Fusion polypeptide and use thereof

Abstract

The present invention relates to the field of viral vectors, and in particular to a fusion polypeptide and a use thereof. Disclosed are a fusion polypeptide and a use thereof. The fusion polypeptide can mediate the budding of a pseudotyped LVV or RVV in packaging cells. Also disclosed is a particle comprising the fusion polypeptide, wherein the non-specific transduction capability of the particle is reduced.

Description

A fusion peptide and its applications Technical Field

[0001] This invention relates to the field of viral vectors, and more specifically to a fusion polypeptide and its applications. Background Technology

[0002] Viral glycoproteins, such as VSV-G and Cocal-G, are widely used in the production of pseudolentiviral vectors (LVVs) or retroviral vectors (RVVs) due to their broad and efficient infectivity. However, precisely because of their broad infectivity, off-target effects when transducing cells using VSV-G / Cocal-G pseudolentiviral LVVs or RVVs can affect their transduction efficiency. Existing technologies have attempted to improve the targeting specificity of VSV-G or Cocal-G by modifying them, for example, by introducing mutations such as K47Q, R354Q, and Y209Q, to inhibit their ability to specifically bind to their widely expressed receptor LDL-R on various cell surfaces. However, the titers of pseudolentiviral LVVs or RVVs containing modified VSV-G or Cocal-G are also significantly reduced. Therefore, the need for a viral glycoprotein whose ability to specifically bind to its receptor is inhibited, but whose packaged pseudolentiviral LVV or RVV titers are effectively maintained, remains unmet. Summary of the Invention

[0003] One aspect of the present invention provides a fusion polypeptide comprising:

[0004] A) Viral glycoproteins or their variants; and

[0005] B) A binding polypeptide that specifically binds to the viral glycoprotein or its variants.

[0006] In some embodiments of the present invention, the binding polypeptide comprises:

[0007] The viral glycoproteins described in A) are selected from: vesicular stomatitis virus glycoproteins, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoproteins, 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 T-cell choriomeningitis virus (LCMV) glycoprotein; and / or,

[0008] The binding polypeptide described in B) comprises a receptor or receptor-binding fragment of the viral glycoprotein, or an antibody against the viral glycoprotein.

[0009] In some embodiments, the vesicular stomatitis virus (VSV) glycoprotein is selected from: VSV Indiana strain glycoprotein, VSV Cocal strain glycoprotein, VSV Maraba strain glycoprotein, VSV Morreton strain glycoprotein, VSV Alagoas strain glycoprotein, VSV New... The following vesicular stomatitis virus (VSV) strains contain glycoproteins: Jersey, Carajas, Chandipura, Eptesicus, Isfahan, Jurona, Malpais, Perinet, Piry, Radi, Rhinolopus, and Yug Bogdanovac.

[0010] In some more specific embodiments, the viral glycoprotein is: vesicular stomatitis virus Indiana strain glycoprotein (VSV-G); or vesicular stomatitis virus Cocal strain glycoprotein (Cocal-G).

[0011] In some embodiments of the present invention, the antibody against the viral glycoprotein is an anti-VSV-G antibody or an anti-Cocal-G antibody.

[0012] In some embodiments of the present invention, the receptor of the viral glycoprotein is LDL-R, and the binding fragment of the LDL-R is selected from one or more of the cysteine-rich domains (CR) of the LDL-R.

[0013] In some embodiments of the present invention, the binding fragments of the LDL-R are CR2 and / or CR3;

[0014] The amino acid sequence of CR2 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:121;

[0015] The amino acid sequence of CR3 has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:122.

[0016] In some embodiments of the present invention, the binding segments of the LDL-R are CR2 and CR3, wherein CR2 is linked to CR3.

[0017] In some embodiments of the present invention, the binding polypeptide further comprises a polypeptide linker, through which CR2 is linked to CR3.

[0018] In some embodiments of the present invention, the viral glycoprotein or a variant thereof is linked to the binding polypeptide.

[0019] In some embodiments of the present invention, the viral glycoprotein or a variant thereof is located at the C-terminus of the binding polypeptide.

[0020] In some embodiments of the present invention, the fusion polypeptide further comprises a polypeptide linker, through which the viral glycoprotein or a variant thereof is linked to the binding polypeptide.

[0021] In some embodiments of the present invention, the fusion polypeptide comprises, from the N-terminus to the C-terminus, the following in sequence: (a) CR2 or CR3, a polypeptide linker, VSV-G or a variant thereof; (b) CR2, CR3, a polypeptide linker, VSV-G or a variant thereof; (c) CR3, CR2, a polypeptide linker, VSV-G or a variant thereof; (d) CR2, a polypeptide linker, CR3, a polypeptide linker, VSV-G or a variant thereof; (e) CR3, a polypeptide linker, CR2, a polypeptide linker, VSV-G or a variant thereof; (f) CR2 or CR3, a polypeptide linker, Cocal-G or a variant thereof; (g) CR2, CR3, a polypeptide linker, Cocal-G or a variant thereof; (h) CR3, CR2, a polypeptide linker, Cocal-G or a variant thereof; (i) CR2, a polypeptide linker, CR3, a polypeptide linker, Cocal-G or a variant thereof; or (j) CR3, a polypeptide linker, CR2, a polypeptide linker, Cocal-G or a variant thereof.

[0022] In some embodiments of the present invention, the viral glycoprotein variant contains mutations that enhance its ability to antagonize complement inactivation.

[0023] In some embodiments of the present invention, the enhanced ability of the viral glycoprotein variant to antagonize complement inactivation is relative to the wild-type viral glycoprotein.

[0024] In some embodiments of the present invention, the viral glycoprotein variant is a VSV-G variant or a Cocal-G variant, which contains mutations that enhance its ability to antagonize complement inactivation.

[0025] In some embodiments of the present invention, the VSV-G or Cocal-G variant comprises one or more mutations selected from the following site mutations:

[0026] (a) Amino acids located at positions 214, 252, 50, and / or 146 of SEQ ID NO:1 or SEQ ID NO:2;

[0027] (b) The amino acid located at positions 214, 252, 50, and / or 146 of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:2.

[0028] Preferably, the mutation at the site is selected from amino acid substitutions, deletions, and insertions;

[0029] More preferably, the mutation at the site is an amino acid substitution.

[0030] In some embodiments of the present invention, the VSV-G or Cocal-G variant comprises a combination of mutations selected from the following sites:

[0031] (a) Substitution of T / K214 and T352 located in SEQ ID NO:1 or SEQ ID NO:2 and T / K214, T352,

[0032] Replacement of K50 and S146; and

[0033] (b) Substitutions of T / K214 and T352 and T / K214, T352, K50 and S146 located at the equivalent of SEQ ID NO:1 or SEQ ID NO:2 after best global alignment with SEQ ID NO:1 or SEQ ID NO:2.

[0034] Preferably, the VSV-G or Cocal-G variant comprises a combination of mutations selected from the following sites:

[0035] (a) T / K214N and T352A located in SEQ ID NO:1 or SEQ ID NO:2,

[0036] K50T and S146T; and

[0037] (b) After best global alignment with SEQ ID NO:1 or SEQ ID NO:2, T / K214N and T352A and T214N, T352A, K50T and S146T are located at the equivalent of SEQ ID NO:1 or SEQ ID NO:2.

[0038] In some embodiments of the present invention, the fusion polypeptide further comprises at least one targeting molecule.

[0039] In some embodiments of the present invention, the targeting molecule may bind to a marker of the target cell, wherein the marker is an endocytic receptor;

[0040] Preferably, the markers are selected from: HER2, CD20, CD19, CD79A, CD79B, CD56, CD22, CD138, CD37, CD98, CD309, CD33, CD163, CD163B, CD169, CD204, CD205, CD209, CD280, CD302, TROP-2, CD19, NECTIN4, 5T4, CD30, TROP2, FRα, STEAP1, ENPP3, GCC, SLC44A4, NaPi2b, CA9, SC-16, CD142, P-Cadherin, PSMA, ED-B, endothelin receptors ETB, TN-C, Collagen. IV, Periostin, CEACAM, c-MET, TDGF1, IGF1R, Mesothelin, TIM1, NCAM1, ZIP6, CD166, GPNMB, SDC1, glycosphingolipid, TfR, Ganglioside, CD74, CLDN18, DPEP3 , SLITRK6, PRL-R, LY75, CD48, MUC1, CDKs, B7-H4, STING, KAAG1, CD70, CDH3, LRRC15, EGFR, ASGPR, CD3, CD3γ, CD3δ, CD3ε, TCRγ, TCRδ, TCRα, TCRβ, CD4, CD5, CD7, CD 8. At least one of the following: CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD57, CD71, CD69, CD94, CD95, 4-1BB (CD137), CD103, CD122, CD127, CD161, OX40 (CD134), ICOS, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), PD-1, CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, IL6ST, P2RX7, TIGIT, TIM-3, and LAG-3;

[0041] More preferably, the marker is selected from at least one of CD3, CD5, CD7, CD19 and MSLN.

[0042] In some embodiments of the present invention, the targeting molecule may bind to a marker of T cells, wherein the marker is an endocytic receptor.

[0043] In some embodiments of the present invention, the T cell markers are selected from: CD3, CD3γ, CD3δ, CD3ε, TCRγ, TCRδ, TCRα, TCRβ, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD57, CD71, CD69, CD94, CD95, 4-1BB (CD137), CD103, CD122, CD1 27. One or more of the following: CD161, OX40 (CD134), ICOS, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), PD-1, CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, IL6ST, P2RX7, TIGIT, TIM-3, and LAG-3;

[0044] Preferably, the T cell markers are selected from one or more of the following: CD3, CD3γ, CD3δ, CD3ε, CD5, CD7, CD28, CD2, CD127, 4-1BB, OX40, ICOS, TCRγ, TCRδ, TCRα, and TCRβ.

[0045] In some embodiments of the present invention, the marker for the T cells is CD7; preferably human CD7.

[0046] In some embodiments of the present invention, the targeting molecule comprises one or more selected from (a) an anti-CD7 antibody or its antigen-binding fragment and (b) a CD7 ligand or its binding fragment.

[0047] In some embodiments of the present invention, the targeting molecules include anti-CD7 scFv and / or VHH;

[0048] Optionally, the anti-CD7 scFv is derived from TH-69 (TH69-scFv), and the amino acid sequences of the HCDR1-3 regions of the TH69-scFv are shown in SEQ ID NO:113-115, respectively; the amino acid sequences of the LCDR1-3 regions of the TH69-scFv are shown in SEQ ID NO:116-118, respectively.

[0049] Optionally, the amino acid sequence of the anti-CD7 VHH (anti-CD7-VHH) has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:101; the amino acid sequences of the HCDR1-3 regions of the anti-CD7-VHH are shown in SEQ ID NO:102-104, respectively.

[0050] In some embodiments of the present invention, the targeting molecule includes an activating molecule.

[0051] In some embodiments of the present invention, the activating molecule may bind to a TCR-CD3 complex or its subunits; the TCR-CD3 complex subunits are selected from TCR subunits and CD3 subunits, the CD3 subunits include CD3γ, CD3δ, CD3ε and CD3ζ; the TCR subunits include TCRγ, TCRδ, TCRα and TCRβ.

[0052] In some embodiments of the present invention, the activating molecule may bind to the CD3 subunit; preferably the human CD3 subunit.

[0053] In some embodiments of the present invention, the activating molecule includes an anti-CD3 antibody or an antigen-binding fragment thereof.

[0054] In some embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment is an anti-CD3 scFv and / or VHH.

[0055] In some embodiments of the present invention, the anti-CD3 antibody is an anti-CD3 scFv derived from UCHT1 (UCHT1-scFv); the amino acid sequences of the HCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NO:59-61, and the amino acid sequences of the LCDR1-3 regions of the UCHT1-scFv are shown in SEQ ID NO:62-64.

[0056] In some embodiments of the present invention, the amino acid sequence of the VH region of the UCHT1-scFv has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 108, 110, or 124; and the amino acid sequence of the VL region of the UCHT1-scFv has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 109, 111, or 125.

[0057] In some embodiments of the present invention, the amino acid sequence of the UCHT1-scFv has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:58, 107, or 123.

[0058] In some embodiments of the present invention, the targeting molecule further includes at least one co-stimulatory molecule.

[0059] In some embodiments of the present invention, the co-stimulatory molecule may bind to CD28, 4-1BB, OX40, or ICOS.

[0060] In some embodiments of the present invention, the co-stimulatory molecule is selected from at least one of anti-CD28 antibody or its antigen-binding fragment, CD80 or its extracellular domain or its functional fragment, CD86 or its extracellular domain or its functional fragment, 4-1BBL or its extracellular domain or its functional fragment, OX40L or its extracellular domain or its functional fragment, and ICOSL or its extracellular domain or its functional fragment.

[0061] In some embodiments of the present invention, the co-stimulatory molecule is selected from one or more of the following: anti-CD28 antibody or its antigen-binding fragment, CD80 extracellular domain, CD86 extracellular domain, 4-1BBL extracellular domain, OX40L extracellular domain, and ICOSL extracellular domain.

[0062] In some embodiments of the present invention, the anti-CD28 antibody or its antigen-binding fragment is an anti-CD28 scFv; the anti-CD28 scFv is derived from 15E8 (15E8-scFv); the amino acid sequences of the HCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO:67-69; the amino acid sequences of the LCDR1-3 regions of the 15E8-scFv are shown in SEQ ID NO:70-72.

[0063] In some embodiments of the present invention, the activating molecule is linked to the co-stimulatory molecule.

[0064] In some embodiments of the present invention, the activating molecule is linked to the co-stimulatory molecule via a polypeptide linker.

[0065] In some embodiments of the present invention, the activating molecule or the co-stimulatory molecule is linked to the binding polypeptide.

[0066] In some embodiments of the present invention, the activating molecule or the co-stimulatory molecule is linked to the binding polypeptide via a polypeptide linker.

[0067] In some embodiments of the present invention, the activating molecule and the co-stimulatory molecule are located at the N-terminus of the binding polypeptide.

[0068] In some embodiments of the present invention, the fusion polypeptide comprises, from the N-terminus to the C-terminus, the following sequence:

[0069] (a) Activating molecules, peptide linkers, co-stimulatory molecules, peptide linkers, CR2 or CR3, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0070] (b) Co-stimulatory molecules, peptide linkers, activating molecules, peptide linkers, CR2 or CR3, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0071] (c) Activating molecules, peptide linkers, co-stimulatory molecules, peptide linkers, CR2, CR3, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0072] (d) Co-stimulatory molecules, peptide linkers, activating molecules, peptide linkers, CR2, CR3, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0073] (e) Activating molecules, peptide linkers, co-stimulatory molecules, peptide linkers, CR3, CR2, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0074] (f) Co-stimulatory molecules, peptide linkers, activating molecules, peptide linkers, CR3, CR2, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0075] (g) Activating molecules, peptide linkers, co-stimulatory molecules, peptide linkers, CR2, peptide linkers, CR3, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0076] (h) Co-stimulatory molecules, peptide linkers, activating molecules, peptide linkers, CR2, peptide linkers, CR3, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0077] (i) Activating molecules, peptide linkers, co-stimulatory molecules, peptide linkers, CR3, peptide linkers, CR2, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0078] (j) co-stimulatory molecules, peptide linkers, activating molecules, peptide linkers, CR3, peptide linkers, CR2, peptide linkers, VSV-G / Cocal-G or variants thereof.

[0079] In some embodiments of the present invention,

[0080] (a) The activating molecule is an anti-CD3 antibody or its antigen-binding fragment; preferably, it is UCHT1-scFv;

[0081] (b) The co-stimulatory molecule is selected from the anti-CD28 antibody or its antigen-binding fragment, CD80 extracellular domain, CD86 extracellular domain, 4-1BBL extracellular domain, OX40L extracellular domain and ICOS extracellular domain; preferably, the anti-CD28 antibody is the 15E8-scFv.

[0082] In some embodiments of the present invention, the targeting molecule further includes an adhesion molecule; preferably, the adhesion molecule can bind CD2; more preferably, the adhesion molecule includes CD58 or its extracellular domain or a functional fragment thereof.

[0083] In some embodiments of the present invention, the targeting molecule is linked to the binding polypeptide.

[0084] In some embodiments of the present invention, the targeting molecule is linked to the binding polypeptide via a polypeptide linker.

[0085] In some embodiments of the present invention, the targeting molecule is located at the N-terminus of the binding polypeptide.

[0086] In some embodiments of the present invention, when the fusion polypeptide contains at least N of the target molecules and N ≥ 2:

[0087] (a) The C-terminus of one of the target molecules is linked to the binding polypeptide, and the N-terminus is linked to another target molecule; and

[0088] (b) Sequential linking of other target molecules.

[0089] In some embodiments of the present invention, the fusion polypeptide comprises, from the N-terminus to the C-terminus, the following sequence:

[0090] (a) Targeting molecules, peptide linkers, CR2 or CR3, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0091] (b) Targeting molecules, peptide linkers, CR2, CR3, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0092] (c) Targeting molecules, peptide linkers, CR3, CR2, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0093] (d) Targeting molecules, peptide linkers, CR2, peptide linkers, CR3, peptide linkers, VSV-G / Cocal-G or variants thereof;

[0094] (e) Targeting molecules, peptide linkers, CR3, peptide linkers, CR2, peptide linkers, VSV-G / Cocal-G or variants thereof.

[0095] In some embodiments of the present invention, the targeting molecule is an anti-CD7 antibody or its antigen-binding fragment;

[0096] Preferably, the anti-CD7 antibody is the anti-CD7-VHH.

[0097] In some embodiments of the present invention, the polypeptide linker is selected from:

[0098] (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;

[0099] (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;

[0100] (c) All or part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2 and CH3 domains;

[0101] (d) The stalk domain of type II C-lectins, wherein the type II C-lectins are selected from the stalk domains of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and

[0102] (e) Flexible linker peptide.

[0103] In some embodiments of the present invention, the polypeptide linker is a flexible linker peptide.

[0104] In some embodiments of the present invention, the flexible linker peptide is selected from (G4S)n linker peptide, linker 1: GSTGSSGKPGSGEGSTKG (SEQ ID NO:89) and linker 2: GSSGGSGGGGSGGGGSGGGGSSG (SEQ ID NO:90); wherein n=1 to 4.

[0105] In some embodiments of the present invention, the flexible linker peptide is a (G4S)n linker peptide, optionally n=3.

[0106] In some embodiments of the present invention, the fusion polypeptide further comprises a leader signal peptide.

[0107] In some embodiments of the present invention, the leader signal peptide is the signal peptide of the viral glycoprotein.

[0108] In some embodiments of the present invention, the leader signal peptide is not particularly limited as long as it can mediate the membrane expression of the fusion polypeptide.

[0109] In some embodiments of the present invention, the leader signal peptide is a VSV-G signal peptide or a Cocal-G signal peptide;

[0110] Preferably, the amino acid sequence of the VSV-G signal peptide has at least 95% identity with SEQ ID NO:3; and the amino acid sequence of the Cocal-G signal peptide has at least 95% identity with SEQ ID NO:4.

[0111] MKCLLYLAFLFIGVNC (SEQ ID NO:3)

[0112] MNFLLLTFIVLPLCSHA (SEQ ID NO: 4).

[0113] In some embodiments of the present invention, the amino acid sequence of the fusion polypeptide has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO: 25, 27, 29, 31, 33, 35, 37, or 39.

[0114] In some embodiments of the present invention, the ability of the fusion polypeptide to specifically bind to the receptor or its binding fragment of the viral glycoprotein is inhibited; preferably, the receptor of the viral glycoprotein is LDL-R.

[0115] In some embodiments of the present invention, the ability of the fusion peptide to specifically bind LDL-R or its binding fragment is inhibited.

[0116] In some embodiments of the present invention, the fusion polypeptide can mediate membrane fusion.

[0117] In some embodiments of the present invention, the fusion polypeptide can mediate endosome / lysosome escape.

[0118] In some embodiments of the present invention, the fusion polypeptide can mediate budding of pseudolentiviral vectors (LVV) or retroviral vectors (LVV) in packaging cells.

[0119] In some embodiments of the present invention, the ability of the viral glycoprotein variant to specifically bind to the receptor or binding fragment of the viral glycoprotein, relative to the wild-type viral glycoprotein, is maintained or not inhibited.

[0120] In another aspect, the present invention also provides a particle comprising any of the aforementioned fusion polypeptides.

[0121] In some embodiments of the present invention, the fusion polypeptide is displayed on the surface of the particles.

[0122] In some embodiments of the present invention, the particles are selected from LNPs, virus-like particles, exosomes, extracellular vesicles, and enveloped virus particles.

[0123] In some embodiments of the present invention, the particles are enveloped viral particles.

[0124] In some embodiments of the present invention, the enveloped viral particles are pseudotyped LVV and / or RVV.

[0125] In some embodiments of the present invention, the particles further comprise one or more targeting molecules displayed on their surface.

[0126] In some embodiments of the present invention, the targeting molecule is displayed on the surface of the particle.

[0127] In some embodiments of the present invention, the targeting molecule is any of the aforementioned targeting molecules.

[0128] In some embodiments of the present invention, the targeting molecule is connected to a transmembrane domain (membrane expression targeting molecule), the transmembrane domain being anchored to the surface of the particle.

[0129] In some embodiments of the present invention, the transmembrane domain is selected from the transmembrane regions of the following proteins:

[0130] 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;

[0131] Preferably, the transmembrane domain is a CD8α transmembrane region;

[0132] More preferably, the CD8α transmembrane region is the human CD8α transmembrane region, and the amino acid sequence of the CD8α transmembrane region has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity with SEQ ID NO:52.

[0133] In some embodiments of the present invention, the targeting molecule is connected to the transmembrane domain via a connecting domain.

[0134] In some embodiments of the present invention, the connection structure domain is selected from:

[0135] (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;

[0136] (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;

[0137] (c) All or part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2 and CH3 domains;

[0138] (d) The stalk domain of type II C-lectins, wherein the type II C-lectins are selected from the stalk domains of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and

[0139] (e) Flexible linker peptides;

[0140] Preferably, the connection structure domain is the CD8α hinge region;

[0141] More preferably, the CD8α hinge region is the human CD8α hinge region, and the amino acid sequence of the human CD8α hinge region has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity with SEQ ID NO:51.

[0142] In some embodiments of the present invention, the particles further comprise at least one exogenous polynucleotide.

[0143] In some embodiments of the present invention, the exogenous polynucleotide comprises at least one of a polynucleotide encoding a chimeric antigen receptor (CAR) and a polynucleotide encoding an engineered TCR.

[0144] In some embodiments of the present invention, the exogenous polynucleotide comprises a polynucleotide encoding a CAR.

[0145] In some embodiments of the present invention, the CAR includes an antigen-binding region, a transmembrane region, and an intracellular signal transduction domain.

[0146] In some embodiments of the present invention, the antigen-binding region binds to disease-related antigens.

[0147] In some embodiments of the present invention, the disease-related antigen is selected from:

[0148] TSHR, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD24, CD25, CD28, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD22, CD126, CD138, DR4, DR5, TAC, TEM1 / CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CDL33, IFNAR1, DLL3, kappa light chain, TIM3, tEGFR, IL-22Ra, IL-2, ErbB3, ErbB4, MUC16, MAGE-A3, MAGE-A6, NKG2DL, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvⅢ, GD2, GD3, BCMA, GPRC5D, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin (MSLN), IL-1Ra, PSCA, PRSS21, VEGFR2, Lewis-Y, CD24, PDGFR-β, SSEA-4, AFP, Folate receptor α, Her2 / neu / ERBB2, MUC1, EGFR, CS1, CD138, NCAM, Claudin18.2. Prostase, PAP, ELF2M, EphrinB2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, FucosylGM1, 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, GP R20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, Bean bud protein, HPVE6 / E7, MAGE-A4, MART-1, WT-1, ETV6-AML, Sperminin 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 / Galectin8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, TMPRSS2 ETS fusion gene / at least one of ERG, NA17, PAX3, androgen receptor, CyclinB1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, murine hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLLI, PD1, PDL1, PDL2, TGFβ, APRIL, GC-C (guanylate cyclase C), and NKG2D.

[0149] In some embodiments of the present invention, the disease-related antigen is selected from at least one of CD19, CD20, BCMA, CD33, HER2, MSLN, GC-C, Claudin18.2, and CEA.

[0150] In some embodiments of the present invention, the antigen-binding region of the CAR includes an antibody or an antigen-binding fragment thereof and / or a ligand or a binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is selected from at least one of immunoglobulin (full-length antibody), hapten, Fab, Fab', F(ab')2, Fv fragment, single-chain variable region fragment (scFv), disulfide bond stable antibody (dsFv), heavy chain variable region (VH) or light chain variable region (VL) of antibody, Fd fragment composed of VH and CH1 domains, linear antibody, and single-domain antibody (nanobody); the binding fragment of the ligand is selected from the extracellular domain of the ligand and the functional fragment of the ligand.

[0151] In some embodiments of the present invention, the extracellular antigen-binding region of the CAR includes scFv and / or VHH.

[0152] In some embodiments of the present invention, the antigen-binding region of the CAR is single-specific, bi-specific, or multi-specific.

[0153] In some embodiments of the present invention, the antigen-binding region of the CAR is derived from an antibody or antigen-binding fragment thereof and / or a ligand or receptor-binding fragment thereof derived from a mouse, rat, monkey, human or humanized antibody.

[0154] In some embodiments of the present invention, the transmembrane region of the CAR is selected from the transmembrane regions of the following proteins:

[0155] 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.

[0156] In some embodiments of the present invention, the transmembrane region of the CAR is the CD8α transmembrane region; preferably the human CD8α transmembrane region.

[0157] In some embodiments of the present invention, the amino acid sequence of the human CD8α transmembrane region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:52.

[0158] 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:

[0159] Intracellular signal transduction domains of proteins containing at least one ITAM, including CD3ε, CD3γ, CD3δ, CD3ζ, CD79a, CD79b, FcεRlγ, FcεRβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, DAP10, DAP12, and other intracellular signal transduction domains.

[0160] In some embodiments of the present invention, the intracellular signal transduction domain of the CAR is the intracellular signal transduction domain of CD3ζ; preferably, it is the intracellular signal transduction domain of human CD3ζ.

[0161] In some embodiments of the present invention, the amino acid sequence of the intracellular signal transduction domain of the human CD3ζ has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:54.

[0162] In some embodiments of the present invention, any of the aforementioned CARs further includes a co-stimulatory signal transduction domain.

[0163] In some embodiments of the present invention, the co-stimulatory signal transduction domain of the CAR is selected from one or more of the co-stimulatory signal transduction domains of the following proteins:

[0164] CD28, 4-1BB, CD27, CD2, CD7, CD8, CD8α, CD8β, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcαRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40 and MyD88.

[0165] In some embodiments of the present invention, the costimulatory signal transduction domain of the CAR is a 4-1BB costimulatory signal transduction domain; preferably, it is a human 4-1BB costimulatory signal transduction domain.

[0166] In some embodiments of the present invention, the costimulatory signal transduction domain of the CAR includes the costimulatory signal transduction domain of human 4-1BB and the costimulatory signal transduction domain of human CD28.

[0167] In some embodiments of the present invention, the amino acid sequence of the co-stimulatory signal transduction domain of the human 4-1BB has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:53.

[0168] In some embodiments of the present invention, any of the aforementioned CARs further includes a hinge area.

[0169] In some embodiments of the present invention, the hinge region of the CAR is sequentially connected to the antigen-binding region of the CAR and the transmembrane region of the CAR.

[0170] In some embodiments of the present invention, the hinge region of the CAR is selected from the hinge regions of the following proteins: CD28, CD8, CD8α, CD8β, CD3, CD45, Ig4, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.

[0171] In some embodiments of the present invention, the hinge region of the CAR is a CD8α hinge region; preferably, it is a CD8α hinge region.

[0172] In some embodiments of the present invention, the amino acid sequence of the human CD8α hinge region has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:51.

[0173] In some embodiments of the present invention, any of the aforementioned CARs further comprises a leader signal peptide; the leader signal peptide is not particularly limited as long as it can mediate CAR exoexpression.

[0174] In some embodiments of the present invention, the CAR leader signal peptide is selected from CD8α signal peptide, CD28 signal peptide and IgG1 signal peptide.

[0175] In some embodiments of the present invention, the CAR signal peptide is the human CD8α signal peptide.

[0176] In some embodiments of the present invention, the amino acid sequence of the human CD8α signal peptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO:50.

[0177] In some embodiments of the present invention, the ability of the particle to specifically bind to the receptor of the viral glycoprotein is inhibited; preferably, the receptor of the viral glycoprotein is LDL-R.

[0178] In some embodiments of the present invention, the ability of the particle to transduce cells expressing the receptor of the viral glycoprotein by specifically binding to the receptor of the viral glycoprotein is inhibited.

[0179] In some embodiments of the present invention, the particles can target and transduce and activate T cells.

[0180] In another aspect, the present invention also provides an isolated polynucleotide encoding any of the aforementioned fusion polypeptides.

[0181] In another aspect, the present invention also provides a nucleic acid vector comprising any of the aforementioned isolated polynucleotides.

[0182] In some embodiments of the present invention, the nucleic acid vector is selected from plasmids, lentiviral vectors, and retroviral vectors.

[0183] In some embodiments of the present invention, the nucleic acid vector is a plasmid.

[0184] In another aspect, the present invention also provides a viral vector packaging system comprising any of the aforementioned isolated polynucleotides.

[0185] In some embodiments of the present invention, the viral vector packaging system further comprises (a) a gag gene, (b) a pol gene, (c) at least one exogenous polynucleotide, and (d) a lentiviral vector backbone gene and a rev gene; or a retroviral vector backbone gene.

[0186] In some embodiments of the present invention, the viral vector packaging system further comprises (a) a gag gene, (b) a pol gene, (c) a polynucleotide encoding any of the aforementioned membrane expression target molecules, (d) at least one exogenous polynucleotide, and (e) a lentiviral vector backbone gene and a rev gene; or a retroviral vector backbone gene.

[0187] In some embodiments of the present invention, the viral vector packaging system comprises one or more nucleic acid vectors, which, when introduced into packaging cells for packaging lentiviral vectors or retroviral vectors, contain nucleic acids necessary for the production, assembly, and / or packaging of lentiviral vectors or retroviral vectors within the packaging cells.

[0188] In some embodiments of the present invention, the viral vector packaging system comprises:

[0189] (a) A nucleic acid vector containing a polynucleotide encoding any of the aforementioned fusion polypeptides;

[0190] (b) Nucleic acid vectors containing the Gag and Pol genes; and

[0191] (c) A nucleic acid vector containing at least one exogenous polynucleotide and the following genes: LVV backbone gene and Rev gene; or RVV backbone gene.

[0192] In some embodiments of the present invention, the nucleic acid vector is a plasmid.

[0193] In some embodiments of the present invention, the viral vector packaging system comprises: at least one packaging plasmid, an envelope plasmid, and a shuttle plasmid;

[0194] (a) The envelope plasmid contains a polynucleotide encoding any of the aforementioned fusion polypeptides;

[0195] (b) The packaging plasmid contains the Gag gene and the Pol gene (Gag / Pol packaging plasmid); and

[0196] (c) The shuttle plasmid contains at least one exogenous polynucleotide and: (1) an LVV backbone gene or (2) an RVV backbone gene.

[0197] In some embodiments of the present invention, the viral vector packaging system comprises: at least one packaging plasmid, an envelope plasmid, and a shuttle plasmid;

[0198] (a) The envelope plasmid comprises a polynucleotide encoding any of the aforementioned fusion polypeptides and a polynucleotide encoding any of the aforementioned membrane expression target molecules;

[0199] (b) The packaging plasmid contains the Gag gene and the Pol gene (Gag / Pol packaging plasmid); and

[0200] (c) The shuttle plasmid contains at least one exogenous polynucleotide and: LVV backbone gene or RVV backbone gene.

[0201] In some embodiments of the present invention, the packaging system comprises: at least one packaging plasmid, a coating plasmid, a shuttle plasmid, and a targeting plasmid;

[0202] (a) The packaging plasmid contains the Gag gene and the Pol gene (Gag / Pol packaging plasmid).

[0203] (b) The envelope plasmid contains a polynucleotide encoding any of the aforementioned fusion polypeptides;

[0204] (c) The shuttle plasmid contains at least one exogenous polynucleotide and: an LVV backbone gene or an RVV backbone gene; and

[0205] (d) The target plasmid contains a polynucleotide encoding any of the aforementioned membrane expression target molecules.

[0206] In some embodiments of the present invention, the viral vector packaging system comprises: at least one packaging plasmid, an envelope plasmid, a shuttle plasmid, and a binding plasmid;

[0207] (a) The packaging plasmid contains the Gag gene and the Pol gene (Gag / Pol packaging plasmid).

[0208] (b) The envelope plasmid contains a polynucleotide encoding any of the aforementioned fusion polypeptides;

[0209] (c) The shuttle plasmid contains at least one exogenous polynucleotide and: an LVV backbone gene or an RVV backbone gene; and

[0210] (d) The binding plasmid contains a polynucleotide encoding the CAR binding molecule.

[0211] In some embodiments of the present invention, the viral vector packaging system comprises: at least one packaging plasmid, an envelope plasmid, and a shuttle plasmid;

[0212] (a) The envelope plasmid comprises a polynucleotide encoding any of the aforementioned fusion polypeptides and a polynucleotide encoding any of the aforementioned membrane expression target molecules;

[0213] (b) The packaging plasmid contains the Gag gene and the Pol gene (Gag / Pol packaging plasmid)

[0214] (c) The shuttle plasmid contains at least one exogenous polynucleotide and: an LVV backbone gene or an RVV backbone gene; and

[0215] (d) The binding plasmid contains a polynucleotide encoding the CAR binding molecule.

[0216] In some embodiments of the present invention, the viral vector packaging system comprises: at least one packaging plasmid, envelope plasmid, shuttle plasmid, targeting plasmid, and binding plasmid;

[0217] (a) The packaging plasmid contains the Gag gene and the Pol gene (Gag / Pol packaging plasmid).

[0218] (b) The envelope plasmid contains a polynucleotide encoding any of the aforementioned fusion polypeptides;

[0219] (c) The shuttle plasmid contains at least one exogenous polynucleotide and: LVV backbone gene or RVV backbone gene;

[0220] (d) The binding plasmid contains a polynucleotide encoding the CAR binding molecule; and

[0221] (e) The targeting plasmid contains a polynucleotide encoding the membrane expression targeting molecule.

[0222] In some embodiments of the present invention, when the packaging system is used to package LVV, the packaging system further includes a Rev packaging plasmid containing the Rev gene.

[0223] In some embodiments of the present invention, the at least one exogenous polynucleotide includes any of the aforementioned exogenous polynucleotides provided by the present invention.

[0224] In some embodiments of the present invention, the at least one exogenous polynucleotide includes any of the aforementioned polynucleotides encoding CAR provided by the present invention.

[0225] In another aspect, the present invention also provides a packaging cell comprising any of the aforementioned viral vector packaging systems.

[0226] In some embodiments of the present invention, the packaging cells are selected from CHO cells, BHK cells, MDCK cells, C3H-10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC-1 cells, BSC-40 cells, BMT-10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, HEK-293 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, and 211 cells;

[0227] Preferably, the packaging cells are HEK-293T cells.

[0228] In some embodiments of the present invention, the packaging cells are configured to produce pseudo-LVV or RVV.

[0229] In another aspect, the present invention also provides a packaging cell line comprising any of the aforementioned packaging cells provided by the present invention.

[0230] In another aspect, the present invention also provides a method for producing pseudo-LVV or RVV, comprising culturing any of the aforementioned packaging cells or packaging cell lines to produce pseudo-LVV or RVV.

[0231] In another aspect, the present invention also provides a pseudo LVV or RVV, which is produced according to any of the foregoing methods for producing LVV or RVV.

[0232] In another aspect, the present invention also provides a pharmaceutical composition comprising any of the aforementioned particles.

[0233] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

[0234] In another aspect, the present invention also provides a method for transducing target cells in vitro in a subject in need, comprising contacting the target cells with any of the aforementioned particles; wherein the subject is an individual to whom the transduced target cells are administered;

[0235] Preferably, the target cell is a lymphocyte, which is selected from B cells, NK cells and T cells; more preferably, the target cell is a T cell.

[0236] In another aspect, the present invention also provides a method for transducing target cells in a subject in need, comprising administering a therapeutically effective amount of any of the aforementioned particles to the subject;

[0237] Preferably, the target cell is a lymphocyte, which is selected from B cells, NK cells and T cells; more preferably, the target cell is a T cell.

[0238] In another aspect, the present invention also provides a method for treating a subject in need, the subject having or suspected of having a disease, comprising administering to the subject a therapeutically effective amount of any of the aforementioned granules.

[0239] In another aspect, the present invention also provides the use of any of the aforementioned particles in the preparation of medicaments for treating diseases.

[0240] In some embodiments of the present invention, the disease is selected from autoimmune diseases and cancer; the cancer is selected from solid tumors and hematologic malignancies.

[0241] 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. One or more 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 center T-cell lymphoma (FTCL).

[0242] In some embodiments of the present invention, the disease is a blood cancer, which is a B-cell malignant tumor, and the B-cell malignant tumor is selected from one or more 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).

[0243] In some embodiments of the present invention, the disease is a solid tumor, which is selected from one or more 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.

[0244] In some embodiments of the present invention, the disease is an autoimmune disease selected from one or more 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.

[0245] In some embodiments of the present invention, the autoimmune disease is an autoimmune disease associated with abnormal B cells that secrete autoantibodies, and the autoimmune disease is selected from one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel 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.

[0246] In some embodiments of the present invention, the administration is selected from one or more of the following methods: intravenous injection, intratumoral injection, subcutaneous injection, intramuscular injection, sternal injection, nodular injection, infusion techniques, oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchyma), intraventricular, intramuscular, intraocular, intraarterial, via portal vein, intralesional, continuous release or secretion system, and implantable device. Beneficial effects

[0247] Similar to common viral glycoproteins, such as VSV-G and Cocal-G, the fusion peptides disclosed in this invention can mediate budding of pseudotype LVV or RVV in packaging cells, and can also mediate membrane fusion and endosome / lysosome escape. However, unlike common viral glycoproteins, the ability of the fusion peptides disclosed in this invention to specifically bind to viral glycoprotein receptors is relatively inhibited, thereby reducing the ability of the particles disclosed in this invention constructed using the fusion peptides to transduce cells expressing the receptors of the viral glycoproteins. Furthermore, when the fusion peptides are constructed on the surface of the pseudotype LVV or RVV or contain one or more targeting molecules, the specificity of the pseudotype LVV or RVV in targeting specific target cells can be effectively improved and non-specific transduction / off-target effects can be reduced. Compared with common pseudotype LVV or RVV containing wild-type VSV-G or Cocal-G, they are more suitable for application in CAR-T cell therapy, especially gene therapy such as in vivo CAR-T cell therapy.

[0248] Definitions and Glossary:

[0249] “TCR-CD3 complex subunit”: The TCR-CD3 complex subunit includes the TCR subunit and the CD3 subunit; the TCR subunit is selected from TCRα, TCRβ, TCRγ and TCRδ; the CD3 subunit is selected from CD3γ, CD3ζ, CD3δ and CD3ε.

[0250] “Inhibition”: When referring to the ability of the viral glycoprotein or its variants, the fusion peptide and / or the particle-specific receptor of the viral glycoprotein being “inhibited”, the term “inhibition” includes the ability to completely eliminate the ability of the viral glycoprotein or its variants, the fusion peptide and / or the particle-specific receptor of the viral glycoprotein, as well as the ability to significantly reduce the specific binding.

[0251] In a specific implementation, "significant reduction" means relative to wild-type viral glycoprotein and / or other control particles containing the wild-type viral glycoprotein but not the fusion polypeptide; "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%.

[0252] 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). Lentiviral and Retroviral Vector Systems. In: Hunt, KK, Vorburger, SA, Swisher, SG (eds) Gene Therapy for Cancer. Cancer Drug Discovery and Development. Humana Press.

[0253] 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 a polynucleotide encoding any of the aforementioned target molecules (including membrane expression target molecules) displayed on the surface of LVV or RVV provided by the present invention.

[0254] The third-generation lentiviral vector 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 to 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.

[0255] In some embodiments of the present invention, the viral vector packaging system further comprises a targeting plasmid containing a polynucleotide encoding any of the aforementioned targeting molecules (including membrane-expressed targeting molecules) displayed on the surface of LVV or RVV provided by the present invention. Exemplary examples include, but are not limited to, pMD2.G, pRSV-rev, pMDLG-pRRE, and pRRL-GOI.

[0256] 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. GagPol packaging plasmids contain the Gag and Pol genes; envelope plasmids typically contain polynucleotides encoding viral glycoproteins, such as VSV-G or Cocal-G.

[0257] In some embodiments of the present invention, the envelope plasmid contains a polynucleotide encoding any of the aforementioned fusion polypeptides disclosed in the present invention.

[0258] In some embodiments of the present invention, the packaging system further comprises a binding nucleic acid vector containing a polynucleotide encoding any of the CAR-binding molecules provided by the present invention. In some embodiments of the present invention, the packaging system further comprises at least one binding plasmid containing a polynucleotide encoding any of the CAR-binding molecules provided by the present invention.

[0259] In some embodiments, production cells are transfected with a defined ratio of transfer plasmid, GagPol plasmid, envelope plasmid, and Rev plasmid. 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 vector. In some embodiments, the mass of each of the transfer plasmid and GagPol plasmid is higher than the mass of each of the envelope plasmid and Rev plasmid. In some embodiments, the defined ratio of transfer plasmid, GagPol plasmid, envelope plasmid, and Rev plasmid is from about 1:1:1:1 to about 9:4:2:2; in some embodiments of the invention, the envelope plasmid may contain nucleic acid encoding the target molecule. In some embodiments, production cells are transfected with a defined ratio of transfer plasmid, GagPol plasmid, envelope plasmid, Rev plasmid, and binding plasmid (containing a polynucleotide encoding a CAR-binding molecule).

[0260] 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 vector or retroviral vector. In some embodiments, the mass of each of the transfer plasmid and GagPol plasmid packaging the lentiviral vector is higher than the mass of each of the envelope plasmid, Rev plasmid, and binding plasmid. In some embodiments, the defined ratio of the transfer plasmid, GagPol plasmid, Rev plasmid, envelope plasmid, and binding plasmid is from about 1:1:1:1:0.1 to about 10:5:4:4:5. In some embodiments, the defined ratio of the transfer plasmid, GagPol plasmid, envelope plasmid, Rev plasmid, and binding plasmid is from about 9:4:2:2:0.1 to about 9:4:2:2:5. In some embodiments of the invention, the envelope plasmid may contain nucleic acid encoding the target molecule displayed on the surface of LVV or RVV.

[0261] In some embodiments, the envelope plasmid comprises a tandem expression cassette encoding either of the aforementioned fusion peptides and a target molecule as disclosed herein, such as an activating molecule and / or a co-stimulatory molecule. In some embodiments, the tandem expression cassette contained in the envelope plasmid comprises 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 either of the aforementioned fusion peptides. In some embodiments, the polynucleotide encoding either of the aforementioned fusion peptides is located at the 5' end of the polynucleotide encoding the target molecule. In other embodiments, the polynucleotide encoding either of the aforementioned fusion peptides is located at the 3' end of the polynucleotide encoding the target molecule. The polynucleotide encoding the target molecule and the polynucleotide encoding either of the aforementioned fusion peptides are separated in the tandem cassette by a polynucleotide encoding IRES, the furin cleavage site, or the viral 2A peptide, which allows co-expression of both proteins by a single mRNA. In some embodiments, the viral 2A peptide is porcine cecilia virus-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 the polynucleotide encoding either of the aforementioned fusion polypeptides and the polynucleotide encoding the target molecule, respectively.

[0262] Administration / “Given”: The route of administration of the pharmaceutical composition is conventional in the art, such as by oral, nasal, intravenous, subcutaneous, intraperitoneal, intracerebral (in the brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein or intralesional route, or by continuous release system or by implanted device.

[0263] For explanations of other terms used herein, please refer to patents WO2025209590A and WO2025011662A. All publications, documents, and patents mentioned herein are hereby incorporated in their entirety by reference, as if each publication, document, or patent not specifically and individually indicated to be incorporated in its entirety by reference were incorporated in its entirety by reference. 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 recommendation of any kind. Section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Attached Figure Description

[0264] Figure 1: Flow cytometry results of GFP expression efficiency detected on Day 2 in Example 1, where m3VSVG LVV, CR2-m3VSVG LVV or CR3-m3VSVG LVV were transduced into Jurkat cells, Nalm6 cells and HEK-293T cells, respectively.

[0265] Figure 2: A schematic diagram of the structure of the dual promoter envelope plasmid in Example 2.

[0266] Figure 3: Flow cytometry results of GFP expression efficiency detected on Day 2 in Example 2, where the A7+CR2-m3VSVG LVV was transduced into Jurkat cells and Nalm6 cells, respectively.

[0267] Figure 4: Flow cytometry results of GFP expression efficiency detected on Day 2 in Example 2, where the A7+CR3-m3VSVG LVV was transduced into Jurkat cells and Nalm6 cells, respectively.

[0268] Figure 5: Flow cytometry results of GFP expression efficiency detected on Day 2 in Example 2, where the A3+CR2-m3VSVG LVV was transduced into Jurkat cells and Nalm6 cells, respectively.

[0269] Figure 6: Flow cytometry results of GFP expression efficiency detected on Day 2 in Example 2, where the A3+CR3-m3VSVG LVV was transduced into Jurkat cells and Nalm6 cells, respectively.

[0270] Figure 7: Flow cytometry results of GFP expression efficiency detected on Day 2 in Example 2, where A19+CR2-m3VSVG LVV was transduced into Jurkat cells and Nalm6 cells, respectively.

[0271] Figure 8: Flow cytometry results of GFP expression efficiency detected on Day 2 in Example 2, where A19+CR3-m3VSVG LVV was transduced into Jurkat cells and Nalm6 cells, respectively.

[0272] Figure 9: Flow cytometry results of GFP expression efficiency detected on Day 2 in Example 2, where the AMSLN+CR3-m3VSVG LVV transduced MSLN-Nalm6 cells and MSLN+Nalm6 cells, respectively.

[0273] Figure 10: Flow cytometry results of GFP expression efficiency detected on Day 2 in Example 3, where the A7VHH-CR3-m3VSVG LVV was transduced into Jurkat cells and Nalm6 cells, respectively.

[0274] Figure 11: Image of the coating plasmid in Example 4.

[0275] Figure 12: Flow cytometry results of GFP expression efficiency detection on Day 2 in Example 4. Embodiments of the present invention Example 1

[0276] The packaging contains a pseudo-LVV of the fusion polypeptide provided by this invention.

[0277] 1. Fake LVV packaging

[0278] A membrane-encapsulated plasmid was constructed based on the pMD2.G plasmid as the backbone, and the packaging surface contained membrane-displayed m3VSVG, fusion peptide CR2-m3VSVG, or fusion peptide CR3-m3VSVG pseudo-LVV: m3VSVG LVV, CR2-m3VSVG LVV, or CR3-m3VSVG LVV;

[0279] The fusion polypeptide CR2-m3 VSVG contains, from N-terminus to C-terminus, the following sequence: CR2, (G4S)3 linker peptide, and m3VSVG;

[0280] The fusion polypeptide CR3-m3VSVG contains, from N-terminus to C-terminus, the following sequence: CR3, (G4S)3 linker peptide, and m3VSVG;

[0281] Compared to wild-type VSV-G (SEQ ID NO:1), m3VSVG (SEQ ID NO:9) contains T214N and T352A to enhance its ability to antagonize complement inactivation, but does not affect its ability to specifically bind to LDL-R;

[0282] (1) The amino acid sequence of the full-length m3VSVG (sp-m3VSVG) containing the VSV-G signal peptide is shown in SEQ ID NO:10;

[0283] (2) The amino acid sequence of CR2 is shown in SEQ ID NO:121;

[0284] (3) The amino acid sequence of CR3 is shown in SEQ ID NO:122;

[0285] (4) The amino acid sequence of the (G4S)3 linker peptide is shown in SEQ ID NO:55;

[0286] (5) The amino acid sequence of the m3VSVG is shown in SEQ ID NO:9;

[0287] (6) The amino acid sequence of the fusion polypeptide CR2-m3VSVG is shown in SEQ ID NO:33;

[0288] (7) The amino acid sequence of the fusion polypeptide CR2-m3VSVG (sp-CR2-m3VSVG), which also contains the VSV-G signal peptide at the N-terminus, is shown in SEQ ID NO:34;

[0289] (8) The amino acid sequence of the fusion polypeptide CR3-m3VSVG is shown in SEQ ID NO:35;

[0290] (9) The amino acid sequence of the fusion polypeptide CR3-m3VSVG (sp-CR3-m3VSVG), which also contains the VSV-G signal peptide at the N-terminus, is shown in SEQ ID NO:36.

[0291] A. Prepare a 4-plasmid packaging system

[0292] Prepare a 4-plasmid packaging system: envelope plasmid, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and lentivirus pGClenti-GFP plasmid (a transfer plasmid containing the GFP gene, transfer plasmid GFP).

[0293] The envelope plasmid contains a polynucleotide encoding the sp-m3VSVG, the fusion polypeptide sp-CR2-m3VSVG, or sp-CR3-m3VSVG; the envelope plasmid is synthesized using conventional molecular cloning methods.

[0294] B. Prepare the HEK-293T cell culture system and package pseudo-LVV.

[0295] Take 56 mL of FBS, filter it into 500 mL of DMEM / high glucose (10% FBS), add 4 mL of P / S (double antibiotic, penicillin × streptomycin), shake well, and place in a carbon dioxide incubator for preheating and neutralization before transfection.

[0296] Day 0: HEK-293T cells were seeded at a density of 4.5 × 10⁴ cells in a 10 cm culture dish. 6 Approximately 48 hours after inoculation, when the cell confluence reached 80-90%, the four plasmids were transfected into HEK-293T packaging cells using PEI reagent, including:

[0297] Add 9 μg of transfer plasmid GFP, 4 μg of pMDLg / pRRE packaging plasmid, 2 μg of pRSV-REV packaging plasmid, and 2 μg of envelope plasmid to 1 mL of Opti-MEM medium. After shaking well, add 64 μL of PEI reagent, mix thoroughly by pipetting, and let stand for 10 minutes. Then add to the HEK-293T cell culture system. Replace the medium after 6 hours. Collect the supernatant 48 hours after transfection, filter through a 0.45 μm filter membrane, centrifuge at 50,000 g for 2.5 h, discard the supernatant, resuspend the m3VSVG LVV, CR2-m3VSVG LVV, or CR3-m3VSVG LVV in 200 μL of F12 medium, and store at -80°C.

[0298] 2. Transducing target cells

[0299] Day 0: Take 50 μL of viral supernatant from each of the m3VSVG LVV, CR2-m3VSVG LVV, or CR3-m3VSVG LVV, in three groups respectively, and add 1×10 5 LDL-R + Jurkat cell culture system, 1×10 5 LDL-R + Nalm6 cell culture system and 1×10 5 LDL-R + 293T cell culture system; the Jurkat cell culture system and Nalm6 cell culture system include 1640 medium + 10% FBS;

[0300] On Day 2, flow cytometry was used to detect the expression of the GFP gene in each cell culture system. The results are shown in Figure 1.

[0301] As shown in Figure 1, the m3VSVG LVV can efficiently transduce LDL-R+ Jurkat cells (GFP expression efficiency of approximately 99.7%), Nalm6 cells (GFP expression efficiency of approximately 99.28%), and HEK-293T cells (GFP expression efficiency of approximately 99.74%) through its unrestricted ability to specifically bind LDL-R; while the CR2-m3VSVG LVV and CR3-m3 VSVG are difficult to transduce LDL-R. + The fusion peptides CR2-m3VSVG and CR3-m3VSVG were used to transduce HEK-293T cells with endocytic capabilities (GFP expression efficiencies of approximately 41.18% and 35.36%, respectively). This demonstrates that by linking m3VSVG, whose ability to specifically bind LDL-R is not inhibited, to CR2 or CR3, the ability of the fusion peptides CR2-m3VSVG and CR3-m3VSVG to specifically bind LDL-R is inhibited, but the ability of the viral glycoprotein variant m3VSVG contained therein to mediate membrane fusion and endosome / lysosome escape is retained. After being internalized by HEK-293T cells with endocytic capabilities, the CR2-m3VSVG LVV and CR3-m3VSVG LVV can effectively transduce HEK-293T cells and deliver the GFP gene.

[0302] Opti-MEMalpha serum-reduced medium, brand: GIBCO; DMEM, brand: GIBCO; FBS, brand: EXCELL; F12 medium, brand: GIBCO; syringe filter, brand: SORFA. Example 2

[0303] The packaging contains a pseudo-LVV containing a fusion peptide provided by the present invention; the pseudo-LVV further contains one or more membrane expression targeting molecules; the ability of the pseudo-LVV to specifically bind to LDL-R is inhibited, but it can be specifically transduced into target cells through the included membrane expression targeting molecules.

[0304] 1. The packaging contains pseudo-LVV expressing anti-CD7 antibodies on a membrane.

[0305] The package contains (i) the fusion peptide CR2-m3VSVG or CR3-m3VSVG and (ii) a pseudo-LVV expressing an anti-CD7 antibody, A7+CR2-m3VSVG LVV or A7+CR3-m3VSVG LVV.

[0306] A. Prepare a 4-plasmid packaging system

[0307] Prepare a 4-plasmid packaging system: dual promoter encapsulation plasmid, pMDLg / pRRE packaging plasmid, pRSV-REV packaging plasmid, and transfer plasmid GFP;

[0308] The dual-promoter envelope plasmid contains (i) a polynucleotide encoding the fusion polypeptide sp-CR2-m3VSVG or sp-CR3-m3VSVG, which is independently expressed by two different promoters, the CAG promoter and the PGK promoter; and (ii) a polynucleotide encoding a membrane-expressing anti-CD7 antibody. A schematic diagram of the structure of the dual-promoter envelope plasmid is shown in Figure 2.

[0309] The polynucleotide encoding the membrane-expressed anti-CD7 antibody comprises, from the 5' end to the 3' end, the following polynucleotides: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding a scFv that specifically binds to human CD7 (a scFv derived from TH-69, TH69-scFv), a polynucleotide encoding the human CD8α hinge region, and a polynucleotide encoding the human CD8α transmembrane region.

[0310] The heavy chain variable region (VH region) of the TH69-scFv is linked to the light chain variable region (VL region) of the TH69-scFv via a (G4S)3 linker peptide.

[0311] (1) The amino acid sequence of the TH69-scFv is shown in SEQ ID NO:112;

[0312] (2) The amino acid sequence of the (G4S)3 linker peptide is shown in SEQ ID NO:55;

[0313] (3) The amino acid sequence of the human CD8α hinge region is shown in SEQ ID NO:51;

[0314] (4) The amino acid sequence of the transmembrane region of human CD8α is shown in SEQ ID NO:52;

[0315] Referring to the method for packaging dummy LVV in Example 1, package A7+CR2-m3VSVG LVV or A7+CR3-m3VSVG LVV.

[0316] B. Transduction of Jurkat and Nalm6 cells

[0317] Day 0: Take 200 μL of viral supernatant from each of the A7+CR2-m3VSVG LVV and A7+CR3-m3VSVG LVV, and add 1×10⁻⁶ ppm of each. 5 LDL-R + Jurkat cell culture system and 1×10 5 LDL-R + Nalm6 cell culture system;

[0318] On Day 2, flow cytometry was used to detect the expression of the GFP gene in each culture system. The results are shown in Figures 3 and 4.

[0319] As shown in Figures 3 and 4, the A7+CR2-m3VSVGLVV and A7+CR3-m3VSVGLVV are difficult to transduce CD7. - Nalm6 cells (GFP expression efficiencies of 0.21% and 0.25%, respectively) were effective in transducing CD7. + Jurkat cells (GFP expression efficiencies of 8.97% and 38.15%, respectively) showed that both A7+CR2-m3VSVG LVV and A7+CR3-m3VSVG LVV effectively and specifically transduced CD7+. + Target cells.

[0320] 2. The packaging contains pseudo-LVV with membrane-expressed anti-CD3 antibodies.

[0321] A. Packaging: A3+CR2-m3VSVG LVV or A3+CR3-m3VSVG LVV

[0322] Referring to the above-described packaging method for A7+CR2-m3VSVG LVV and A7+CR3-m3VSVG LVV, the packaging comprises (i) the fusion peptide CR2-m3VSVG or CR3-m3VSVG; and (ii) a membrane-expressed pseudo-LVV, A3+CR2-m3VSVG LVV or A3+CR3-m3VSVG LVV, expressing an anti-CD3 antibody;

[0323] The polynucleotide encoding the membrane-expressed anti-CD3 antibody comprises, from the 5' end to the 3' end, the following: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding a scFv specifically binding to human CD3ε (a scFv derived from UCHT1, UCHT1-scFv), a polynucleotide encoding the human CD8α hinge region, and a polynucleotide encoding the human CD8α transmembrane region.

[0324] The VH region of the UCHT1-scFv is connected to the VL region of the UCHT1-scFv via a (G4S)3 linker peptide.

[0325] The amino acid sequence of the UCHT1-scFv is shown in SEQ ID NO:58.

[0326] B. Transduction of Jurkat and Nalm6 cells

[0327] Following the method described above for transducing Jurkat and Nalm6 cells with A7+CR2-m3VSVG LVV, on Day 0, the A3+CR2-m3VSVG LVV or A3+CR3-m3VSVG LVV was added to the Jurkat cell culture system and the Nalm6 cell culture system, respectively. On Day 2, the expression of the GFP gene in each group of cells was detected, and the results are shown in Figures 5 and 6, respectively.

[0328] As shown in Figures 5 and 6, both A3+CR2-m3VSVG LVV and A3+CR3-m3VSVG LVV can effectively transduce CD3 by expressing anti-CD3 antibodies through the membranes displayed on their surfaces. + Jurkat cells (GFP expression efficiencies of approximately 16.99% and 24.58%, respectively) were unable to effectively transduce CD3. - Nalm6 cells (GFP expression efficiencies of 0.36% and 0.06%, respectively); the A3+CR2-m3VSVG LVV and A3+CR3-m3VSVG LVV can effectively and specifically transduce CD3 + Target cells.

[0329] 3. The packaging contains pseudo-LVV with membrane-expressed anti-CD19 antibody.

[0330] A. Packaging: A19+CR2-m3VSVG LVV or A19+CR3-m3VSVG LVV

[0331] Referring to the above-described packaging method for A7+CR2-m3VSVG LVV and A7+CR3-m3VSVG LVV, the packaging comprises (i) the fusion polypeptide CR2-m3VSVG or CR3-m3VSVG; and (ii) a membrane-expressed pseudo-LVV, A19+CR2-m3VSVG LVV or A19+CR3-m3VSVG LVV, expressing an anti-CD19 antibody;

[0332] The polynucleotide encoding the membrane-expressed anti-CD19 antibody comprises, from the 5' end to the 3' end, the following: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding a scFv specifically binding to human CD19 (a scFv derived from FMC63, FMC63-scFv), a polynucleotide encoding the human CD8α hinge region, and a polynucleotide encoding the human CD8α transmembrane region.

[0333] The amino acid sequence of the FMC63-scFv is shown in SEQ ID NO:49.

[0334] B. Transduction of Jurkat and Nalm6 cells

[0335] Following the method described above for transducing Jurkat and Nalm6 cells with A7+CR2-m3VSVG LVV, on Day 0, A19+CR2-m3VSVG LVV or A19+CR3-m3VSVG LVV was added to the Jurkat and Nalm6 cell culture systems, respectively. On Day 2, the expression of the GFP gene in each group of cells was detected, and the results are shown in Figures 7 and 8, respectively.

[0336] As shown in Figures 7 and 8, both A19+CR2-m3VSVG LVV and A19+CR3-m3VSVG LVV can effectively transduce CD19 by expressing anti-CD19 antibodies through the membranes displayed on their surfaces. + Nalm6 cells (GFP expression efficiencies of approximately 2.75% and 2.09%, respectively) were not effectively transduced with CD19. - Jurkat cells (GFP expression efficiencies of 0.10% and 0.17%, respectively); the A19+CR2-m3VSVG LVV and A19+CR3-m3VSVG LVV can effectively and specifically transduce CD19. + Target cells.

[0337] 4. The packaging contains pseudo-LVV expressing anti-MSLN antibodies on a membrane.

[0338] A. Packaging AMSLN+CR2-m3VSVG LVV or AMSLN+CR3-m3VSVG LVV

[0339] Referring to the above-described packaging method for A7+CR2-m3VSVG LVV and A7+CR3-m3VSVG LVV, the packaging comprises (i) the fusion peptide CR2-m3VSVG or CR3-m3VSVG; and (ii) a pseudo-LVV expressing an anti-MSLN antibody, AMSLN+CR2-m3VSVG LVV or AMSLN+CR3-m3VSVG LVV;

[0340] The polynucleotide encoding the membrane-expressing anti-MSLN antibody comprises, from the 5' end to the 3' end, the following polynucleotides: a polynucleotide encoding the human CD8α signal peptide, a polynucleotide encoding a scFv (derived from PE38, PE38-scFv) that specifically binds to human MSLN (Mesothelin), a polynucleotide encoding the human CD8α hinge region, and a polynucleotide encoding the human CD8α transmembrane region;

[0341] The amino acid sequence of the PE38-scFv is shown in SEQ ID NO:92.

[0342] B. Transduction of Nalm6 cells and MSLN overexpressing MSLN + Nalm6 cells

[0343] Referring to the above method of transducing Nalm6 cells with A7+CR2-m3VSVG LVV, on Day 0, the AMSLN+CR2-m3VSVG LVV or AMSLN+CR3-m3VSVG LVV was added to the Nalm6 cell culture system and MSLN overexpressing MSLN, respectively. + In the Nalm6 cell culture system (constructing MSLN overexpressing MSLN), + The method for using the Nalm6 cell line is well known to those skilled in the art.

[0344] Day 2, the expression of the GFP gene in each group of cells was detected; the results of AMSLN+CR3-m3VSVG LVV transduction of each group of cells are shown in Figure 9.

[0345] As shown in Figure 9, the AMSLN+CR3-m3VSVG LVV can effectively transduce MSLN overexpressing MSLN by expressing anti-MSLN antibodies through the membrane on its surface. + Nalm6 cells (GFP expression efficiency of approximately 2.08%), but they were difficult to effectively transduce MSLN. - Nalm6 cells (GFP expression efficiency approximately 0.09%); the AMSLN+CR3-m3VSVG LVV can effectively and specifically transduce MSLN. + Target cells. Example 3

[0346] A fusion peptide containing a targeting molecule was constructed, and a pseudo-LVV containing the fusion peptide was packaged.

[0347] 1. Constructing a fusion peptide containing an anti-CD7 nanobody

[0348] A fusion polypeptide A7VHH-CR3-m3VSVG comprising an anti-CD7 nanobody (VHH) was constructed, wherein the fusion polypeptide A7VHH-CR3-m3VSVG comprises, from the N-terminus to the C-terminus, the anti-CD7 nanobody, the (G4S)3 linker peptide, the CR3, the (G4S)3 linker peptide, and the m3VSVG.

[0349] The amino acid sequence of the anti-CD7 nanobody is shown in SEQ ID NO:101; the amino acid sequences of the HCDR1-3 regions of the anti-CD7 nanobody are shown in SEQ ID NO:102-104 respectively; the amino acid sequence of the A7VHH-CR3-m3VSVG is shown in SEQ ID NO:105.

[0350] The N-terminus also contains the amino acid sequence of the fusion polypeptide A7VHH-CR3-m3VSVG (sp-A7VHH-CR3-m3VSVG), which is a fusion polypeptide of the VSV-G signal peptide, as shown in SEQ ID NO:106.

[0351] 2. Fake LVV packaging

[0352] Referring to the method of packaging the CR3-m3VSVG LVV in Example 1, the packaging surface contains a pseudo-LVV of the fusion polypeptide A7VHH-CR3-m3VSVG, A7VHH-CR3-m3VSVG LVV; wherein, the envelope plasmid contains a polynucleotide encoding the fusion polypeptide sp-A7VHH-CR3-m3VSVG.

[0353] 3. Transduction of Jurkat and Nalm6 cells

[0354] Following the method described above for transducing Jurkat and Nalm6 cells with A7+CR3-m3VSVG LVV, on Day 0, the A7VHH-CR3-m3VSVG LVV was added to the Jurkat and Nalm6 cell culture systems, respectively; on Day 2, the expression of the GFP gene in each group of cells was detected, and the results are shown in Figure 10.

[0355] As shown in Figure 10, the A7VHH-CR3-m3VSVG LVV can effectively transduce CD7 through the anti-CD7 nanobody contained in the fusion peptide displayed on its surface. + LDL-R + Jurkat cells (GFP expression efficiency of approximately 84.27%) were used, but CD7 expression was difficult to transduce effectively. - LDL-R + Nalm6 cells (GFP expression efficiency approximately 0.29%); the A7VHH-CR3-m3VSVG LVV can effectively and specifically transduce CD7+. + Target cells. Example 4

[0356] 1. Fake LVV packaging

[0357] Referring to Example 1, an envelope plasmid was constructed based on the pMD2.G plasmid (encapsulated plasmid). The packaging surface contained a pseudo-LVV displaying TH69-scFv and a fusion peptide (CR2-Cocal-G or fusion peptide CR3-Cocal-G).

[0358] The fusion polypeptide CR2-Cocal-G comprises, from N-terminus to C-terminus, CR2, (G4S)3 linker peptide, and Cocal-G;

[0359] The fusion polypeptide CR3-Cocal-G contains, from N-terminus to C-terminus, the following sequence: CR3, (G4S)3 linker peptide, and Cocal-G;

[0360] The amino acid sequence of the Cocal-G is shown in SEQ ID NO:2. Taking CR3-Cocal-G as an example, the plasmid map of CR3-Cocal-G is shown in Figure 11.

[0361] A. Prepare a 4-plasmid packaging system according to Example 1, including an envelope plasmid, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a lentivirus pGClenti-GFP plasmid.

[0362] B. Prepare the HEK-293T cell culture system according to Example 1, and package the pseudo-LVV.

[0363] C. Transduction target cells

[0364] Day 0: The viruses prepared above (membrane-expressing CR2-Cocal-G and CR3-Cocal-G) were used to infect Jurkat cells and Nalm6 cells respectively at MOI=1;

[0365] On Day 2, flow cytometry was used to detect the expression of the GFP gene in each cell culture system. The results are shown in Figure 12.

[0366] As shown in Figure 12, the A7+CR2-CocalV and A7+CR3-CR2-Cocal are difficult to transduce CD7. - Nalm6 cells, but can effectively transduce CD7 + Jurkat cells, both A7+CR2-CocalV and A7+CR3-CR2-Cocal can effectively and specifically transduce CD7. + Target cells.

Claims

1. A fusion polypeptide, characterized in that, The fusion polypeptide comprises: A) a viral glycoprotein or a variant thereof; and / or, B) a binding polypeptide that specifically binds to the viral glycoprotein or a variant thereof.

2. The fusion polypeptide according to claim 1, characterized in that... , The viral glycoproteins described in A) are selected from: vesicular stomatitis virus glycoproteins, Nipah virus (NiV) glycoprotein G, measles virus glycoprotein H, lentivirus glycoproteins, 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 T-cell choriomeningitis virus (LCMV) glycoprotein; and / or, The binding polypeptide described in B) comprises a receptor or receptor-binding fragment of the viral glycoprotein, or an antibody against the viral glycoprotein.

3. The fusion polypeptide according to claim 2, characterized in that, The vesicular stomatitis virus glycoproteins are selected from: Indiana vesicular stomatitis virus strain glycoprotein, Cocal vesicular stomatitis virus strain glycoprotein, Maraba vesicular stomatitis virus strain glycoprotein, Morreton vesicular stomatitis virus strain glycoprotein, Alagoas vesicular stomatitis virus strain glycoprotein, and New vesicular stomatitis virus strain glycoprotein. The following vesicular stomatitis virus (VSV) strains contain glycoproteins: Jersey, Carajas, Chandipura, Eptesicus, Isfahan, Jurona, Malpais, Perinet, Piry, Radi, Rhinolopus, and Yug Bogdanovac.

4. The fusion polypeptide according to claim 3, characterized in that, The viral glycoprotein is either the Indiana strain vesicular stomatitis virus glycoprotein (VSV-G) or the Cocal strain vesicular stomatitis virus glycoprotein (Cocal-G).

5. The fusion polypeptide according to claim 4, characterized in that, The receptor for the viral glycoprotein is LDL-R, and the binding fragment of LDL-R is selected from one or more of the cysteine-rich domains ("CR") of LDL-R. Preferably, the binding fragments of the LDL-R are CR2 and / or CR3; more preferably, the binding fragments of the LDL-R are CR2 and CR3, wherein CR2 is linked to CR3; Optionally, the CR2 is linked to the CR3 via a polypeptide linker.

6. The fusion polypeptide according to claims 1-5, characterized in that, The viral glycoprotein or its variant described in A) is linked to the binding polypeptide described in B); Optionally, the viral glycoprotein or a variant thereof is linked to the binding polypeptide via a polypeptide linker.

7. The fusion polypeptide according to claim 6, characterized in that, The fusion polypeptide further comprises: C) at least one targeting molecule; Optionally, the target molecule described in C) is linked to the binding peptide described in B); preferably, the target molecule described in C) is linked to the binding peptide described in B) via a peptide linker.

8. The fusion polypeptide according to claim 7, characterized in that, The targeting molecule described in C) can bind to a marker on the target cell, which is an endocytic receptor; Preferably, the marker is selected from:

9. More preferably, the marker is selected from at least one of CD3, CD5, CD7, CD19 and MSLN.

10. The fusion polypeptide according to claim 7, characterized in that, The targeting molecule described in C) can bind to a marker on T cells, wherein the marker is an endocytic receptor; The T cell markers were selected from: CD3, CD3γ, CD3δ, CD3ε, TCRγ, TCRδ, TCRα, TCRβ, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD44, CD45RA, CD45RB, CD45RO, CD57, CD71, CD69, CD94, CD95, 4-1BB (CD137), CD103, CD122, CD127, CD1 61. One or more of the following: OX40 (CD134), ICOS, CD183 (CXCR3), CD184 (CXCR4), CD185 (CXCR5), PD-1, CD193 (CCR3), CD194 (CCR4), CD195 (CCR5), CD196 (CCR6), CD197 (CCR7), CCR10, IL6ST, P2RX7, TIGIT, TIM-3, and LAG-3; Preferably, the T cell markers are selected from one or more of the following: CD3, CD3γ, CD3δ, CD3ε, CD5, CD7, CD28, CD2, CD127, 4-1BB, OX40, ICOS, TCRγ, TCRδ, TCRα, and TCRβ.

11. The polypeptide according to any one of claims 7-9, characterized in that, The target molecules mentioned in C) include activating molecules; Optionally, the activating molecule may bind to a TCR-CD3 complex or its subunits; the TCR-CD3 complex subunits are selected from TCR subunits and CD3 subunits, the CD3 subunits include CD3γ, CD3δ, CD3ε and CD3ζ; the TCR subunits include TCRγ, TCRδ, TCRα and TCRβ.

12. The fusion polypeptide according to claim 10, characterized in that, The targeting molecule further includes at least one co-stimulatory molecule, and the activating molecule is linked to the co-stimulatory molecule; optionally, the activating molecule is linked to the co-stimulatory molecule via a linker peptide; optionally, the co-stimulatory molecule may bind to CD28, 4-1BB, OX40, or ICOS.

13. The fusion polypeptide according to claim 11, characterized in that, The targeting molecule also includes an adhesion molecule; preferably, the adhesion molecule can bind to CD2; more preferably, the adhesion molecule includes CD58 or its extracellular domain or a functional fragment thereof.

14. The fusion polypeptide according to any one of claims 1-12, characterized in that, The polypeptide linker is selected from one or more of the following: 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 part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2 and CH3 domains; d) The stalk domain of type II C-lectins, wherein the type II C-lectins are selected from the stalk domains of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and e) Flexible linker peptides.

15. The fusion polypeptide according to claim 13, characterized in that, The fusion polypeptide also includes a leader signal peptide.

16. A type of particle, characterized in that, The particle comprises the fusion polypeptide of any one of claims 1-14, the fusion polypeptide being displayed on the surface of the particle; Preferably, the particles are selected from LNPs, virus-like particles, exosomes, extracellular vesicles, and enveloped virus particles; More preferably, the particle is an enveloped viral particle; Optionally, the enveloped viral particles are pseudotyped LVV and / or RVV.

17. The particles according to claim 15, characterized in that, The particles also contain one or more targeted molecules displayed on their surface.

18. The particles according to claim 16, characterized in that, The targeting molecule can bind to a marker on the target cell, which is an endocytic receptor.

19. The particles according to claim 16, characterized in that, The targeting molecule can bind to a marker on T cells, which is an endocytic receptor.

20. The particles according to any one of claims 16-18, characterized in that, The target molecules include activating molecules.

21. The particles according to claim 19, characterized in that, The targeting molecule also includes at least one co-stimulatory molecule.

22. The particles according to claims 16-20, characterized in that, The targeting molecule is connected to a transmembrane domain (membrane expression targeting molecule), which is anchored to the surface of the particle.

23. The particles according to claim 21, characterized in that, The transmembrane domain is selected from the transmembrane regions of the following proteins: CD2, CD3, TCR, CD4, CD5, CD7, CD8, CD8α, CD8β, CD9, CD16, CD22, CD27, CD28, CD28H, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD84, CD154, CD166, CD226, CD244, 4-1BB, OX40 , ICOS, ICAM-1, CTLA-4, PD-1, LAG-3, GITR, HVEM, DAP10, DAP12, TIM-1, LIGHT, ICOS, OX40, 2B4, B TLA, DNAM-1, DR3, FcERIγ, IL7, IL12, IL15, SLAM, KIR2DL4, KIR2DS1, KIR2DS2, NKG2C, NKG2D and CS1.

24. The particles according to claim 21, characterized in that, The target molecule is connected to the transmembrane domain via a connecting 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 part of the Fc domain, wherein the Fc domain is selected from one or more of the CH1, CH2 and CH3 domains; d) The stalk domain of type II C-lectins, wherein the type II C-lectins are selected from the stalk domains of CD23, CD69, CD72, CD94, NKG2A, and NKG2D; and e) Flexible linker peptides.

25. The particles according to any one of claims 15-23, characterized in that, The particles also contain at least one exogenous polynucleotide. Optionally, the exogenous polynucleotide comprises at least one of a polynucleotide encoding a chimeric antigen receptor (CAR) and a polynucleotide encoding an engineered TCR; Preferably, the CAR includes an antigen-binding region, a transmembrane region, and an intracellular signal transduction domain; Optionally, the antigen-binding region binds to disease-related antigens.

26. The particles according to claim 24, characterized in that, The CAR also includes a co-stimulatory signal transduction domain, a hinge region, and / or a leader signal peptide.

27. An isolated polynucleotide, characterized in that, The isolated polynucleotide encodes the fusion polypeptide according to any one of claims 1-14.

28. A nucleic acid vector, characterized in that, The nucleic acid vector comprises the isolated polynucleotide as described in claim 26; Preferably, the nucleic acid vector is selected from plasmids, lentiviral vectors, and retroviral vectors.

29. A viral vector packaging system, characterized in that, The viral vector packaging system comprises the isolated polynucleotides as described in claim 26.

30. The viral vector packaging system according to claim 28, characterized in that, The viral vector packaging system further comprises (a) the gag gene, (b) the pol gene, (c) at least one exogenous polynucleotide, and (d) a lentiviral vector backbone gene and a rev gene; or a retroviral vector backbone gene.

31. A type of packaging cell, characterized in that, The packaging cells comprise the viral vector packaging system of claim 28 or 29.

32. A drug combination, characterized in that, The pharmaceutical composition comprises particles according to any one of claims 15-25. The pharmaceutical composition further comprises pharmaceutically acceptable excipients or carriers.

33. A method for treating a subject in need, said subject having or suspected of having a disease, characterized in that, The treatment includes administering to the subject a therapeutically effective amount of any one of claims 15-25 or the drug combination of claim 31; Preferably, the disease is selected from autoimmune diseases and cancer; the cancer is selected from solid tumors and hematologic malignancies.

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