembrane domains; preferably, it is the human CD8α transmembrane domain.

[0078] In some embodiments of the present invention, the linker domain of the membrane-expressing antibody is any of the aforementioned linker domains; preferably, it is the human CD8α hinge region.

[0079] In some embodiments of the present invention, the membrane-expressed antibody further comprises a signal peptide.

[0080] In some embodiments of the present invention, the signal peptide of the membrane-expressing antibody is any of the aforementioned signal peptides; preferably, it is the human CD8α signal peptide.

[0081] In some embodiments of the present invention, the modified viral vector weakens the ability of the viral glycoprotein to bind to the receptor of the viral glycoprotein on the cell surface by mixing with at least one of the receptor and antibody of the viral glycoprotein.

[0082] Preferably, the receptor and antibody of the viral glycoprotein are artificially synthesized and expressed;

[0083] More preferably, the receptor and antibody of the viral glycoprotein are artificially synthesized, expressed, and purified.

[0084] In some embodiments of the present invention, the modified viral vector further comprises exogenous payload genes;

[0085] Preferably, the exogenous burden gene comprises a polynucleotide (CAR gene) encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen-binding region, a transmembrane region, and an intracellular signal transduction domain;

[0086] More preferably, the CAR further includes a hinge region and a co-stimulatory signal transduction domain; the hinge region connects the extracellular antigen-binding region and the transmembrane region.

[0087] In some embodiments of the present invention, the extracellular antigen-binding region of the CAR is selected from:

[0088] 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, soybean protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, spermin 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate-specific protein, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1. At least one of the following: BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, muthsp70-2, CD79α, CD79β, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, NKG2D, TCRα, TCRβ, TCRγ, and TCRδ.

[0089] In some embodiments of the present invention, the extracellular antigen-binding region of the CAR 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.

[0090] In some embodiments of the present invention, the extracellular antigen-binding region of the CAR binds to CD19; preferably human CD19 (UniProtKB number: P15391).

[0091] In some embodiments of the present invention, the extracellular antigen-binding region of the CAR binds to human CD19, and the extracellular antigen-binding region of the CAR contains an anti-C19 antibody or an antigen-binding fragment thereof.

[0092] In some embodiments of the present invention, the extracellular antigen-binding region of the CAR binds to human CD19, and the extracellular antigen-binding region of the CAR contains an anti-CD19 antibody or its antigen-binding fragment, wherein the anti-CD19 antibody or its antigen-binding fragment is a scFv derived from the monoclonal antibody FMC-63 (FMC63-scFv), and the amino acid sequences of its HCDR1-3 regions are shown in SEQ ID NO:15-17, and the amino acid sequences of its LCDR1-3 regions are shown in SEQ ID NO:18-20, respectively.

[0093] In some embodiments of the present invention, the transmembrane region of the CAR is selected from the transmembrane regions of the following proteins:

[0094] 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.

[0095] In some embodiments of the present invention, the transmembrane region of the CAR includes the transmembrane region of CD8α or CD28.

[0096] In some embodiments of the present invention, the transmembrane domain of the CAR is the transmembrane domain of human CD8α, and its amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:13.

[0097] In some embodiments of the present invention, the intracellular signal transduction domain of the CAR is selected from the intracellular signal transduction domains of the following proteins:

[0098] Intracellular signal transduction domains of proteins containing at least one ITAM, including 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 signal transduction domains.

[0099] In some embodiments of the present invention, the intracellular signal transduction domain of the CAR includes the intracellular signal transduction domain of CD3ζ.

[0100] In some embodiments of the present invention, the intracellular signal transduction domain of the CAR is the intracellular signal transduction domain of human CD3ζ, and its amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity with SEQ ID NO:21.

[0101] In some embodiments of the present invention, the co-stimulatory signal transduction domain of the CAR is selected from the co-stimulatory signal transduction domains 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-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88.

[0103] In some embodiments of the present invention, the costimulatory signal transduction domain of the CAR includes the costimulatory signal transduction domains of 4-1BB and / or CD28.

[0104] In some embodiments of the present invention, the costimulatory signal transduction domain of the CAR is the costimulatory signal transduction domain of 4-1BB; preferably human 4-1BB (UniProtKB number: Q07011).

[0105] In some embodiments of the present invention, the costimulatory signal transduction domain of the CAR is the costimulatory signal transduction domain of human 4-1BB, and its amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:22.

[0106] In some embodiments of the present invention, the hinge region of the CAR originates from the hinge region of at least one of the following proteins:

[0107] CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154.

[0108] In some embodiments of the present invention, the hinge region of the CAR includes the hinge region of CD8α or CD28.

[0109] In some embodiments of the present invention, the hinge region of the CAR is the human CD8α hinge region, whose amino acid sequence has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:12.

[0110] In a second aspect, the present invention also provides a method for reducing the ability of a viral vector to infect a cell by binding to receptors of the viral glycoprotein on the cell surface via viral glycoprotein contained on its surface, the method comprising reducing the binding sites of the viral glycoprotein that are available to bind to receptors of the viral glycoprotein on the cell surface.

[0111] In some embodiments of the present invention, the method includes, prior to the viral vector infecting cells, causing the viral glycoprotein to bind to at least one of its receptor and antibody.

[0112] In some embodiments of the present invention, the viral vector is a pseudotype LVV or RVV.

[0113] In some embodiments of the present invention, the method includes:

[0114] (a) Packaging the pseudotyped LVV or RVV using a packaging system containing a viral glycoprotein binding molecule gene; and / or

[0115] (b) After packaging the pseudo-LVV or RVV, the pseudo-LVV or RVV is bound to a viral glycoprotein binding molecule;

[0116] The viral glycoprotein binding molecule is selected from at least one of the receptor and antibody of the viral glycoprotein.

[0117] In some embodiments of the present invention, after packaging the pseudo-LVV or RVV, the pseudo-LVV or RVV is mixed with the viral glycoprotein binding molecule.

[0118] In some embodiments of the present invention, the viral glycoprotein is selected from vesicular stomatitis virus strains glycoprotein, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibberish leukemia virus glycoprotein (GaLV), ditropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein;

[0119] The vesicular stomatitis virus (VSV) glycoproteins include: VSV Indiana strain glycoprotein, VSV Cocal strain glycoprotein, VSV Maraba strain glycoprotein, VSV Morreton strain glycoprotein, VSV Alagoas strain glycoprotein, VSV New Jersey strain glycoprotein, VSV Carajas strain glycoprotein, VSV Chandipura strain glycoprotein, and vesicular stomatitis virus (VSV) glycoprotein. The glycoproteins of the following strains of vesicular stomatitis virus (VSV) are: Eptesicus, Isfahan, Jurona, Malpais, Perinet, Piry, Radi, Rhinolopus, and YugBogdanovac.

[0120] In some embodiments of the present invention, the viral glycoprotein is VSV-G or Cocal-G, the receptor includes LDL-R, and the antibody includes VSV-G antibody or Cocal-G antibody.

[0121] In some embodiments of the present invention, the viral glycoprotein is VSV-G or Cocal-G, and after packaging the pseudotyped LVV or RVV, the pseudotyped LVV or RVV is mixed with at least one of the following viral glycoprotein binding molecules: (a) LDL-R and (b) (i) anti-VSV-G antibody or (ii) anti-Cocal-G antibody.

[0122] Thirdly, the present invention also provides a pseudo-LVV or RVV packaging system, the packaging system comprising a viral glycoprotein binding molecule gene, wherein the viral glycoprotein binding molecule is selected from at least one of viral glycoprotein receptors and antibodies.

[0123] In some embodiments of the present invention, the viral glycoprotein is selected from vesicular stomatitis virus strains glycoprotein, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibberish leukemia virus glycoprotein (GaLV), ditropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein;

[0124] The vesicular stomatitis virus (VSV) glycoproteins include: VSV Indiana strain glycoprotein, VSV Cocal strain glycoprotein, VSV Maraba strain glycoprotein, VSV Morreton strain glycoprotein, VSV Alagoas strain glycoprotein, VSV New Jersey strain glycoprotein, VSV Carajas strain glycoprotein, VSV Chandipura strain glycoprotein, and vesicular stomatitis virus (VSV) glycoprotein. The glycoproteins of the following strains of vesicular stomatitis virus (VSV) are: Eptesicus strain, Isfahan strain, Jurona strain, Malpais strain, Perinet strain, Piry strain, Radi strain, Rhinolopus strain, and YugBogdanovac strain.

[0125] Preferably, the viral glycoprotein is VSV-G or Cocal-G, the receptor includes LDL-R, and the antibody includes VSV-G antibody or Cocal-G antibody;

[0126] More preferably, the amino acid sequences of the HCDR1-3 regions of the VH region of the anti-VSV-G antibody are shown in SEQ ID NO:30-32, respectively; and the amino acid sequences of the LCDR1-3 regions of the VL region of the anti-VSV-G antibody are shown in SEQ ID NO:33-35, respectively.

[0127] In some embodiments of the present invention, the amino acid sequence of the VH region of the anti-VSV-G antibody is shown in SEQ ID NO:36; and the amino acid sequence of the VL region of the anti-VSV-G antibody is shown in SEQ ID NO:37.

[0128] In some embodiments of the present invention, any of the aforementioned packaging systems comprises one or more nucleic acid vectors, which, when introduced into packaging cells for packaging LVV or RVV, contain nucleic acids necessary for the production, assembly, and / or packaging of LVV or RVV within the packaging cells.

[0129] In some embodiments of the present invention, any of the aforementioned packaging systems further comprises:

[0130] (a) Gag and Pol genes; and

[0131] (b)

[0132] (i) LVV backbone genes and Rev genes; or

[0133] (ii) RVV skeletal genes;

[0134] (c) viral glycoproteins or their variant genes; and

[0135] (d) Shuttle genes.

[0136] In some embodiments of the present invention, any of the aforementioned packaging systems further comprises:

[0137] (a) Gag and Pol genes; and

[0138] (b)

[0139] (i) LVV backbone genes and Rev genes; or

[0140] (ii) RVV skeletal genes;

[0141] (c) Viral glycoproteins or their variant genes;

[0142] (d) shuttle genes; and

[0143] (e) Targeting molecular genes.

[0144] In some embodiments of the present invention, the viral glycoprotein or its variant is any of the aforementioned viral glycoproteins or a variant thereof containing any of the aforementioned complement inactivation mutations.

[0145] In some embodiments of the present invention, the targeting molecule is any of the aforementioned targeting molecules.

[0146] In some embodiments of the present invention, any of the aforementioned packaging systems comprises: at least one packaging plasmid, a coating plasmid, a shuttle plasmid, and at least one binding plasmid;

[0147] The binding plasmid contains the viral glycoprotein binding molecule gene;

[0148] The shuttle plasmid comprises (a) the shuttle gene and (b): (i) the LVV backbone gene or (ii) the RVV backbone gene; preferably, the shuttle gene comprises the CAR gene.

[0149] In some embodiments of the present invention, any of the aforementioned packaging systems comprises: at least one packaging plasmid, a coating plasmid, a shuttle plasmid, and a binding plasmid;

[0150] The binding plasmid contains a polynucleotide encoding an antibody that encodes the viral glycoprotein, wherein the antibody is scFv.

[0151] In some embodiments of the present invention, any of the aforementioned packaging systems comprises: at least one packaging plasmid, a coating plasmid, a shuttle plasmid, and a binding plasmid;

[0152] The binding plasmid contains a polynucleotide encoding an antibody that encodes the viral glycoprotein, and the antibody is a membrane-expressed antibody.

[0153] In some embodiments of the present invention, any of the aforementioned packaging systems comprises: at least one packaging plasmid, a coating plasmid, a shuttle plasmid, and at least two binding plasmids;

[0154] The at least two binding plasmids comprise an antibody light chain plasmid and an antibody heavy chain plasmid; the antibody light chain plasmid comprises a light chain polynucleotide encoding an antibody of the viral glycoprotein; and the antibody heavy chain plasmid comprises a heavy chain polynucleotide encoding an antibody of the viral glycoprotein.

[0155] In some embodiments of the present invention,

[0156] (a) The packaging plasmid contains the Gag gene and the Pol gene (Gag / Pol packaging plasmid);

[0157] (b) The envelope plasmid comprises (i) the viral glycoprotein or a variant thereof and (ii) the targeting molecule gene; and

[0158] (c) The shuttle plasmid contains (i) a shuttle gene and (ii) either (1) an LVV backbone gene or (2) an RVV backbone gene.

[0159] In some embodiments of the present invention, when used for packaging LVV, the pseudo-LVV packaging system further comprises a Rev packaging plasmid containing the Rev gene.

[0160] In some embodiments of the present invention, the shuttle gene comprises any of the aforementioned CAR genes.

[0161] In a fourth aspect, the present invention also provides a packaging cell comprising the pseudo-LVV or RVV packaging system provided in the third aspect of the present invention;

[0162] Preferably, the packaging cells are 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;

[0163] More preferably, the packaging cells are HEK-293T cells.

[0164] Fifthly, the present invention also provides a method for preparing pseudo-LVV or RVV, the method comprising culturing the packaging cells provided in the fourth aspect of the present invention, and isolating and collecting the pseudo-LVV or RVV.

[0165] In a sixth aspect, the present invention also provides a pseudo LVV or RVV, said pseudo LVV or RVV being prepared by the method provided in the fifth aspect of the present invention.

[0166] In a seventh aspect, the present invention also provides an engineered cell prepared from cells transfected with the modified viral vector containing the exogenous load gene provided in the first aspect of the present invention;

[0167] Preferably, the cells include immune cells;

[0168] More preferably, the immune cells are selected from at least one of T cells and B cells.

[0169] In some embodiments of the present invention, the exogenous load gene is any of the aforementioned CAR genes.

[0170] In some embodiments of the present invention, the immune cells are T cells, and the engineered cells are CAR-T cells.

[0171] In an eighth aspect, the present invention also provides the use of the modified viral vector provided in the first aspect of the present invention, the pseudo-LVV or RVV provided in the sixth aspect of the present invention, or the engineered cells provided in the seventh aspect of the present invention in the preparation of a disease treatment drug;

[0172] Preferably, the disease is selected from autoimmune diseases and cancer, including solid tumors and hematologic malignancies;

[0173] More preferably, the cancer is a blood cancer.

[0174] In some embodiments of the present invention, the disease is a blood cancer selected from: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), and transplant-unsuitable leukemia. At least one of the following: 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, 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 Embryonic centrifugal T-cell lymphoma (FTCL).

[0175] In some embodiments of the present invention, the hematologic malignancy is a B-cell malignant tumor, which is selected from at least one of the following: 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).

[0176] In some embodiments of the present invention, the disease is a solid cancer, which 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.

[0177] In some embodiments of the present invention, the disease is solid cancer, 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 mellitus, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis, and scleroderma.

[0178] In a ninth aspect, the present invention also provides a method for transducing target cells and delivering an exogenous load gene, the method comprising contacting the target cells with the modified viral vector containing the exogenous load gene provided in the first aspect of the present invention;

[0179] Preferably, the target cells include immune cells;

[0180] More preferably, the immune cells are selected from at least one of T cells and B cells.

[0181] In some embodiments of the present invention, the exogenous load gene is any of the aforementioned CAR genes.

[0182] In some embodiments of the present invention, the immune cells are T cells, and the engineered cells are CAR-T cells.

[0183] In a tenth aspect, the present invention also provides a composition comprising a pharmaceutically acceptable carrier or excipient and the modified viral vector provided in the first aspect of the present invention, the pseudo-LVV or RVV provided in the sixth aspect of the present invention, or the engineered cells provided in the seventh aspect of the present invention.

[0184] In an eleventh aspect, the present invention also provides a method for treating a disease suffered by a subject or killing diseased cells of a subject, the method comprising administering to the subject or the diseased cells of the subject the modified viral vector comprising the exogenous load gene provided in the first aspect of the present invention;

[0185] Preferably, the disease is selected from autoimmune diseases and cancer, including solid tumors and hematologic malignancies;

[0186] More preferably, the cancer is a blood cancer.

[0187] In some embodiments of the present invention, the disease is any of the aforementioned autoimmune diseases.

[0188] In some embodiments of the present invention, the disease is any of the aforementioned blood cancers.

[0189] In some embodiments of the present invention, the disease is any of the aforementioned solid cancers.

[0190] In some embodiments of the present invention, the administration is selected from at least one of oral, nasal, intravenous, intraperitoneal, intracerebral (brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, continuous release system and implanted device administration.

[0191] In a twelfth aspect, the present invention also provides a kit, characterized in that the kit comprises the modified viral vector provided in the first aspect of the present invention, the packaging cells provided in the fourth aspect of the present invention, the pseudo-LVV or RVV provided in the sixth aspect of the present invention, or the engineered cells provided in the seventh aspect of the present invention.

[0192] Beneficial effects:

[0193] This invention provides a modified viral vector containing viral glycoproteins, such as VSV-G, with reduced binding sites for receptors such as LDL-R. This significantly reduces the ability of the modified viral vector to transduce cells by binding to receptors on the cell surface of viral glycoproteins. While the modified viral vector provided by this invention reduces the ability of viral glycoproteins to bind to their receptors, it does not contain any mutations that inhibit the ability of these viral glycoproteins to bind to their receptors. Compared to existing technologies that mutate viral glycoproteins to inhibit their receptor-binding ability, the production process of the modified viral vector provided by this invention is more controllable, and it carries less risk and is safer in clinical applications.

[0194] definition:

[0195] "Attenuation": As used herein, when referring to the ability of a viral glycoprotein contained in a modified viral vector to bind to a receptor for the viral glycoprotein on the cell surface, the term "attenuation" includes both the complete elimination of the binding of the viral glycoprotein to the receptor for the viral glycoprotein on the cell surface and a significant reduction in the binding. In a specific embodiment, "significant reduction" means relative to a viral glycoprotein contained in an unmodified viral vector (and which viral glycoprotein does not contain any mutations that could inhibit its ability to bind to its receptor); "reduction" is selected from reductions 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%.

[0196] "Complement": As used in this article, "complement (C)" exists in the serum, tissue fluid, and cell membrane surface of normal humans and animals. After activation, it possesses enzymatic activity and can undergo complex cascade reactions. The complement system is composed of a series of proteins and is part of the innate immune system. The complement system is initiated by a series of enzymes cleaving each other, ultimately forming a pore-like membrane attack complex on the target microorganism, causing the microorganism to rupture and die. Complement components can be activated by antigen-antibody complexes or antibodies, clearing immune complexes through cytolysis, opsonization, phagocytosis, and mediating inflammatory responses, exhibiting corresponding biological functions. Complement is widely involved in the body's defense responses against microbial infections and immune regulation, and also mediates immunopathological damage responses, making it an important effector system and effector mechanism system in the body. VSV-G or Cocal-G may be recognized and inactivated by complement after entering serum. By mutating viral glycoproteins such as VSV-G, the ability of viral glycoprotein variants to antagonize complement inactivation is enhanced, making the modified pseudo-LVV or RVV containing the mutated viral glycoprotein more suitable for in vivo infection of target cells.

[0197] "Receptors and / or antibodies expressed or displayed on the viral membrane": as used herein, this includes receptors and / or antibodies against viral glycoproteins expressed on the cell membrane of packaging cells such as HEK-293T cells that package the modified viral vector (including, but not limited to, pseudo-LVV or RVV) and which are transferred to the envelope of the viral vector during budding, including, but not limited to, LDL-R or membrane-expressed anti-VSV-G antibodies.

[0198] "Free receptors and / or antibodies": as used herein, including but not limited to receptors (including but not limited to LDL-R) and / or antibodies (including but not limited to anti-VSV-G antibodies) of viral glycoproteins expressed in the cytoplasm of packaging cells such as HEK-293T cells.

[0199] "Antibody" refers to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or the variable regions of the immunoglobulin light chain, enabling it to specifically bind to an antigen. In this article, "antibody" encompasses various forms and structures, as long as they exhibit the desired antigen-binding activity.

[0200] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0201] In this article, "antigen-binding fragment" refers to a part or a variant of a complete antibody that does not possess the full structure of the complete antibody, but only contains a part or a variant of the complete antibody, and the part or variant has the ability to bind antigens.

[0202] For example, in this document, "antibody or its antigen-binding fragment" includes, but is not limited to, immunoglobulins (full-length antibodies), such as IgG, IgA and IgM, haptens, Fab, Fab', F(ab')2, Fv fragments, single-chain variable region fragments (scFv), disulfide bond stable antibodies (dsFv), the heavy chain variable region (VH) or light chain variable region (VL) of an antibody, Fd fragments composed of VH and CH1 domains, linear antibodies and single-domain antibodies (nanobodies); the heavy chain (VH) of the scFv is linked to the light chain (VL) via a linker peptide.

[0203] In some embodiments of the present invention, the "antibody" may be a membrane-expressing antibody, which includes a transmembrane domain (which may further include an extracellular antigen-binding fragment, such as scFv, which is a membrane-expressing antibody, and a linker domain, such as a hinge region, that provides flexibility) and can be expressed on a membrane under the action of a signal peptide.

[0204] "Ligand": In receptor-ligand binding, the ligand is typically a molecule that binds to a site on the receptor to generate a signal. This binding usually leads to a conformational change in a complex structure, thereby inducing related physiological activities.

[0205] "Receptor-binding fragment" refers to a fragment that does not possess the complete structure of the ligand, but only contains a portion or a variant of the complete ligand, which has the ability to bind to a receptor. For example, "receptor-binding fragment" herein includes, but is not limited to, the extracellular domain or variable region of the ligand.

[0206] "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. It includes at least (1) an extracellular antigen-binding domain, such as scFv or VHH; (2) a transmembrane domain that anchors the CAR molecule into immune effector cells; and (3) an intracellular signal transduction domain. The extracellular structure of the CAR may further include a hinge region, and the intracellular structure may further include one or more co-stimulatory signal transduction domains. The CAR molecule can use the extracellular antigen-binding domain to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.

[0207] “Nucleic acid” refers to any compound and / or substance including polymers containing nucleotides, such as polynucleotides. In this document, “nucleic acid,” “polynucleotide,” and “gene” are used synonymously. Each nucleotide consists 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, nucleic acid molecules are 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 represented as 5' to 3'. In this document, 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 containing mixtures of two or more of these molecules. “Nucleic acid” can be linear or circular. Furthermore, “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 acid” described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include nucleotide bases modified with derived sugars, phosphate backbones, or chemically modified residues.

[0208] "Exogenous" refers to any molecule originating from outside the organism, including nucleic acids, proteins, polypeptides, or small molecule compounds. In contrast, the term "endogenous" refers to any molecule originating from within the organism (i.e., naturally produced by the organism).

[0209] "Viral envelope": This 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.). As viruses travel through host cells, the viral envelope protects their genetic material throughout their life cycle. Not all viruses have a viral envelope. Many human pathogenic viruses are encased in a lipid bilayer; they infect target cells by fusing their viral envelope with the cell membrane. Viruses with a viral envelope include retroviruses, among others.

[0210] "Retrovirus": A retrovirus is a double-stranded RNA enveloped virus, characterized by its ability to "reverse transcribe" its genome from RNA to DNA. Viral particles are measured to be 100-120 nm in diameter and contain a dimer genome of the same positive RNA strand complexed with nucleocapsid proteins. The genome is encapsulated in a protein capsid that also contains enzymes—reverse transcriptase, integrase, and protease—required for viral infection. Matrix proteins form a layer outside the capsid core, interacting with the lipid bilayer derived from the host cell membrane surrounding the viral core particle. Anchored to this bilayer are viral envelope glycoproteins responsible for recognizing specific receptors on the host cell and initiating the infection process. These envelope (glyco)proteins consist of two subunits: one anchoring the protein to the transmembrane (TM) lipid membrane, and the other binding to the surface (SU) of the cell receptor.

[0211] Retroviruses contain reverse transcriptase and integrase. Upon entering a target cell, the retrovirus uses its reverse transcriptase to transcribe its RNA molecules into DNA molecules. Subsequently, the integrase integrates the DNA molecule into the host cell's genome. The sequence derived from the retrovirus after integration into the host cell's genome is called a provirus (e.g., a proviral sequence or proviral sequence).

[0212] Based on their genomic structure, retroviruses are classified into simple retroviruses, such as MLV and murine leukemia virus, and complex retroviruses, such as HIV and EIAV. Retroviruses contain four genes: gag (group-specific antigen), pro (protease), pol (polymerase), and env (env). The gag sequence encodes three main 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 reverse transcriptase and integrase; the former catalyzes the reverse transcription of the viral genome from RNA to DNA during infection, while the latter is responsible for integrating proviral DNA into the host cell genome. The env sequence encodes the SU and TM subunits of the envelope glycoprotein. In addition, retroviral genomes contain non-coding cis-acting sequences, such as two LTRs (long terminal repeats) containing elements necessary for driving gene expression, reverse transcription, and integration into the host cell chromosome; a sequence called the packaging signal (ψ) required for the specific packaging of viral RNA into newly formed virions; and a polypurine tract (PPT) that serves as the site for initiating positive-strand DNA synthesis during reverse transcription. Besides 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 in infected cells, assembly of infected particles, and control of viral replication.

[0213] Lentivirals are complex retroviruses that contain not only the common retroviral genes gag, pol, and env, but also other genes with regulatory or structural functions. This high complexity allows viruses to regulate their life cycle, as they do during latent infection. Lentivirals belong to the 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 simultaneous immunodeficiency virus (SIV).

[0214] "Lentiviral vector (LVV) and retroviral vector (RVV)": As used herein, the term "lentiviral vector (LVV) or retroviral vector (RVV)" is intended to refer to self-inactivated viral particles that include a viral envelope, possess one or more characteristics of lentiviruses or retroviruses, are capable of invading target cells and delivering the gene of interest (GOI), and are not capable of self-replication.

[0215] LVV and RVV can stably integrate exogenous load genes, such as CAR genes, into the chromosomes of target cells, allowing target cells to express the delivered transgenes long-term, providing a significant advantage for gene therapy. Furthermore, they do not transfer viral genes, thus avoiding the problem of transduced cells that can be destroyed by cytotoxic T cells. They also possess relatively large cloning capacity, sufficient to meet most anticipated clinical applications.

[0216] "Packaging system": as used herein, refers to a carrier system containing one or more nucleic acid vectors that, when introduced into packaging cells / packaging cell lines for packaging lentiviral or retroviral vectors, contain nucleic acids necessary for the production, assembly, and / or packaging of the lentiviral or retroviral vectors in the packaging cells / packaging cell lines.

[0217] 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 packaging cell lines and (b) signals providing the signals required for key functions such as viral replication, packaging, reverse transcription, and integration.

[0218] Commonly used lentiviral vector packaging systems include the so-called third-generation lentiviral vector packaging systems. Third-generation lentiviral vector packaging systems include four types of plasmids, typically including a transfer plasmid and three packaging plasmids: a transfer plasmid / master plasmid containing transgenic / shuttle genes encoding the target gene / GOI, such as the CAR 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).

[0219] Typically, a "shuttle / transfer vector" contains the lentiviral backbone genes, transgenes, and signals necessary for key functions such as viral replication, packaging, reverse transcription, and integration. Transfer vectors usually contain one or more transgenes flanked by long terminal repeats (LTRs), which facilitate the integration of transgenes, such as CAR genes, contained in the transfer vector into the host genome. LTRs are responsible for the reverse transcription and integration processes of the viral genome. Through these sequences, lentiviruses can integrate transgenes into the host cell's genome. For safety reasons, transfer vectors are often engineered to prevent the resulting viral vector from self-replicating; for example, the transfer vector may lack the genetic elements necessary to produce an infectious lentiviral vector in the host cell. Furthermore, transfer plasmids can be engineered to lack the 3'LTR, thereby achieving "self-inactivating" of the virus. Compared to traditional second-generation pseudotyped lentiviral vector packaging systems (typically containing a single packaging plasmid and a separate envelope plasmid encoding nucleic acids for Gag, Pol, Rev, and Tat), the TAT gene can be eliminated from third-generation pseudotyped lentiviral vector packaging systems by adding a chimeric 5'LTR fused to a heterologous promoter (e.g., CMV or RSV promoter) to the transfer plasmid. Transfer plasmids typically contain a Ψ sequence (Psi sequence, also known as the Ψ packaging signal) downstream of the 5'LTR, responsible for packaging the transgenic RNA into the viral vector. The Ψ sequence ensures that only transgenic RNA is packaged into the viral vector. Optionally, transfer plasmids may also include an internal ribosome entry site. Sites (“IRES”) are used to allow simultaneous translation of two or more open reading frames (ORFs) on a single mRNA, thereby enabling multigene expression. Some transfer plasmids, such as the master plasmid / transfer plasmid used in some embodiments of the present invention, may also contain selection marker genes, such as antibiotic resistance genes (e.g., PuroR, encoding puromycin resistance) or fluorescent protein genes (e.g., GFP), for screening or tracking transduced cells.

[0220] 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. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, each of which is incorporated herein by reference in its entirety. Generally, transfer plasmids contain basic nucleic acid sequences configured to carry for selecting vector-containing cells, for incorporating foreign nucleic acids into lentiviral particles, and for transferring nucleic acids to target cells.

[0221] Lentiviral backbone genes typically refer 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 provide the signals required for key functions such as viral replication, packaging, reverse transcription, and integration. Specifically, lentiviral backbone genes typically include: long terminal repeats (LTRs): located at both ends of the genome, containing promoters, enhancers, and terminators that regulate viral gene transcription and integration; packaging signals (Ψ): determining which RNA molecules can be recognized and encapsulated into viral particles; central polypurine region (cPPT) and central termination signal (CTS): contributing to improved reverse transcription efficiency and nuclear transport; and Rev response elements (RREs): binding to Rev proteins and regulating the transport of viral RNA from the nucleus to the cytoplasm.

[0222] When constructing transfer plasmids, to ensure safety and efficiency, only the essential backbone sequences are typically retained in the transfer plasmid, while sequences encoding viral structural proteins and enzymes (such as gag, pol, env, etc.) are removed and provided by the packaging system in helper plasmids. This design not only ensures the functionality of the vector but also reduces the risk of generating replicating viruses.

[0223] In some embodiments of the invention, the "backbone gene" is intended to include a nucleic acid encoding a lentiviral or retroviral cis-nucleotide sequence required for genome packaging. The backbone gene may also encode other cis-nucleotide sequences beneficial for gene delivery, including, for example, cis-sequences required for reverse transcription, proviral integration, or genome transcription. Therefore, the exact composition of the backbone gene will depend on the genetic material desired to be introduced into the target cell. Thus, the backbone gene may encode, for example, additional polypeptides or functions besides those required for packaging, reverse transcription, integration, or transcription. Such functions typically include cis-elements encoding those required for the expression of the target nucleic acid / shuttle gene.

[0224] 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. Patent Nos. 6,013,516 and 5,994,136, each of which is incorporated herein by reference in its entirety.

[0225] For a comparison and discussion of the packaging systems of lentiviral vectors and retroviral vectors, and the transfer plasmids they contain, please see: Stripecke, R., Kasahara, N. (2007). Lentivira and Retroviral Vector Systems. In: Hunt, KK, Vorburger, SA, Swisser, SG (eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

[0226] The packaging system of third-generation lentiviral vectors typically includes three packaging plasmids: GagPol plasmid, Rev plasmid, and envelope plasmid. The envelope plasmid usually carries a viral envelope glycoprotein gene; for example, wild-type VSV-G or Cocal-G are commonly used viral glycoproteins. The viral glycoprotein gene is operatively linked to a promoter, typically a CMV promoter, to initiate transcription of the viral glycoprotein gene. In some embodiments of the present invention, the envelope plasmid contains an encoding of any of the aforementioned targeting molecules provided by the present invention.

[0227] The third-generation lentiviral vector packaging system also includes two packaging plasmids: one containing genes encoding the Gag and Pol proteins (GagPol packaging plasmid), and the other containing a gene encoding the Rev protein (Rev plasmid) as a further safety feature, an improvement over the single packaging plasmid of the so-called second-generation packaging system. The Gag gene encodes the Gag polyprotein precursor, which contains 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, containing a protease, reverse transcriptase, and integrase; the Rev gene encodes the Rev protein, which binds Rev response elements (RREs) to allow the nuclear export of unspliced ​​and single-spliced ​​HIV RNA during viral replication. The Gag and Pol polyprotein precursors are cleaved during viral vector preparation. The Rev protein binds to the Rev response element (RRE) sequence on the viral RNA, facilitating the transport of incompletely cleaved viral RNA from the nucleus to the cytoplasm through interaction with the host cell's nuclear export mechanism. This unspliced ​​RNA can then be translated into viral structural proteins and enzymes in the cytoplasm, or assembled into new viral vectors.

[0228] For example, the packaging plasmids include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0229] Compared to LVV packaging systems, RVV packaging systems typically do not contain Rev plasmids. This is because the genomic RNA from retroviruses such as Moloney Murine Leukemia Virus (MMLV) can be naturally transported from the nucleus to the cytoplasm for translation and assembly, thus eliminating the need for specific nuclear export mechanisms such as Rev proteins. RVV packaging systems typically contain one transfer plasmid and two packaging plasmids: an envelope plasmid and a GagPol packaging plasmid. The transgenic sequence contained in the transfer plasmid is flanked by long terminal repeats (LTRs), which facilitate the integration of the transfer plasmid sequence into the host genome. Generally, during viral transduction, sequences between and including LTRs are integrated into the host genome. The genomes of MMLV or Murine Stem Cell Virus (MSCV), containing their respective LTRs, are often used to construct the transfer plasmid in the RVV packaging system. The GagPol packaging plasmid contains the Gag and Pol genes; the envelope plasmid typically contains a polynucleotide encoding a viral glycoprotein, such as VSV-G or Cocal-G. In some embodiments of the present invention, the envelope plasmid may also contain a polynucleotide encoding any of the aforementioned target molecules.

[0230] In some embodiments of the present invention, the packaging system further comprises a binding nucleic acid vector containing a binding molecule encoding any of the viral glycoproteins provided by the present invention, including but not limited to a receptor for the viral glycoprotein and / or a polynucleotide for an antiviral glycoprotein antibody.

[0231] In some embodiments of the present invention, the packaging system further comprises at least one binding plasmid containing a polynucleotide encoding any of the viral glycoprotein binding molecules provided by the present invention.

[0232] In some embodiments, production cells are transfected with transfer plasmids, GagPol plasmids, envelope plasmids, Rev plasmids, and binding plasmids in defined ratios. In some embodiments, the ratio of each plasmid is determined by mass and is not particularly limited as long as it can package a biologically active non-integrating lentiviral or retroviral vector. In some embodiments, the mass of each of the transfer plasmids and GagPol plasmids packaging the lentiviral vector is higher than the mass of each of the envelope plasmids, Rev plasmids, and binding plasmids. In some embodiments, the defined ratio of transfer plasmids, GagPol plasmids, Rev plasmids, envelope plasmids, and binding plasmids is from about 1:1:1:1:0.1 to about 10:5:4:4:4. In some embodiments, the defined ratio of transfer plasmids, GagPol plasmids, envelope plasmids, Rev plasmids, and binding plasmids is from about 9:4:2:2:0.1 to about 9:4:2:2:2. In some embodiments of the present invention, the envelope plasmid may contain nucleic acids encoding target molecules on the surface of the pseudotype LVV or RVV.

[0233] In some embodiments, the viral glycoprotein binding molecule is a membrane-expressing antibody of the viral glycoprotein.

[0234] In some embodiments, the viral glycoprotein binding molecule is an antibody against the viral glycoprotein, and the packaging system further comprises an antibody light chain plasmid and an antibody heavy chain plasmid, the antibody light chain plasmid containing a polynucleotide encoding the light chain of the antibody, and the antibody heavy chain plasmid containing a polynucleotide encoding the heavy chain of the antibody; in the packaging system, the defined ratio of the transfer plasmid, GagPol plasmid, Rev plasmid, envelope plasmid, antibody light chain plasmid, and antibody heavy chain plasmid is from about 1:1:1:1:0.1:0.1 to about 10:5:4:4:4:4. In some embodiments, the defined ratio of the transfer plasmid, GagPol plasmid, Rev plasmid, envelope plasmid, antibody light chain plasmid, and antibody heavy chain plasmid is from about 9:4:2:2:0.1:0.1 to about 9:4:2:2:2:2.

[0235] In some embodiments, the envelope plasmid contains a targeting molecule encoding the surface of any of the aforementioned pseudotypes of LVV or RVV. In some embodiments, the tandem expression cassette contained in the envelope plasmid contains a polynucleotide encoding a first signal peptide, a polynucleotide encoding the target molecule, a polynucleotide encoding one of the internal ribosome entry site (IRES), a furin cleavage site, or viral 2A peptide, a polynucleotide encoding a second signal peptide, and a polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof. In some embodiments, the polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof is located at the 5' end of the polynucleotide encoding the target molecule. In other embodiments, the polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof is located at the 3' end of the polynucleotide encoding the target molecule. The polynucleotide encoding the target molecule and the polynucleotide encoding any of the aforementioned viral glycoproteins or variants thereof are separated in the tandem cassette by the polynucleotide encoding IRES, the furin cleavage site, or viral 2A peptide, which allows co-expression of both proteins by a single mRNA. In some embodiments, the viral 2A peptide is porcine cyclovir-1 (P2A), *Thosea asigna* virus (T2A), equine rhinovirus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. In some embodiments, the viral 2A peptide includes derivatives thereof, such as derivatives of the T2A peptide, T2A cleavage sites with furin cleavage sites and GSG linkers, and FT2A peptides. In some embodiments, at least two different promoters independently drive the expression of polynucleotides encoding any of the aforementioned viral glycoproteins or variants thereof and polynucleotides encoding the target molecule, respectively.

[0236] "Packaging cell lines": The use of packaging systems for lentiviral / retroviral vectors depends on "packaging cells" and "packaging cell lines." Generally, a packaging cell line is a cell line whose cells, when introduced with a packaging system including, for example, a transfer plasmid and one or more packaging plasmids, are capable of producing lentiviral or retroviral vectors that do not have self-replication capabilities and can infect / transduce target cells. An overview of available packaging lines is provided in Cold Spring Harbour Laboratory Press, 1997, p. 447, by JM Coffin, SM Hughes, et al., and is incorporated herein by reference in its entirety.

[0237] For example, various plasmids can be introduced into packaging cell lines using transfection methods including chemically mediated transfection, physically mediated transfection, or biologically mediated transfection. For instance, chemically mediated transfection methods include transfection using chemical reagents such as calcium phosphate, DEAE-glucan, or PEI (polyethylenimine transfection reagent), while physically mediated transfection methods include transfection methods such as electroporation.

[0238] The production / host / packaging cells that can be used to prepare the LVV or RVV provided by this 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.

[0239] For example, the packaging / production cells are 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;

[0240] Preferably, the packaging / production cells are HEK-293T cells.

[0241] 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; the other ways include, but are not limited to, adding genes, deleting genes, and introducing point mutations into genes.

[0242] "Pharmaceutically acceptable excipients or carriers": Pharmaceutically acceptable excipients or carriers include, but are not limited to, diluents, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipients are preferably non-toxic or substantially non-toxic to the recipient at the dosage and concentration used.

[0243] “Subject”: As used herein, the terms “subject,” “patient,” and “individual” are used synonymously, including, but not limited to, mammals, such as humans or non-human mammals, such as livestock, agricultural animals, or wild animals, as well as birds and aquatic animals. “Patient” is a subject who suffers from a disease, condition, or illness, or is at risk of developing a disease, condition, or illness, or otherwise requires any of the pseudo-LVV or RVV, engineered immune cells, compositions, or treatments provided herein.

[0244] “Treatment” refers to the use of the treatment methods described in this article to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor size, a decrease in the rate at which cancer cells infiltrate surrounding organs, or a decrease in the rate of tumor metastasis or growth). Effective treatment methods for patients can vary depending on various factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.

[0245] As used in this article, “treatment” includes any beneficial or desired effect associated with treatment. “Treatment” does not necessarily mean the complete eradication or cure of a disease or condition, or its associated symptoms.

[0246] "And / or": should be understood as referring to one or two alternatives.

[0247] "Comprising": In this document, unless the context otherwise requires, the word "comprising" will be understood to mean including the specified steps, elements, or groups of steps or elements, but not excluding any other steps, elements, or groups of steps or elements. In some embodiments of the invention, the terms "comprising," "having," "containing," and "including" are used synonymously.

[0248] "Embodiments": Throughout this specification, references to "some embodiments" and "some embodiments," or combinations thereof, refer to specific features, structures, or characteristics described in connection with embodiments that are included in at least one embodiment of the invention. Therefore, the appearance of the foregoing phrase in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.

[0249] “Transduction”: As used herein, the terms “transfection,” “transformation,” and “transduction” are used synonymously and refer to the process of transferring or introducing exogenous nucleic acids into host cells, packaging cells, or target cells. “Transfected,” “transformed,” or “transduced” cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include primary subject cells and their progeny. In the context of using viral vectors to deliver exogenous polynucleotides, “infection,” “transfection,” “transformation,” and “transduction” are used synonymously and refer to the process of transferring or introducing exogenous nucleic acids into host cells, packaging cells, or target cells via viral vectors, including but not limited to pseudotyped LVV or RVV.

[0250] Methods for introducing vectors, such as viral particles or isolated polynucleotides, into mammalian cells are known in the art. The described vectors can be transferred to immune effector cells by physical, chemical, or biological methods.

[0251] Physical methods for introducing vectors or isolated polynucleotides into immune effector cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art (see Sambrook, J., Fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.). In some embodiments of the present invention, the vector is introduced into cells via electroporation. In some embodiments of the present invention, the vector is introduced into cells via a PEI transfection reagent.

[0252] All publications, documents, and patents mentioned herein are hereby incorporated in their entirety by reference, as are each individual publication, document, or patent not specifically and individually indicated to be incorporated herein by reference in its entirety. In case of conflict, this application (including any definitions herein) shall prevail. However, any references, articles, publications, patents, patent publications, and patent applications cited herein are not and should not be construed as an admission or suggestion of any kind, but constitute valid prior art or are part of common general knowledge in any country of the world.

[0253] The section headings used in this article are for organizational purposes only and are not intended to limit the topics described. Attached Figure Description

[0254] Figure 1This is a bar chart showing the results of detecting the efficiency of LVV1-1ng, LVV1-10ng, LVV1-100ng, LVV1-1000ng and LVV2-1000ng in transducing Jurkat cells in the experimental groups in Example 1.

[0255] Figure 2 This is a bar chart showing the results of transducing Jurkat cells with LVV7-0.5, LVV7-1, LVV7-2 and LVV8-2 in Example 4.

[0256] Figure 3 : This is the plasmid map of the pGClenti-GFP plasmid;

[0257] Figure 4 This is a bar chart showing the OD value detection results of each positive clone selected for ELISA titer detection in Example 2.

[0258] Figure 5 This is a bar chart showing the GFP positivity rate of each group of cells after transducing Jurkat cells in the experimental groups LVV3, LVV4, LVV5 and LVV6 in Example 2.

[0259] Figure 6 The bar chart shows the results of detecting the GFP positivity rate of each group after transducing Jurkat cells with the experimental groups LVV9, LVV10, LVV11 and LVV12 in Example 3. Detailed Implementation

[0260] The present invention and its technical effects will be clearly and completely described below with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments; other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the protection scope of the present invention.

[0261] Experimental methods not specifically described in the following examples were performed according to conventional methods and conditions known in the art, or according to the product instructions. Reagents and raw materials not specifically described in this invention are all commercially available.

[0262] Example 1

[0263] 1. Packaging control group LVV1

[0264] A. Prepare the following 4 plasmid packaging systems:

[0265] pMD2.G plasmid, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and pGClenti-GFP plasmid; the plasmid map of the pGClenti-GFP plasmid is shown below. Figure 3 As shown.

[0266] B. Packaging control group LVV1

[0267] (1) Mix the four plasmids and transfect them into HEK-293T cells using PEI reagent to package control LVV1 cells; the specific steps are as follows:

[0268] On Day 0, 9 μg of pGClenti-GFP plasmid, 4 μg of pMDLg / pRRE packaging plasmid, 2 μg of pRSV-REV packaging plasmid, and 2 μg of pMD2.G plasmid were added to 1 mL of Opti-MEM medium (Opti-MEM alpha serum-reduced medium, brand: GIBCO, catalog number: #SP0272). After shaking well, 64 μL of PEI reagent was added, and the mixture was incubated for 10 minutes. Then, it was added to the HEK-293T cell culture system. The medium was replaced after 6 hours. 48 hours after transfection, the supernatant was collected, filtered through a 0.45 μm filter, centrifuged at 50,000 g for 2.5 h, and the supernatant was discarded. The lentiviral vector was resuspended in 200 μL of F12 medium and stored at -80 °C.

[0269] HEK-293T cell culture system: DMEM + 10% FBS; DMEM: Brand: GIBCO, Catalog No.: #C12430500BT; FBS: Brand: EXCELL, Catalog No.: #FSP500; F12 medium: Brand: GIBCO, Catalog No.: #C11330500BT; Needle filter: Brand: SORFA, Catalog No.: #622120.

[0270] 2. Packaging control group LVV2

[0271] Referring to the method used for the control group LVV1, the packaging surface contains a targeting molecule, namely the control group LVV2 which contains membrane-expressed anti-CD7 antibody;

[0272] In this embodiment, the pMD2.G plasmid is replaced with a plasmid containing a polynucleotide encoding a membrane-expressing anti-CD7 antibody and a wild-type VSV-G gene (A7 envelope plasmid); the method for synthesizing the A7 envelope plasmid using conventional molecular cloning methods is well known to those skilled in the art;

[0273] The wild-type VSV-G contains the amino acid sequence shown in SEQ ID NO:1;

[0274] KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:1)

[0275] The amino acid sequence of the wild-type VSV-G full-length protein is shown in SEQ ID NO:7.

[0276] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEA VIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAAAR FPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPNGVL RTSSGYKFPLYMIGGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK(SEQ ID NO:7)

[0277] Among them, the sequence MKCLLYLAFLFIGVNC contained in sites 1-16 is the amino acid sequence of the signal peptide of wild-type VSV-G.

[0278] Construct membrane-expressed anti-CD7 antibody:

[0279] Anti-CD7 antibodies bind to the endocytosis receptor CD7, triggering endocytosis. This allows lentiviral vectors containing anti-CD7 antibodies in their viral envelope to enter and infect CD7 cells via endocytosis. + Cells such as T cells.

[0280] The structure of the membrane-expressed anti-CD7 antibody from the N-terminus to the C-terminus is as follows: human CD8α signal peptide, CD7-binding antigen-binding region, human CD8α hinge region, and human CD8α transmembrane domain; the CD7-binding antigen-binding region includes scFv TH-69 (TH69-scFv), and the heavy chain variable region (VH region) of TH-69 is connected to the light chain variable region (VL region) of TH-69 through a (G4S)3 linker;

[0281] (1) The amino acid sequence of the human CD8α signal peptide is shown in SEQ ID NO:8;

[0282] (2) The amino acid sequence of the VH region of TH-69 is shown in SEQ ID NO:9;

[0283] (3) The amino acid sequence of the (G4S)3Linker is shown in SEQ ID NO:10;

[0284] (4) The amino acid sequence of the VL region of TH-69 is shown in SEQ ID NO:11;

[0285] (5) The amino acid sequence of the TH69-scFv is shown in SEQ ID NO:29; the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NO:23-25; the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NO:26-28.

[0286] (6) The amino acid sequence of the human CD8α hinge region is shown in SEQ ID NO:12;

[0287] (7) The amino acid sequence of the human CD8α transmembrane domain is shown in SEQ ID NO:13.

[0288] 3. Preparation of LDL-R infection via VSV-G binding to LDL-R + The experimental group with weakened LVV cells

[0289] A. Preparation of experimental group LVV1

[0290] On Day 0, the control group LVV1 from the 4E5 TU (Transducing Unit) was mixed with 1 ng (packaged LVV1-1ng), 10 ng (packaged LVV1-10ng), 100 ng (packaged LVV1-100ng), or 1 μg / 1000 ng (LVV1-1000ng group) of anti-VSV-G antibody (trade name: 1E9F9, brand: KERAFAST, catalog number: #Kf-Ab01402-2.0) and treated at 37°C for 1 hour to prepare the experimental groups LVV1-10ng, LVV1-100ng, and LVV1-1000ng.

[0291] B. Preparation of experimental group LVV2

[0292] The control group LVV2 described in 4E5 TU was mixed with 1 μg / 1000ng (packaged LVV2-1000ng) of anti-VSV-G antibody and treated at 37℃ for 1 h to prepare the experimental group LVV2.

[0293] 4. Detection of LVV transduction LDL-R in each group + and CD7 + Jurkat cell capacity (MOI=4)

[0294] At MOI=4, 1×10⁻⁶ lentiviral vectors were transduced into the control group LVV1, control group LVV2, LVV1-1ng group, LVV1-10ng group, LVV1-100ng group, LVV1-1000ng group, and LVV2-1000ng group, respectively. 5 LDL-R + and CD7 + Jurkat cells were cultured at 37°C and 5% CO2. The Jurkat cell culture system included 1640 (brand: Elgbio, catalog number: EH80809) medium and 10% FBS.

[0295] Day 2, flow cytometry was used to detect the positivity rate of GFP molecules in Jurkat cells of each group. The results are as follows: Figure 1 As shown.

[0296] Depend on Figure 1 It can be seen that, compared to the control group LVV1 and 2 (i.e., not mixed with the VSV-G antibody), Figure 1 The experimental groups treated with a mixture of "VSVG" and "VSVG-antiCD7" antibody and VSV-G antibody showed LVV1 ( Figure 1 The experimental groups of LVV1 ("VSVG+1E9F9-1ng", "VSVG+1E9F9-10ng", "VSVG+1E9F9-100ng", "VSVG+1E9F9-1000ng") contain VSV-G on their surface that has been pre-bound to anti-VSV-G antibodies. The viral envelope does not contain membrane-expressing anti-CD7 antibodies. These LVV1 cells transduced LDL-R by binding to it. + The ability of Jurkat cells to react with the increased amount of anti-VSV-G antibody mixed with them was significantly reduced;

[0297] The experimental group LVV2 (i.e. Figure 1 When "VSVG-antiCD7+1E9F9-1000ng" is mixed with 1000ng of VSV-G antibody, even though the VSV-G on its surface has been pre-bound to the VSV-G antibody, it causes the transduction of LDL-R by binding to LDL-R. + The ability of Jurkat cells to infect was significantly weakened; however, because its viral envelope contains membrane-expressing anti-CD7 antibodies, the LVV2 in the experimental group could still enter and infect CD7 cells via endocytosis induced by binding to the endocytosis receptor CD7. + Jurkat cells were used, and the infection efficiency was comparable to that of the control groups LVV1 and 2.

[0298] Example 2

[0299] 1. Immunize mice to prepare anti-VSV-G antibodies.

[0300] In this embodiment, mice were immunized with VSV-G antigen, and mouse spleen cells were used for hybridoma fusion. Positive clones were selected, and after ELISA titer detection, the positive clones with the highest OD values ​​were selected for sequencing to obtain the anti-VSV-G antibody sequence. The specific operations included:

[0301] A. Antigen preparation and immunization:

[0302] VSV-G antigen was prepared using a recombinant expression system. A polynucleotide encoding the extracellular domain of VSV-G was cloned into the pET-28a expression vector, and a 6×His tag (VSV-G-His antigen) was attached to the C-terminus of the VSV-G extracellular domain for purification and coating. The VSV-G-His antigen was expressed in *E. coli* and purified by Ni-NTA affinity chromatography to a concentration of approximately 0.5 mg / mL. BALB / c mice were immunized intraperitoneally with a mixture of VSV-G-His antigen and Freund's complete adjuvant, every two weeks for a total of three times. After confirming serum titer (10000) by ELISA using tail vein blood collection, the spleens of the mice were harvested for subsequent hybridoma fusion.

[0303] The amino acid sequence of the VSV-G-His antigen is shown in SEQ ID NO:43.

[0304] MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPP QSCGYATVTDAEAAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEM ADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVE IGPNGVLRTSSGYKFPLYMIGGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK HHHHHH (SEQ IDNO:43).

[0305] B. Cell fusion and hybridoma screening

[0306] Mouse spleen cells and SP2 / 0 myeloma cells were mixed at a ratio of 5:1 and chemically fused using 50% PEG1500. The fused cells were seeded in 96-well plates containing HAT selective medium. After 7–10 days of culture, positive clones that specifically bound the VSV-G antigen were selected from the supernatant; a total of 8 groups were used for ELISA detection.

[0307] C. ELISA screening clone titer

[0308] The purified VSV-G-His antigen was coated onto a 96-well ELISA plate at a concentration of 0.5 μg / mL and incubated overnight at 4°C. The plate was then blocked with 5% BSA. A 1:10 dilution of hybridoma supernatant was added, followed by incubation. HRP-labeled anti-mouse IgG secondary antibody was then added, and TMB was used for colorimetric development. OD values ​​were read at 450 nm. Results are as follows: Figure 4 As shown.

[0309] Depend on Figure 4It can be seen that clone1, clone2, clone5, clone6, and clone7 showed obvious positive results, among which clone6 had the highest OD value and was selected as the target for subsequent sequencing and recombination expression.

[0310] D. Sequencing anti-VSV-G antibodies

[0311] RNA was extracted from clone6 cells, and the variable region sequences of the heavy and light chains were amplified by RT-PCR. The heavy and light chain constant regions were ligated using MouseIgG2ak heavy chain constant region and κ chain constant region primers, respectively. The complete gene sequence of the anti-VSV-G antibody was obtained by sequencing.

[0312] The amino acid sequence of the HCDR1 region of the anti-VSV-G antibody is shown in SEQ ID NO:30.

[0313] IRQPSWK (SEQ ID NO:30)

[0314] The amino acid sequence of the HCDR2 region of the anti-VSV-G antibody is shown in SEQ ID NO:31.

[0315] TISKDTSSSQVFLKFASVDAADT(SEQ ID NO:31)

[0316] The amino acid sequence of the HCDR3 region of the anti-VSV-G antibody is shown in SEQ ID NO:32.

[0317] YYFGTSFFDY(SEQ ID NO:32)

[0318] The amino acid sequence of the LCDR1 region of the anti-VSV-G antibody is shown in SEQ ID NO:33.

[0319] KSLQSLLNSRARKNYLA(SEQ ID NO:33)

[0320] The amino acid sequence of the LCDR2 region of the anti-VSV-G antibody is shown in SEQ ID NO:34.

[0321] WWSTRES (SEQ ID NO:34)

[0322] The amino acid sequence of the LCDR3 region of the anti-VSV-G antibody is shown in SEQ ID NO:35.

[0323] CKQSYFLFT(SEQ ID NO:35)

[0324] The amino acid sequence of the VH region of the anti-VSV-G antibody is shown in SEQ ID NO:36.

[0325] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSWKGLEWLAHIWWDDAKHYNPA LKSRLTISKDTSSSQVFLKFASVDAADTASYFCARIPYYFGTSFFDYWGQGTTLTVSS(SEQ ID NO:36)

[0326] The amino acid sequence of the VL region of the anti-VSV-G antibody is shown in SEQ ID NO:37.

[0327] DIVMSQSPSSLTVSAGEKVTMSCKSLQSLLNSRARKNYLAWYQQKPGQSPKLLIYWWSTRESGV PDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYFLFTFGSGTKLEIK(SEQ ID NO:37)

[0328] E. Synthesize the light and heavy chains of the antibody separately.

[0329] The heavy and light chains of an anti-VSV-G antibody that can be secreted and expressed in HEK-293T cells under the action of a signal peptide are constructed, comprising: linking the N-terminus of the VL region and VH region of the anti-VSV-G antibody to the signal peptide (SEQ ID NO:38), and linking the C-terminus to the light chain constant region (CL region) and heavy chain constant region (CH region) of mouse IgG2ak, respectively.

[0330] The polynucleotide encoding the heavy chain of the anti-VSV-G antibody sequentially encodes from the 5' end to the 3' end: signal peptide (SEQ ID NO:38), the VH region of the anti-VSV-G antibody (SEQ ID NO:36), and the CH region of mouse IgG2ak (SEQ ID NO:39);

[0331] The polynucleotide encoding the light chain of the anti-VSV-G antibody sequentially encodes, from the 5' end to the 3' end, the following: signal peptide (SEQ ID NO: 38), the VL region of the anti-VSV-G antibody (SEQ ID NO: 37), and the CL region of mouse IgG2ak (SEQ ID NO: 40).

[0332] The amino acid sequence of the signal peptide is shown in SEQ ID NO:38.

[0333] MGILPSPGMPALLSLVSLLSVLLMGCVA(SEQ ID NO:38)

[0334] The amino acid sequence of the CL region of the mouse IgG2ak is shown in SEQ ID NO:40.

[0335] RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDS TYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:40)

[0336] The amino acid sequence of the CH region of the mouse IgG2ak is shown in SEQ ID NO:39.

[0337] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEV HTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:39)

[0338] The amino acid sequence of the anti-VSV-G antibody heavy chain is shown in SEQ ID NO:41.

[0339] MGILPSPGMPALLSLVSLLSVLLMGCVAQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSWKGLEWLAHIWWDDAKHYNPALKSRLTISKDTSSSQVFLKFASVDAADTA SYFCARIPYYFGTSFFDYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTK VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPA PIERTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:41)

[0340] The amino acid sequence of the light chain of the anti-VSV-G antibody is shown in SEQ ID NO:42.

[0341] MGILPSPGMPALLSLVSLLSVLLMGCVADIVMSQSPSSLTVSAGEKVTMSCKSLQSLLNSRARKNYLAWYQQKPGQSPKLLIYWWSTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQS YFLFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:42)

[0342] The gene sequences encoding the heavy and light chains of the anti-VSV-G antibody were cloned into the pcDNA3.4 expression vector, and anti-VSV-G antibody heavy chain plasmid and anti-VSV-G antibody light chain plasmid were synthesized respectively.

[0343] 2. Packaging experimental groups: LVV3, LVV4, LVV5, and LVV6

[0344] Referring to the method for packaging the control group LVV1 described above, 0.5 μg, 1 μg, or 2 μg of anti-VSV-G antibody heavy chain plasmid and light chain plasmid were added to the 4-plasmid packaging system to package the experimental groups LVV3, LVV4, and LVV5, respectively.

[0345] Referring to the method used for packaging the control group LVV2, 2 μg of anti-VSV-G antibody heavy chain plasmid and anti-VSV-G antibody light chain plasmid were added to the 4-plasmid packaging system. The packaged viral envelope also contained the experimental group LVV6, which expressed anti-CD7 antibody.

[0346] After the anti-VSV-G antibody heavy chain plasmid and light chain plasmid are secreted and expressed separately by packaging cells, they form a complete anti-VSV-G antibody to bind to the VSV-G expressed in the packaging cells, thereby enabling the release of VSV-G expressed on the cell membrane of the packaging cells to bind LDL-R. + The reduction in LDL-R binding sites on the cell surface decreases, thereby reducing the ability of LVV3, LVV4, LVV5, and LVV6 in the experimental group to transduce LDL-R by binding to it. + The cell's ability.

[0347] 3. Transducing Jurkat cells

[0348] Following the transduction method for Jurkat cells in experimental groups LVV1 and 2, on Day 0, LVV3, LVV4, LVV5, and LVV6 in experimental groups were transduced with 1×10⁻⁵ TU of the same method. 5 Jurkat cells (i.e., MOI = 4) were transduced with 1 × 10⁶ Jurkat cells, while the control group consisted of LVV1 and LVV2 cells transduced with the control group. 5 One Jurkat cell;

[0349] Day 2, flow cytometry was used to detect the positivity rate of GFP molecules in Jurkat cells of each group. The results are as follows: Figure 5 As shown.

[0350] Depend on Figure 5 It can be seen that, compared to the control group LVV1 and 2 (i.e., those without the addition of the VSV-G antibody heavy chain plasmid and light chain plasmid in the packaging system), Figure 5 The experimental groups LVV3, LVV4, and LVV5 (i.e., groups "VSVG" and "VSVG-antiCD7") were packaged by adding 0.5 μg, 1 μg, or 2 μg of anti-VSV-G antibody heavy chain plasmid and light chain plasmid to the 4-plasmid packaging system. Figure 5 (0.5μg, 1μg, and 2μg groups) LDL-R transduction +The ability of Jurkat to bind was significantly reduced, demonstrating that adding heavy and light chain plasmids containing the VSV-G antibody to the packaging system to express anti-VSV-G antibodies in packaging cells successfully reduced the number of available binding sites for LDL-R on the viral envelope of VSV-G expressed and translocated to LVV in packaging cells, thereby reducing LVV transduction of LDL-R by binding to LDL-R. + The ability of cells such as Jurkat cells and some cancer cells.

[0351] The experimental group LVV6 (i.e. Figure 5 The "VSVG-antiCD7+2μg" group transduced CD7 + LDL-R + Jurkat cells did not exhibit a significant decrease in their ability compared to the control groups LVV1 and LVV2. This demonstrates that even though the VSV-G on their surface has already bound to anti-VSV-G antibodies in packaging cells, leading to LDL-R transduction via binding, this indicates that even if the VSV-G on their surface has already bound to anti-VSV-G antibodies in packaging cells, this does not necessarily mean that the cells can transduce LDL-R. + The ability of Jurkat cells to infect CD7 was significantly reduced; however, because its viral envelope contains membranes expressing anti-CD7 antibodies, the experimental group of LVV6 could still enter and infect CD7 cells via endocytosis induced by binding to the endocytosis receptor CD7. + Jurkat cells.

[0352] Example 3

[0353] 1. Construct a membrane expression system for anti-VSV-G antibodies

[0354] A plasmid (anti-VSV-G antibody plasmid) containing a polynucleotide encoding a membrane-expressing anti-VSV-G antibody gene was synthesized using conventional molecular cloning methods. The membrane-expressing anti-VSV-G antibody gene sequentially encodes from the 5' end to the 3' end: the human CD8α signal peptide (SEQ ID NO:8), the VL region of the anti-VSV-G antibody (SEQ ID NO:37), the (G4S)3Linker (SEQ ID NO:10), the VH region of the anti-VSV-G antibody (SEQ ID NO:36), the human CD8α hinge region (SEQ ID NO:12), and the human CD8α transmembrane region (SEQ ID NO:13).

[0355] Referring to the method for packaging the control group LVV1 described above, 0.5 μg, 1 μg, or 2 μg of the membrane-expressing anti-VSV-G antibody plasmid was added to the 4-plasmid packaging system to package the experimental groups LVV9, LVV10, and LVV11, respectively.

[0356] Referring to the method described above for packaging the control group LVV2, 2 μg of the membrane-expressing anti-VSV-G antibody plasmid was added to the 4-plasmid packaging system, and the packaged viral envelope also contained the experimental group LVV12, which expressed the membrane-expressing anti-CD7 antibody.

[0357] 2. Transducing Jurkat cells

[0358] Following the transduction methods for LVV1 and LVV2 cells in the experimental groups described above, on Day 0, LVV9, LVV10, LVV11, and LVV12 cells in the experimental groups described above were transduced with 1×10⁻⁶ tbsp solution. 5 Jurkat cells (i.e., MOI = 4) were used as the control group, which received 1 × 10⁶ Jurkat cells transduced with LVV1 and LVV2, respectively. 5 One Jurkat cell;

[0359] Day 2, flow cytometry was used to detect the positivity rate of GFP molecules in Jurkat cells of each group. The results are as follows: Figure 6 As shown.

[0360] Depend on Figure 6 It can be seen that, compared to the control group LVV1 and 2 (i.e., those without the membrane expressing anti-VSV-G antibody plasmid added to the packaging system), Figure 6 The experimental groups LVV9, LVV10, and LVV11 (i.e., groups "VSVG" and "VSVG-antiCD7") were packaged by adding 0.5 μg, 1 μg, or 2 μg of membrane-expressed anti-VSV-G antibody plasmids to the 4-plasmid packaging system. Figure 6 (0.5μg, 1μg, and 2μg groups) LDL-R transduction + The ability of Jurkat cells to express anti-VSV-G antibodies was significantly reduced, demonstrating that adding a plasmid containing the membrane-expressing anti-VSV-G antibody plasmid to the packaging system to express the anti-VSV-G antibody in packaging cells successfully reduces the number of available binding sites for LDL-R on the viral envelope of VSV-G that expresses and translocates to LVV in packaging cells, thereby reducing LVV transduction of LDL-R by binding to LDL-R. + The ability of cells such as Jurkat cells and some cancer cells.

[0361] The experimental group LVV12 (i.e. Figure 6 The "VSVG-antiCD7+2μg" group transduced CD7 + LDL-R +Jurkat cells did not exhibit a significant decrease in their ability compared to the control groups LVV1 and LVV2. This demonstrates that even though the VSV-G on their surface has already bound to anti-VSV-G antibodies in packaging cells, leading to LDL-R transduction via binding, this indicates that even if the VSV-G on their surface has already bound to anti-VSV-G antibodies in packaging cells, this does not necessarily mean that the cells can transduce LDL-R. + The ability of Jurkat cells to infect CD7 was significantly reduced; however, because its viral envelope contains a membrane expressing anti-CD7 antibodies, the experimental group of LVV12 could still enter and infect CD7 cells via endocytosis induced by binding to the endocytosis receptor CD7. + Jurkat cells.

[0362] Example 4

[0363] Referring to the packaging methods of control groups LVV1 and 2 described above, experimental group LVV7, whose viral envelope does not contain the membrane expressing anti-CD7 antibodies, and experimental group LVV8, whose viral envelope contains the membrane expressing anti-CD7 antibodies;

[0364] 1. Packaging Experimental Group LVV7

[0365] The experimental group LVV7 was packaged as follows: referring to the method for packaging the control group LVV1, 0.5 μg (packaging LVV7-0.5), 1 μg (packaging LVV7-1), or 2 μg (packaging LVV7-2) of LDL-R plasmid was added to the 4-plasmid packaging system containing the pMD2.G plasmid, so that the LDL-R was overexpressed in the packaging cells HEK-293T cells;

[0366] The LDL-R plasmid was synthesized using conventional molecular cloning methods. It contains a polynucleotide encoding LDL-R, the amino acid sequence of which is shown in SEQ ID NO:14.

[0367] 2. Packaging Experimental Group LVV8

[0368] The packaging experimental group LVV8 was operated as follows: referring to the packaging method of the control group LVV2, 2 μg of the LDL-R plasmid (packaging LVV8-2) was added to the 4 plasmid packaging system containing the A7 envelope plasmid, so that the LDL-R was overexpressed in the packaging cells HEK-293T cells.

[0369] 3. LVV transduction of Jurkat cells in each group

[0370] Following the method described above for transducing Jurkat cells with LVV1 and LVV2 in the experimental groups, on Day 0, at MOI = 4, 1×10⁻⁶ cells were transduced using the lentiviral vectors LVV7-0.5, LVV7-1, LVV7-2, and LVV8-2, respectively. 5Jurkat cells; the control group consisted of LVV1 and LVV2 transduced with 1 × 10⁶ cells each; 5 Jurkat cells; on Day 2, flow cytometry was used to detect the positivity rate of GFP molecules in each group of Jurkat cells, and the results are as follows. Figure 2 As shown.

[0371] Depend on Figure 2 It can be seen that, compared to the control group LVV1 and 2 (i.e., packaged by a packaging system without the added LDL-R plasmid), Figure 2 The "VSVG" and "VSVG-antiCD7" groups, because their surface-containing VSV-G had already been pre-bound to LDL-R in HEK-293T cells, did not contain the membrane-expressing anti-CD7 antibodies in the experimental groups LVV7-0.5, 1, and 2 (i.e., VSVG and VSVG-antiCD7 groups). Figure 2 The drugs “VSVG+LDLR-0.5μg”, “VSVG+LDLR-1μg”, and “VSVG+LDLR-2μg” transduce LDL-R by binding to it. + The ability of Jurkat cells to produce this effect was significantly reduced with increasing amounts of LDL-R plasmids added to their packaging system;

[0372] Because its viral envelope contains membrane-expressing anti-CD7 antibodies, the experimental group LVV8-2 (i.e. Figure 2 The "VSVG-antiCD7+LDLR-2μg" can still enter and infect CD7 cells through endocytosis triggered by binding to the CD7 endocytic receptor. + Jurkat cells were used, and the infection efficiency was comparable to that of the control groups LVV1 and 2.

Claims

1. A modified viral vector, characterized in that, The modified viral vector surface contains viral glycoproteins, which have a reduced ability to bind to receptors on the cell surface.

2. The modified viral vector according to claim 1, characterized in that, The number of binding sites for the viral glycoprotein to bind to receptors on the cell surface is reduced.

3. The modified viral vector according to claim 1 or 2, characterized in that, The viral glycoprotein has bound to at least one of its receptor and antibody, thereby weakening the ability of the viral glycoprotein to bind to its receptor on the cell surface. Preferably, the receptor or antibody to which the viral glycoprotein has bound is a free receptor or antibody that is expressed on the viral membrane or displayed on the viral membrane.

4. The modified viral vector according to any one of claims 1-3, characterized in that, The modified viral vector is a pseudotyped lentiviral vector (LVV) or a retroviral vector (RVV). Preferably, the viral glycoprotein binds to receptors and / or antibodies on the packaging cells and / or cell membranes of the pseudotype LVV or RVV.

5. The modified viral vector according to claim 4, wherein the viral glycoprotein is selected from vesicular stomatitis virus strain glycoprotein, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibberish leukemia virus glycoprotein (GaLV), ditropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein; The glycoproteins of the vesicular stomatitis virus strain include: The following are glycoproteins of vesicular stomatitis virus (VSV) strains: Indiana, Cocal, Maraba, Morreton, Alagoas, New Jersey, Carajas, Chandipura, and Ept. The glycoproteins of the following strains are listed: esicus, Isfahan, Jurona, Malpais, Perinet, Piry, Radi, Rhinolopus, and YugBogdanovac.

6. The modified viral vector according to claim 5, characterized in that, The viral glycoprotein is either the Indiana strain glycoprotein of vesicular stomatitis virus (VSV-G) or the Cocal strain glycoprotein of vesicular stomatitis virus (Cocal-G), the receptor includes LDL-R, and the antibody includes VSV-G antibody or Cocal-G antibody. Preferably, the amino acid sequences of the HCDR1-3 regions of the VH region of the anti-VSV-G antibody are shown in SEQ ID NO:30-32, respectively; and the amino acid sequences of the LCDR1-3 regions of the VL region of the anti-VSV-G antibody are shown in SEQ ID NO:33-35, respectively.

7. The modified viral vector according to any one of claims 1-6, characterized in that, The viral glycoprotein contains mutations that enhance or prevent the viral glycoprotein from being inactivated by complement.

8. The modified viral vector according to claim 7, characterized in that, The viral glycoprotein is VSV-G or Cocal-G, and the mutation includes mutations at at least one of the following sites: (a) The 214th amino acid located 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, the amino acid located at the 214th amino acid position corresponding to SEQ ID NO:1 or SEQ ID NO:2; (c) The 352nd amino acid located in SEQ ID NO:1 or SEQ ID NO:2; (d) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at position 352, which corresponds to SEQ ID NO:1 or SEQ ID NO:2; (e) The 50th amino acid located in SEQ ID NO:1 or SEQ ID NO:2; (f) After optimal global alignment with SEQ ID NO:1 or SEQ ID NO:2, the amino acid located at the 50th amino acid corresponding to SEQ ID NO:1 or SEQ ID NO:2; (g) the amino acid located at 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, the amino acid located at the 146th amino acid position corresponding to SEQ ID NO:1 or SEQ ID NO:2; Preferably, the mutation at the site is selected from amino acid substitutions, deletions, and insertions; More preferably, the mutation at the site is an amino acid substitution.

9. The modified viral vector according to claim 7 or 8, characterized in that, The mutation includes mutations at at least one of the following sites: (a) The amino acid at position 214 of SEQ ID NO:1 is replaced by threonine T with asparagine N (T214N), the amino acid at position 352 is replaced by threonine T with alanine A (T352A), the amino acid at position 50 is replaced by lysine K with threonine T (K50T), and the amino acid at position 146 is replaced by serine S with threonine T (S146T); (b) After best global alignment with SEQ ID NO:1, T214N, T352A, K50T, and S146T are located at the equivalent of SEQ ID NO:1; (c) The amino acid at position 214 of SEQ ID NO:2 is replaced by lysine K with asparagine N (K214N), T352A, K50T, or S146T; and (d) After best global alignment with SEQ ID NO:2, it is located at K214N, T352A, K50T, and S146T, which are equivalent to SEQ ID NO:

2.

10. The modified viral vector according to claim 8, characterized in that, The mutation includes any combination of mutations at the following sites: (a) Substitution of (1) T214 and T352 located in SEQ ID NO:1; or (2) Substitution of T214, T352, K50 and S146; (b) After best global alignment with SEQ ID NO:1, the substitutions located at (1) T214 and T352 of SEQ ID NO:1; or (2) substitutions of T214, T352, K50 and S146; (c) Substitution of (1) K214 and T352 located in SEQ ID NO:2; or (2) Substitution of K214, T352, K50 and S146; and (d) After best global alignment with SEQ ID NO:2, the substitutions located at (1) K214 and T352 in SEQ ID NO:2; or (2) K214, T352, K50 and S146; Preferably, the mutation includes a combination of any of the following site mutations: (a) (1) T214N and T352A located in SEQ ID NO:1; or (2) T214N, T352A, K50T and S146T; (b) After best global alignment with SEQ ID NO:1, it is located at (1) T214N and T352A, or (2) T214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:1; (c) K214N and T352A located in SEQ ID NO:2; or (2) K214N, T352A, K50T and S146T; and (d) After best global alignment with SEQ ID NO:2, it is located at (1) K214N and T352A, or (2) K214N, T352A, K50T and S146T, which are equivalent to SEQ ID NO:

2.

11. The modified viral vector according to any one of claims 1-10, characterized in that, The modified viral vector surface also contains a targeting molecule that binds to at least one of the following antigens: 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, soybean protein, HPV E6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, spermin 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostate-specific protein, survival protein and telomerase, PCTA-1 / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2-ETS fusion gene / ERG, NA17, PAX3, androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B 1. At least one of the following: BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, muthsp70-2, CD79α, CD79β, ASGPR, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, MSLN, NKG2D, TCRα, TCRβ, TCRγ, and TCRδ. Preferably, the targeting molecule binds to at least one of CD3, CD5, CD7, CD28, TCRα, TCRβ, TCRγ, and TCRδ.

12. The modified viral vector according to claim 11, characterized in that, The targeting molecule includes an antigen-binding region that can bind the antigen, and the antigen-binding region includes at least one of (a) an antibody or an antigen-binding fragment thereof and (b) a ligand or a receptor-binding fragment thereof.

13. The modified viral vector according to claim 12, characterized in that, The targeting molecule further includes a transmembrane domain, and the antigen-binding region is directly or indirectly connected to the transmembrane domain. Preferably, the transmembrane domain is selected from the transmembrane domains of the following proteins: CD28, CD2, CD4, CD8α, CD5, CD3ε, CD3δ, CD3ζ, CD9, CD16, CD22, CD25, CD27, CD33, CD37, CD40, CD45, CD64, CD79A, CD79B, CD80, CD86, CD 95(Fas), CD134(OX40), CD137(4-1BB), CD150(SLAMF1), CD152(CTLA4), CD154(CD40L), CD200R, CD223(LAG3), CD270(HVEM), CD272( BTLA), CD273(PD-L2), CD274(PD-L1), CD278(ICOS), CD279(PD-1), CD300, CD357(GITR), A2aR, DAP10, FcRα, FcRβ, FcRγ, Fyn, GAL9, K IR, Lck, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PTCH2, ROR2, Ryk, Slp76, SIRPα, pTα, TCRα, TCRβ, TIM3, TRIM, LPA5 and Zap70; More preferably, the transmembrane domain includes the CD8α transmembrane domain.

14. The modified viral vector according to claim 13, characterized in that, The targeting molecule further includes a linker domain, through which the antigen-binding region is indirectly connected to the transmembrane domain; Preferably, the connection structure domain is selected from: (a) Immunoglobulin hinge region, wherein the immunoglobulin hinge region is selected from wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA and IgD hinge regions; (b) Hinge region, wherein the hinge region is selected from the wild-type or modified hinge regions of the following proteins: CD28, CD7, CD8, CD8α, CD8β, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154; (c) All or a portion of the Fc domains, wherein the Fc domains are selected from one or more of the CH1, CH2, and CH3 domains; and (d) Stem regions of type II C-lectins, wherein the type II C-lectins are selected from the stem regions of CD23, CD69, CD72, CD94, NKG2A and NKG2D; More preferably, the connection structure domain includes the CD8α hinge region.

15. The modified viral vector according to any one of claims 1-14, characterized in that, The modified viral vector, by mixing with at least one of the viral glycoprotein's receptor and antibody, weakens the ability of the viral glycoprotein to bind to the viral glycoprotein's receptor on the cell surface.

16. The modified viral vector according to any one of claims 1-15, characterized in that, The modified viral vector also contains exogenous payload genes; Preferably, the exogenous burden gene contains a polynucleotide (CAR gene) encoding a chimeric antigen receptor (CAR), wherein the CAR contains an extracellular antigen-binding region, a transmembrane region, and an intracellular signal transduction domain; More preferably, the CAR further includes a hinge region and a co-stimulatory signal transduction domain; the hinge region connects the extracellular antigen-binding region and the transmembrane region.

17. A method for reducing the ability of a viral vector to infect a cell by binding to receptors of the viral glycoprotein on the cell surface via a viral glycoprotein contained on its surface, characterized in that, The method includes reducing the binding sites of the viral glycoprotein on cell surfaces that are available for binding to receptors of the viral glycoprotein.

18. The method according to claim 17, characterized in that, The method includes, prior to infecting cells with the viral vector, causing the viral glycoprotein to bind to at least one of its receptor and antibody.

19. The method according to claim 17 or 18, characterized in that, The viral vector is a pseudotype LVV or RVV.

20. The method according to claim 19, characterized in that, The method includes: (a) Packaging the pseudotyped LVV or RVV using a packaging system containing a viral glycoprotein binding molecule gene; and / or (b) After packaging the pseudo-LVV or RVV, the pseudo-LVV or RVV is bound to a viral glycoprotein binding molecule; The viral glycoprotein binding molecule is selected from at least one of the receptor and antibody of the viral glycoprotein.

21. The method according to claim 20, characterized in that, After packaging the pseudo-LVV or RVV, the pseudo-LVV or RVV is mixed with viral glycoprotein binding molecules.

22. The method according to any one of claims 17-21, characterized in that, The viral glycoproteins are selected from vesicular stomatitis virus strains glycoprotein, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibberish leukemia virus glycoprotein (GaLV), ditropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein; The vesicular stomatitis virus (VSV) glycoproteins include: VSV Indiana strain glycoprotein, VSV Cocal strain glycoprotein, VSV Maraba strain glycoprotein, VSV Morreton strain glycoprotein, VSV Alagoas strain glycoprotein, VSV New Jersey strain glycoprotein, VSV Carajas strain glycoprotein, VSV Chandipura strain glycoprotein, and vesicular stomatitis virus (VSV) glycoprotein. The glycoproteins of the following strains of vesicular stomatitis virus (VSV) are: Eptesicus, Isfahan, Jurona, Malpais, Perinet, Piry, Radi, Rhinolopus, and YugBogdanovac. Preferably, the viral glycoprotein is VSV-G or Cocal-G, the receptor includes LDL-R, and the antibody includes VSV-G antibody or Cocal-G antibody; More preferably, the amino acid sequences of the HCDR1-3 regions of the VH region of the anti-VSV-G antibody are shown in SEQ ID NO:30-32, respectively; and the amino acid sequences of the LCDR1-3 regions of the VL region of the anti-VSV-G antibody are shown in SEQ ID NO:33-35, respectively.

23. A pseudo-LVV or RVV packaging system, characterized in that, The pseudotyped LVV or RVV packaging system contains a viral glycoprotein binding molecule gene, wherein the viral glycoprotein binding molecule is selected from at least one of viral glycoprotein receptors and antibodies.

24. The pseudo-LVV or RVV packaging system according to claim 23, characterized in that, The viral glycoproteins are selected from vesicular stomatitis virus strains glycoprotein, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoprotein, rabies virus glycoprotein (RVG), gibberish leukemia virus glycoprotein (GaLV), ditropic murine leukemia virus glycoprotein (MLV-A), feline endogenous virus (RD114) glycoprotein, avian plague virus (FPV) glycoprotein, Ebola virus (EboV) glycoprotein, and lymphocytic choriomeningitis virus (LCMV) glycoprotein; The vesicular stomatitis virus (VSV) glycoproteins include: VSV Indiana strain glycoprotein, VSV Cocal strain glycoprotein, VSV Maraba strain glycoprotein, VSV Morreton strain glycoprotein, VSV Alagoas strain glycoprotein, VSV New Jersey strain glycoprotein, VSV Carajas strain glycoprotein, VSV Chandipura strain glycoprotein, and vesicular stomatitis virus (VSV) glycoprotein. The glycoproteins of the following strains of vesicular stomatitis virus (VSV) are: Eptesicus strain, Isfahan strain, Jurona strain, Malpais strain, Perinet strain, Piry strain, Radi strain, Rhinolopus strain, and YugBogdanovac strain. Preferably, the viral glycoprotein is VSV-G or Cocal-G, the receptor includes LDL-R, and the antibody includes VSV-G antibody or Cocal-G antibody; More preferably, the amino acid sequences of the HCDR1-3 regions of the VH region of the anti-VSV-G antibody are shown in SEQ ID NO:30-32, respectively; and the amino acid sequences of the LCDR1-3 regions of the VL region of the anti-VSV-G antibody are shown in SEQ ID NO:33-35, respectively.

25. The pseudo-LVV or RVV packaging system according to claim 23 or 24, characterized in that, The pseudo-LVV or RVV packaging system comprises one or more nucleic acid vectors that, when introduced into packaging cells for packaging LVV or RVV, contain nucleic acids necessary for the production, assembly, and / or packaging of LVV or RVV within the packaging cells.

26. The pseudo-LVV or RVV packaging system according to any one of claims 23-25, characterized in that, The pseudo-LVV or RVV packaging system also includes: (a) Gag and Pol genes; and (b) (i) LVV backbone genes and Rev genes; or (ii) RVV skeletal genes; (c) viral glycoproteins or their variant genes; and (d) Shuttle genes.

27. The pseudo-LVV or RVV packaging system according to any one of claims 23-26, characterized in that, The pseudo-LVV or RVV packaging system also includes: (a) Gag and Pol genes; and (b) (i) LVV backbone genes and Rev genes; or (ii) RVV skeletal genes; (c) Viral glycoproteins or their variant genes; (d) shuttle genes; and (e) Targeting molecular genes.

28. The pseudo-LVV or RVV packaging system according to any one of claims 23-27, characterized in that, The pseudo-LVV or RVV packaging system comprises: at least one packaging plasmid, a coating plasmid, a shuttle plasmid, and at least one binding plasmid; The binding plasmid contains the viral glycoprotein binding molecule gene; The shuttle plasmid comprises (a) the shuttle gene and (b): (i) the LVV backbone gene or (ii) the RVV backbone gene; preferably, the shuttle gene comprises the CAR gene.

29. The pseudo-LVV or RVV packaging system according to claim 28, characterized in that, (a) The packaging plasmid contains the Gag gene and the Pol gene (Gag / Pol packaging plasmid); (b) The envelope plasmid comprises (i) the viral glycoprotein or a variant thereof and (ii) the targeting molecule gene; and (c) The shuttle plasmid comprises (i) the shuttle gene and (ii) (1) the LVV backbone gene or (2) the RVV backbone gene.

30. The pseudo-LVV or RVV packaging system according to claim 28 or 29, characterized in that, When used to package LVV, the pseudo-LVV packaging system also includes a Rev packaging plasmid containing the Rev gene.

31. A type of packaging cell, characterized in that, The packaging cells comprise any one of the pseudo-LVV or RVV packaging systems according to any one of claims 23-30; Preferably, the packaging cells are 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; More preferably, the packaging cells are HEK-293T cells.

32. A method for preparing pseudo-LVV or RVV, characterized in that, The method includes culturing the packaging cells of claim 31, isolating and collecting the pseudo-LVV or RVV.

33. A pseudo-LVV or RVV, characterized in that, The pseudo-LVV or RVV is prepared by the method described in claim 32.

34. An engineered cell, characterized in that, The engineered cells are prepared by transfecting cells with the modified viral vector as described in claim 16; Preferably, the cells include immune cells; More preferably, the immune cells are selected from at least one of T cells and B cells.

35. The use of the modified viral vector of any one of claims 1-16, the pseudo-LVV or RVV of claim 33, or the engineered cell of claim 34 in the preparation of a disease therapeutic agent; Preferably, the disease is selected from autoimmune diseases and cancer, including solid tumors and hematologic malignancies; More preferably, the cancer is a blood cancer.

36. The application according to claim 35, characterized in that, The disease described is a blood cancer, specifically selected from: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), and transplant-unsuitable leukemia. At least one of the following: 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, 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 Embryonic centrifugal T-cell lymphoma (FTCL).

37. The application according to claim 35, characterized in that, The hematologic malignancy is a B-cell malignancy selected from at least one of the following: non-Hodgkin's 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).

38. The application according to claim 35, characterized in that, The disease is a solid tumor, which is selected from at least one of the following: mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer, and gastrointestinal cancer.

39. The application according to claim 35, characterized in that, The disease is a solid tumor, and the disease is an autoimmune disease 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 mellitus, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis, and scleroderma.

40. A method for transducing target cells and delivering exogenous load genes, characterized in that, The method includes contacting the target cells with the modified viral vector of claim 16; Preferably, the target cells include immune cells; More preferably, the immune cells are selected from at least one of T cells and B cells.

41. A composition, characterized in that, The composition comprises a pharmaceutically acceptable carrier or excipient and a modified viral vector according to any one of claims 1-16, a pseudo-LVV or RVV according to claim 33, or an engineered cell according to claim 34.

42. A method for treating a disease suffered by a subject or killing diseased cells of a subject, characterized in that, The method includes administering the modified viral vector of claim 16 to the subject or the subject's diseased cells; Preferably, the disease is selected from autoimmune diseases and cancer, including solid tumors and hematologic malignancies; More preferably, the cancer is a blood cancer.

43. The method according to claim 42, characterized in that, The disease described is a blood cancer, specifically selected from: non-Hodgkin's lymphoma (NHL), acute B-cell lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), large B-cell lymphoma (LBCL), and transplant-unsuitable leukemia. At least one of the following: 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, 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 Embryonic centrifugal T-cell lymphoma (FTCL).

44. The method according to claim 42, characterized in that, The hematologic malignancy is a B-cell malignancy selected from at least one of the following: non-Hodgkin's 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).

45. The method according to claim 42, characterized in that, The disease is a solid tumor, which is selected from at least one of the following: mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, biliary tract cancer, and gastrointestinal cancer.

46. ​​The method according to claim 42, characterized in that, The disease is a solid tumor, and the disease is an autoimmune disease 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 mellitus, diffuse toxic goiter, Addison's disease, autoimmune vasculitis, pernicious anemia, dermatomyositis, polymyositis, and scleroderma.

47. The method according to any one of claims 42-46, characterized in that, The administration is selected from at least one of the following methods: oral, nasal, intravenous, intraperitoneal, intracerebral (within brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, intralesional, continuous release system, and implanted device.

48. A reagent kit, characterized in that, The kit comprises the modified viral vector of any one of claims 1-16, the packaging cells of claim 31, the pseudo-LVV or RVV of claim 33, or the engineered cells of claim 34.

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