Targeting vectors and their manufacturing methods and uses

Abstract

A targeting vector and a method for targeting its host cell are provided. The vector comprises a first molecule that binds to an endocytic receptor of a target cell and a second molecule that promotes the release of a substance carried by the targeting vector into the cytoplasm. When the targeting vector is a viral vector, the first molecule is not part of a viral envelope protein, and the second molecule promotes endosomal or lysosomal escape of the targeting vector. By designing the first molecule based on the endocytic receptor of the target cell, different types of cells can be targeted. After rational mutation of the vector, it is possible to avoid infection of cells that do not require targeting and improve the precision of the vector.

Description

Cross-reference to Related Applications

[0001] This application claims the priority of a Chinese patent application filed with the China National Intellectual Property Administration on September 30, 2022, with the application number 202211217795.2 and the invention title "Targeting Vector and Its Manufacturing Method and Use", and all of its contents are incorporated herein by reference in their entirety.

[0002] [Incorporation of the Sequence Listing Submitted in Electronic Format] This application includes a sequence listing in XML format submitted in electronic form, which is incorporated herein by reference in its entirety. This sequence listing was created on September 28, 2023, named "JYSW-PA-PCT-NO-05-1-seql.xml", and the file size is 1.56 MB.

Technical Field

[0003] The present invention belongs to the field of vector delivery, and specifically relates to a vector having targeting properties and a method for targeting its host cells.

Background Art

[0004] Currently, drug delivery employs two main methods: (1) viral vectors, such as adenoviruses, adeno-associated viruses (AAVs), retroviruses, and lentiviruses; and (2) non-viral vectors, such as liposome nanoparticles (LNPs), exosomes, virus-like particles (VLPs), and antibody-drug conjugates (ADCs). Here, adenoviruses, adeno-associated viruses, virus-like particles, and antibody-drug conjugates are delivery vectors without liposome envelopes, while retroviruses, lentiviruses, nanoliposomes, and exosomes are delivery vectors with liposome envelopes. Viral vectors with an envelope can target and recognize specific receptor proteins on the cell membrane via their envelope protein. For example, the vesicular stomatitis virus envelope protein (VSVG) can target and recognize the low-density lipoprotein receptor (LDL-R), and the baboon endogenous retrovirus (BaEV) envelope glycoprotein can target and recognize the ASCT1 and ASCT2 receptors, thereby mediating the entry of the viral vector into target cells expressing these receptors.

[0005] Currently, lentiviral skeletons and VSVG envelope protein-based vectors are already widely used in clinical treatment. For example, in chimeric antigen receptor T-cell therapy (CAR-T), lentiviruses carrying VSVG encapsulate CAR molecular genes, thereby infecting T cells and producing CAR-T cells.

[0006] Lentiviral vectors are viral vectors that modify the human immunodeficiency virus (HIV). They are a type of retrovirus with an RNA genome. Their toxic genes have already been removed and replaced with exogenous target genes, and they belong to the pseudotype virus class. By using reverse transcriptase to integrate exogenous genes into the genome, stable expression can be achieved, and they have the characteristic of infecting both mitotic and non-mitotic cells. The original HIV virus carries a gp120 and gp41 complex, and can promote HIV infection of cells by recognizing the CD4 molecule. Modified lentiviral vectors do not express gp120 and gp41, but express the VSVG envelope protein. Because VSVG can target and recognize LDL-R, and LDL-R is widely expressed, lentiviruses carrying VSVG can widely infect various types of cells, including T cells, hepatocytes, cardiomyocytes, neurons, endothelial cells, stem cells, and various tumor cells.

[0007] The advantages of lentiviral vectors are as follows: (1) Long expression time: Lentiviruses can integrate foreign genes into the host cell genome, enabling stable gene expression over long periods. These genes are not deleted during cell division and passaging, making them the first choice for cell experiments and frequently used to construct stable cell lines. (2) High safety: Lentiviral vectors are not pathogenic, and lentiviral vector-modified T cells are used in CAR-T cell therapy. (3) Low immunogenicity: Direct injection into living tissue is unlikely to trigger an immune response, making them suitable for animal experiments. The disadvantages are that LDL-R is expressed in a broad spectrum across many types, resulting in weak targeting, and many cells are difficult to infect with lentiviruses, such as resting hematopoietic stem cells, resting T cells, NK cells, and B cells.

[0008] Currently, the development of targeted lentiviruses has already been reported. For example, (1) CD3 / CD28 antibody-based targeted lentiviruses can promote the activation of viral particles and infection of T cells by expressing CD3 and CD28 antibodies with transmembrane sequences or other antibodies that can activate T cells on the surface of the lentivirus. This method can effectively promote the in vivo infection of T cells by lentiviral vectors. The presence of CD3 antibodies can give this virus a certain degree of targeting, but the presence of wild-type envelope proteins such as VSVG or Cocal (cocal virus) means that it can still infect other types of cells, and its targeting ability still needs improvement. (2) Fusion-based targeted viruses. Unlike envelope proteins such as VSVG and BaEV, which simultaneously link receptor recognition and envelope fusion, the measles virus (MV) and Nipah virus (NiV) envelope glycoproteins are H / G and F proteins. Here, the H / G protein is responsible for receptor recognition, and after H recognizes the receptor, the F protein (Fusogen) undergoes a conformational change to mediate the fusion of the viral envelope to the cell membrane. By linking the H / G protein with an antibody, for example, a CD4 or CD8 antibody, and promoting the extracellular delivery of the antibody, the virus can specifically recognize CD4+ or CD8+ T cells. Increasing the amount of antibody on the H / G protein increases the steric hindrance of the H / G protein, which may make it difficult for the F protein to reach the target cell membrane after allosteric conversion, thus hindering its ability to mediate the fusion of the virus to the envelope.

[0009] Beyond lentiviruses, AAV vectors have a wide range of applications in the delivery of gene therapy drugs. Adeno-associated viruses are a type of single-stranded DNA virus, and the current consensus in the scientific community is that they do not cause any human diseases. The protein capsid carried by recombinant adeno-associated virus (rAAV), the vector for gene therapy, is almost identical to that of wild-type AAV, except that the coding viral protein portion of the genome within the capsid has been completely deleted and replaced with therapeutic recombinants. The uniquely retained portion within the AAV genome is the ITRs, which play a role in guiding genome replication and viral vector assembly. Currently, the majority of AAV clinical trials are focused on four major organs / tissues: the eyes, liver, muscles, and the central nervous system. Intravenous gene therapy targeting the liver has shown that it can address metabolic and hematological disorders. Parkinson's disease, hemophilia A, and hemophilia B are the three indications with the most clinical trials.

[0010] The advantages of AAV vectors are as follows: (1) They are non-pathogenic, and the vectors are derived from non-pathogenic viruses; (2) The particles are small and can penetrate tissues; and (3) They are easy to manufacture and have an extremely high packaging titer. Furthermore, while AAV vectors of different serotypes have different infection preferences, their targeting capabilities are still insufficient.

[0011] Other vectors, including LNP and ADC drug delivery systems, are already widely used in clinical practice.

[0012] Messenger ribonucleic acid (mRNA) encapsulated in LNPs is used in the production of COVID-19 vaccines, and extensive research is being conducted in the field of oncology vaccines. Because the lipid bilayer does not possess targeting properties, it is difficult to target and infect specific cells. By linking CD5 antibodies with LNPs, mRNA expressing CAR-T molecules can be targeted and delivered to T cells, thereby producing CAR-T cells. However, LNPs are readily taken up naturally by dendritic cells (DCs) and macrophages, and are easily enriched in the liver. Furthermore, the biomolecular crown formed on LNPs after administration contains hundreds of types of biomolecules, such as high-density lipoprotein HDL, which affects their targeting properties. Therefore, even with antibodies that target and modify LNPs, their targeting properties remain weak.

[0013] ADC drugs link antibodies to cell-specific endocytosis receptors with a linker. Because the linker is stable in the blood, the drug is not released during intravenous infusion. However, after the antibody binds to the cell-specific endocytosis receptor, it is encapsulated in a lysosome. At this point, the linker is degraded by enzymes in the lysosome, thereby releasing the drug. The drug is not degraded by lysosomes and can exert its effects without relying on lysosomal escape. Although ADC drugs have strong targeting properties, it is difficult to deliver other drugs, especially biomacromolecules, to cells based on conventional ADC delivery methods because biomacromolecules such as proteins and nucleic acids are easily degraded by lysosomes.

[0014] In summary, conventional delivery vectors generally struggle to deliver substances to specific cells, and it is particularly difficult to deliver biomacromolecules to specific cells and allow them to exert their functions. [Overview of the project] [Means for solving the problem]

[0015] A first aspect of the present invention provides a targeting vector comprising a first molecule that binds to an endocytosis receptor of a target cell and a second molecule that facilitates the release of a substance carried on the targeting vector into the cytoplasm, wherein, if the targeting vector is a viral vector, the first molecule is not part of a viral envelope protein.

[0016] Targeting vectors can take various forms, including enveloped vectors and non-enveloped vectors. Enveloped vectors include, for example, retroviral vectors, lentiviral vectors, and lipid nanoparticles, while non-enveloped vectors include, for example, adenoviruses, adeno-associated viruses, and virus-like particles.

[0017] Endocytosis receptors are membrane proteins expressed on the cell surface that, after binding to antibodies, ligands, or specific substances, can induce endocytosis (also known as cell cytosis). Examples include CD7, CD5, HER2, and mesothelin. Generally, endocytosis receptors contain domains such as YXXPhi, [D / E]XXXL[L / I], and FXNPXY. However, some membrane proteins, such as the CD8 molecule, do not possess an endocytosis receptor domain and are therefore generally considered to have weak endocytic ability. For this reason, screening for endocytosis domains often involves using CD8 molecules with random sequences chimerically added to their intracellular end.

[0018] Different cells express different specific endocytosis receptors, and the first molecule is designed based on the endocytosis receptor of the cell to be targeted. By causing the vector to be endocytotic by the target cell via an antibody or ligand (part of the first molecule) that specifically binds to the endocytosis receptor, the vector can be made capable of infecting specific cells but not other cells.

[0019] The first molecule, after binding to the endocytosis receptor, induces receptor-mediated endocytosis. Receptor-mediated endocytosis is the process by which cells specifically take up extracellular proteins or other compounds in a manner dependent on receptors on their cell surface. Cell surface receptors have a high degree of specificity and bind to the corresponding ligand to form a complex. The cell membrane at this point then indents to form a coated pit, and the pit and cell membrane detach to form a coated vesicle, which takes in extracellular material into the cell. After the coated vesicle enters the cell, it sheds its outer layer and binds to endosomal vesicles to form a larger endosome or endosome.

[0020] The second molecular action is to facilitate endosomal escape or lysosomal escape of the targeting vector after endocytosis in the target cell, thereby enabling it to enter the cytoplasm. If endosomal or lysosomal escape does not occur after endocytosis, the targeting vector will ultimately be degraded by lysosomes. Therefore, for the substance carried by the vector to be effectively delivered to the cytoplasm, the vector must possess the ability to undergo endosomal or lysosomal escape.

[0021] Common methods of endosome / lysosome escape include: (1) disrupting endosomes / lysosomes, for example polyethyleneimine, which swells and ruptures the endosome / lysosome through the "proton sponge" effect, allowing the carried substance to escape and enter the cytoplasm; (2) reducing the stability of the lysosomal membrane by utilizing the interaction between the positive charge on the vector surface and the negatively charged lysosomal membrane; and (3) achieving escape through membrane fusion between the vector and the endosome / lysosome, for example, by gala modification of the fusion peptide in vector expression, or by having a viral envelope protein with membrane fusion capability, such as VSVG or its variants, in the vector. In one specific approach, the second molecule is a viral envelope protein and / or a non-viral envelope protein.

[0022] In one specific solution, the viral envelope protein is selected from at least one of vesicular stomatitis virus envelope glycoprotein VSVG and its variants, baboon endogenous retrovirus envelope glycoprotein BaEV and its variants, feline endogenous retrovirus envelope glycoprotein RD114 and its variants, and gibbon ape leukemia virus envelope glycoprotein GALV and its variants, and the non-viral envelope protein is selected from at least one of VP1 of adeno-associated virus AAV and its variants, VP2 of adeno-associated virus AAV and its variants, and polyethyleneimine.

[0023] Viral envelope proteins, such as VSVG, can promote the release of substances carried by the vector by promoting the fusion of the viral envelope and the endosome / lysosome membrane in the endosome / lysosome. Non-viral envelope proteins, such as VP1 and VP2 of AAV, can undergo conformational changes under acidic conditions and form pores on the endosome / lysosome to release the substances carried by the vector. Polyethyleneimine can exert a proton sponge effect in the endosome / lysosome, that is, when the pH in the lysosome decreases, PEI can capture a large amount of protons, cause the influx of chloride ions, increase the osmotic pressure in the endosome / lysosome, and finally cause the endosome / lysosome to rupture and promote the release of the substances carried by the vector.

[0024] A variant is a mutant having at least 75% identity with the amino acid sequence of the non-mutant (wild type), and "at least 75% identity" means 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% the same as the amino acid sequence of the non-mutant (wild type).

[0025] Since the receptor LDL-R of VSVG is widely expressed in various cells such as T cells, hepatocytes, cardiomyocytes, endothelial cells, stem cells, etc. after activation, and various tumor cells, pseudotyped lentiviruses carrying VSVG or its variants can infect various types of cells. VSVG-based pseudotyped viruses have broad-spectrum infectivity, but many cells do not express or express low levels of the receptor LDL-R of VSVG, such as NK cells, resting T cells, etc. At this time, VSVG-based pseudotyped viruses are difficult to infect. In addition, since LDL-R is widely expressed in various cells, the targeting of VSVG-based pseudotyped viruses is insufficient. For example, various cells all express LDL-R, and it is difficult for lentiviruses to accurately transfect only one of them.

[0026] This solution utilizes the endosome / lysosome escape ability of lentiviruses, modifies it, links a first molecule to the lentivirus, and the obtained targeting vector can be flexibly applied to different scenarios for different cells. The first molecule is designed based on the cells to be targeted, the application range is greatly expanded, and the targeting accuracy is also improved.

[0027] In one specific solution, the second molecule is selected from VSVG and its variants.

[0028] In one specific solution, the first molecule includes a transmembrane peptide stretch, an antibody or ligand that binds to the endocytosis receptor of the target cell. In some solutions, the first molecule further includes an extracellular hinge region. Membrane-expressed proteins generally require a hinge region, which is beneficial for the extension of membrane proteins, and the commonly used one is the CD8 hinge region.

[0029] As long as it does not affect its function, the amino acid sequence of the first molecule is not limited, and for example, when the first molecule contains a CD33 antibody, it may be a protein that is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% the same as the amino acid sequence of the CD33 antibody.

[0030] Endocytosis receptors differ depending on the target cell. Common endocytosis receptors include, for example, HER2, CD20, CD19, CD79A, CD79B, CD56, CD22, CD138, CD37, CD98, CD309, CD33, CD163, CD163B, CD5, CD7, CD169, CD204, CD205, CD209, CD280, CD302, TROP-2, CD19, NECTIN4, 5T4, CD30, FRα, STEAP1, ENPP3, GCC, SLC44A4, NaPi2b, CA9, SC-16, CD142, P-Cadherin, PSMA, ED-B, endothelin receptors, ETB, TN-C, and Collagen receptors. IV, Periostin, CEACAM, c-MET, TDGF1, IGF1R, Mesothelin, TIM1, NCAM1, ZIP6, CD166, GPNMB, SDC1, glycosphingolipid, TfR, Gang lioside, CD74, CLDN18, DPEP3, SLITRK6, PRL-R, LY75, CD48, MUC1, CDKs, B7-H4, STING, KAAG1, CD70, CDH3, LRRC15, EGFR, ASGPR.

[0031] In one specific solution, the targeting vector is a lentiviral vector, the first molecule expressed by the lentiviral vector is a transmembrane protein, and the second molecule expressed by the lentiviral vector is a viral envelope protein, the viral envelope protein having the ability to promote endosomal or lysosomal escape. If the target cell does not express a viral envelope protein receptor, the virus can infect the target cell by the first molecular-mediated endocytosis.

[0032] In one specific scheme, the transmembrane protein is selected from CD7 antibody, CD19 antibody, CD33 antibody, ASGRP antibody or ligand, Mesothelin antibody, HER2 antibody, and the transmembrane protein comprises at least one amino acid sequence from the CD8 signal peptide, TH-69 heavy chain (VH), GS linker peptide (linker), TH-69 light chain (VL), CD8 hinge region, and CD8 transmembrane region of the CD7 antibody, and at least about 75%, 80%, and 85% of the amino acid sequence. The protein may be identical to %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5%, and the transmembrane protein is identical to at least one amino acid sequence of the CD33 antibody, specifically the CD8 signal peptide, Gemtuzumab light chain (VL), GS linker peptide, Gemtuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region, by at least approximately 75%, 80%, 85%, 90%, and 91%. The protein may be 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical, and the transmembrane protein is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to at least one amino acid sequence of the CD19 antibody, specifically the CD8 signal peptide, FMC-63 heavy chain (VH), GS linker peptide, FMC-63 chain (VL), CD8 hinge region, and CD8 transmembrane region. The protein may be 97%, 98%, 99%, or 99.5% identical to the transmembrane protein, and the transmembrane protein is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identical to the amino acid sequence of at least one of the following: the CD8 signal peptide, ASGRP light chain B11 (VL), GS linker peptide, ASGRP heavy chain (VH), CD8 hinge region, or CD8 transmembrane region of the ASGRP antibody.The protein may be 5% identical, and the transmembrane protein is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 99.5% identical to the amino acid sequence of at least one of the following: the CD8 signal peptide, Pertuzumab light chain (VL), GS linker peptide, Pertuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region of the HER2 antibody. The transmembrane protein may be a protein whose amino acid sequence is at least approximately 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to at least one of the amino acid sequences of the mesothelin antibody's CD8 signal peptide, PE38 heavy chain (VH), GS linker peptide, PE38 light chain (VL), CD8 hinge region, or CD8 transmembrane region.

[0033] In one specific approach, the viral envelope protein undergoes mutations that weaken its receptor recognition ability, for example, reducing or eliminating its ability to bind to LDL-R, while retaining only its ability to escape from endosomal or lysosomal regions, thereby exerting its targeting effect entirely dependent on the first molecule. By carrying this mutated envelope protein, the targeting ability of the vector is further enhanced, allowing it to infect only specific target cells.

[0034] In one specific scheme, the first molecule contains an antibody or ligand, and after the antibody binds to the corresponding antigen (endocytosis receptor) on the target cell, endocytosis occurs. The second molecule employs mutated VSVG, which disrupts the ability of VSVG to bind to LDL-R, but at the same time does not affect the lysosomal escape ability of VSVG. Mutations involve insertion, deletion, or substitution. For example, the envelope glycoprotein VSVG of the vesicular stomatitis virus Indiana strain has undergone H8 mutations, N9 mutations, Q10 mutations, K47 mutations, K50 mutations, and A51 mutations. This includes mutations in the following locations: differentiating, S183 mutation, S179 mutation, N180 mutation, I182 mutation, M184 mutation, Y209 mutation, I347 mutation, T350 mutation, T352 mutation, E353 mutation, R354 mutation, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353.

[0035] Furthermore, VSVG includes one or more combinations of mutations at the following sites: H8 substitution, N9 substitution, Q10 substitution, K47 substitution, K47 deletion, K50 substitution, A51 substitution, S183 substitution, S179 substitution, N180 substitution, I182 substitution, M184 substitution, Y209 substitution, I347 substitution, T350 substitution, T352 substitution, E353 substitution, R354 substitution, amino acid deletions from positions 1 to 18, amino acid deletions from positions 19 to 36, amino acid deletions from positions 37 to 51, amino acid deletions from positions 314 to 384, amino acid deletions from positions 321 to 374, amino acid deletions from positions 331 to 364, amino acid deletions from positions 344 to 354, and amino acid deletions from positions 345 to 353.

[0036] Furthermore, mutations may occur in the amino acid sequence of VSVG, such as the substitution of lysine K for glutamine Q at the 47th amino acid of VSVG, and / or the substitution of arginine R for glutamine Q at the R354 amino acid, or, for example, the deletion of the 47th amino acid of VSVG.

[0037] In one specific plan, the viral envelope protein is selected from the following: vesicular stomatitis virus Indian strain envelope glycoprotein VSVG, Cocal virus envelope glycoprotein, Maraba virus envelope glycoprotein, Morreton virus envelope glycoprotein, Alagoa virus envelope glycoprotein, New Jersey virus envelope glycoprotein, and Carajas virus envelope glycoprotein.

[0038] In one specific example, when compared across the entire region with the vesicular stomatitis virus Indiana strain envelope glycoprotein VSVG, the Cocal virus envelope glycoprotein, Maraba virus envelope glycoprotein, Morreton virus envelope glycoprotein, Alagoa virus envelope glycoprotein, New Jersey virus envelope glycoprotein, and Carajas virus envelope glycoprotein exhibit substitutions / deletions at H8, N9, Q10, K47, K50, A51, S183, S179, N180, I182, M184, Y209, I347, and T35. Mutations occur at sites corresponding to the following amino acid deletions: substitution / deletion of 0, substitution / deletion of T352, substitution / deletion of E353, substitution of R354, deletion of amino acids 1-18, deletion of amino acids 19-36, deletion of amino acids 37-51, deletion of amino acids 314-384, deletion of amino acids 321-374, deletion of amino acids 331-364, deletion of amino acids 344-354, and deletion of amino acids 345-353.

[0039] Amino acid mutations include amino acid deletions, substitutions, and insertions. Most studies and experiments focus on amino acid substitutions, while research on amino acid insertions and deletions remains largely neglected (Savino S, et al., Insertions and deletions in protein evolution and engineering. Biotechnol Adv. (2022)).

[0040] Substitutions or deletions of amino acids at the same site can lead to unpredictable and entirely different effects.

[0041] The 47th amino acid in the extracellular domain of VSV-G is substituted from lysine K to glutamine Q (K47Q), and the 354th amino acid is substituted from arginine R to glutamine Q (R354Q). Both substitutions can weaken, and even lead to the loss of, VSV-G's ability to specifically bind to LDL-R. The result that constructing an envelope glycoprotein using VSV-G containing the K47Q or R354Q mutation in the extracellular domain, and that the viral envelope contains the first molecule, i.e., that the lentivirus, a transmembrane protein, can still infect target cells in a target molecule-specific manner, does not necessarily predict that constructing an envelope glycoprotein using VSV-G containing the K47 deletion or R354 deletion in the extracellular domain, and that the viral envelope contains the first molecule, will still possess the ability to specifically infect target cells.

[0042] When an envelope glycoprotein was constructed using VSV-G containing an R354 deletion in the extracellular region, the lentivirus, whose viral envelope contained membrane-expressed anti-CD7 antibodies, could not effectively infect CD7+ Jurkat cells. However, when an envelope glycoprotein was constructed using VSV-G containing a K47 deletion in the extracellular region, the lentivirus, whose viral envelope contained membrane-expressed anti-CD7 antibodies, could effectively infect CD7+ Jurkat cells. This further demonstrates that changes in protein function due to amino acid deletions are unpredictable.

[0043] Taking lentivirus-infected T cells as an example, the lentivirus envelope expresses mutated VSVG and CD7 antibodies. After the CD7 antibodies bind to the CD7 antigen on the T cell surface, they can mediate lentivirus endocytosis, and the mutated VSVG then completes the targeted infection of the T cells by the lentivirus by mediating lysosomal escape. The lentivirus infects only T cells that express CD7 and has no ability to infect cells that do not express CD7.

[0044] The substance supported on the targeting vector is not limited and may be selected as needed, including, for example, small molecule compounds, proteins, polypeptides, RNA, or DNA, such as chimeric antigen receptors (CARs) and TCRs. The CAR type is not limited, and the CAR includes an antigen-binding domain. In some embodiments, the antigen-binding domain is a single-stranded variable fragment (scFv) containing a heavy chain and a light chain variable region, which specifically bind to the required antigen. The scFv is selected from monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, human antibodies, nanoantibodies, and synthetic antibodies. In some embodiments, the CAR further includes a transmembrane domain (e.g., a CD8 transmembrane domain) and a signaling domain (e.g., CD3ζ) containing one or more activation motifs (ITAMs) based on immunoreceptor tyrosine. In some embodiments, the CAR includes one or more co-stimulatory domains. The CAR is not limited to the type of co-stimulatory domain. In practice, any known co-stimulatory domain in this field may be used, including but not limited to CD28, 4-1BB, DAP10, and DAP12.

[0045] Small molecule compounds are organic compound molecules with a molecular weight less than 900 daltons, and are particularly small molecules that can be used as drugs.

[0046] In one specific plan, the target cells are lymphocytes, bone marrow cells, hematopoietic stem cells / hematopoietic progenitor cells, or non-blood cells, particularly bone marrow cells, hematopoietic stem cells / hematopoietic progenitor cells, or non-blood cells, and include normal cells and tumor cells.

[0047] In one specific solution, the endocytosis receptor is an endocytosis receptor on bone marrow cells, an endocytosis receptor on hematopoietic stem cells / hematopoietic progenitor cells, or an endocytosis receptor on non-blood cells.

[0048] In one specific approach, the endocytosis receptor is not CD80, TCR, BCR, CD19, CD20, or IL-13Rα.

[0049] In one specific approach, the endocytosis receptor is not a lymphocyte-specific protein.

[0050] Lymphocyte-specific proteins are specific proteins of B cells, T cells, or NK cells. T cell-specific proteins include CD3, CD28, CD80, 4-1BB, AhR, CD2, CD7, CD4, CD8, CD25, CD44, CD45RA, CD47, CD62L, CD69, CD94, CD95, CD127, CD161, CD183 (CXCR3), and CD184 (C XCR4), CD185(CXCR5), CD193(CCR3), CD194(CCR4), CD195(CCR5), CD196(CCR6), CD197(CCR7), CCR10 , PD-1, TCRa / b, CD5, CD27, CD45RO, CD45RB, CD57, CD103, CD122, P2RX7, TIGIT, LAG-3, TIM-3, IL6ST, gd Contains TCR (TCRγ, TCRδ), Vdeltal, Vdelta2, NKG2D (KLRK1, CD314), TCR (Va24-Jal8), CD185 (CXCR5), CXCR6, IL-21R, Va7.2, Ja33, CXCR6, IL-18R, KLRB1 (CD161), and VLA4.

[0051] B cell-specific proteins include CD19, CD20, CD21, CD22, CD24, CD38, CD40, CD72, CD32b, CD268, CD269, CD267, CD86, CD80, CD52, CD138, CD27, CD28, CD23, CD84, CD257, CD270, CD37, CD74, and CD269.

[0052] NK cell-specific proteins include CD56, NKp46, CD16, KIR(s), NKG2 proteins (NKG2D, KLRK1, CD314), KLRB1 (CD161), KLRDl (CD94), IL2Rb (CD122), IL-21R, SLAMF6 (CD352), SLAMF7 (CD319), and IL-18R.

[0053] In another embodiment of the present invention, a method for producing a targeting vector is: The first step is to design the molecule based on the endocytosis receptor of the target cell, The step of selecting a second molecule, The method includes the step of assembling a first molecule, a second molecule, and a substance to be supported on a vector to produce a targeting vector.

[0054] In the first case, by designing a first molecule, that is, modifying the viral surface with an antibody / ligand capable of targeting and recognizing a specific receptor, and selecting a wild-type viral envelope protein as the second molecule, if the target cell expresses the envelope protein receptor carried by the virus, the presence of the first molecule can promote the infection of the targeting vector into the target cell.

[0055] In the second case, by designing the first molecule—that is, modifying the viral surface with an antibody / ligand capable of targeting and recognizing a specific receptor—and selecting the wild-type viral envelope as the second molecule, if the target cell has low or no expression of the envelope protein receptor carried by the virus, the virus can still complete infection of the target cell by the first molecule.

[0056] In the third case, by designing the first molecule—that is, modifying the viral surface with an antibody / ligand capable of targeting and recognizing a specific receptor—and selecting a mutant viral envelope protein as the second molecule, which has restricted binding to the receptor but possesses the ability to escape endosomes / lysosomes, the virus can target and infect target cells via the first molecule, regardless of whether the target cells express the envelope protein receptor carried by the virus. Furthermore, the virus significantly improves the accuracy of targeting without infecting other cells.

[0057] Methods for assembling vectors with and without envelopes: In the assembly of enveloped vectors, using VSVG as an example, a target expression vector can be constructed by mixing a plasmid expressing an antibody / ligand that binds to an endosomal receptor, a lentiviral packaging plasmid such as psPAX2 and pMD.2G (VSVG or a VSVG mutant), and a lentiviral expression vector.

[0058] In the case of AAV, which is an example of an envelope-less vector, the construction of an AAV targeting vector can be achieved by linking an antibody / ligand that binds to the endocytosis receptor to the AAV envelope protein via transmembrane peptide stretching.

[0059] Methods by which enveloped and non-enveloped vectors enter cells: Enveloped vectors, such as VSVG-based pseudotyped lentiviral vectors, bind to LDL-R on the cell membrane surface via VSVG and enter the cell through clathrin-mediated endocytosis. After the endosomes formed by endocytosis are acidified, the VSVG conformation changes, and the viral envelope fuses with the endosomal membrane, allowing the virus to detach from the endosome / lysosome and enter the cell nucleus through the nuclear pore.

[0060] For vectors without an envelope, recombinant AAV virus particles enter the cell via clathrin-mediated endocytosis by binding to glycation receptors expressed on the host cell surface. After the endosomes formed by endocytosis are acidified, the conformation of the VP1 / VP2 portion of the viral capsid changes, causing the virus to detach from the endosome and enter the cell nucleus through the nuclear pore.

[0061] In another embodiment of the present invention, the targeting vector may be used for drug or vaccine delivery, and in particular for the delivery of small molecule compounds, proteins, polypeptides, RNA, or DNA.

[0062] In another embodiment of the present invention, a method for introducing a substance into cells, the method comprising contacting the cells with a targeting vector.

[0063] In one specific plan, the cells are mammalian cells.

[0064] In one specific plan, the cells are either normal cells or cancer cells.

[0065] In one specific plan, the cells are T cells, NK cells, B cells, macrophages, granulocytes, dendritic cells, hematopoietic stem cells, hepatocytes, pancreatic islet cells, nerve cells, and muscle cells.

[0066] In one specific plan, the contact may be performed in vivo or in vitro, for example, by administration intravenously, intraperitoneally, intratumorally, intraosseously, or intranodulely, to introduce the targeting vector in vivo and bring it into contact with target cells, or to directly infect target cells with the target virus in vitro.

[0067] The term "mammal" refers to any mammalian species, such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, and domesticated animals.

[0068] The term "viral envelope protein" refers to naturally occurring viral envelope proteins, such as VSV-G, BaEV, and RD114. Viral envelope proteins play crucial roles in viral packaging and infection of host cells.

[0069] Another aspect of the present invention is a composition comprising a targeting vector, which may be used as a drug, which may be used in the manufacture of drugs for gene therapy, immunotherapy, cell therapy, treatment of gene deficiency diseases, treatment of autoimmune diseases, treatment of infectious diseases, and treatment of cancer, wherein cancer includes hematological cancers and solid tumors.

[0070] Another aspect of the present invention is a method for treating a disease in a subject, the method comprising administering a therapeutically effective amount of a targeting vector or composition to the subject.

[0071] The routes of administration of the drug composition are common in this art, and may include injection via, for example, oral, nasal, intravenous, intraperitoneal, intracerebral (intracerebral parenchymal), intraventricular, intramuscular, intraocular, intraarterial, portal vein, or intrafocal routes, and may also be administered via a continuous-release system or implantable device.

[0072] "Treatment" means employing the treatment methods described herein on a subject to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a reduction in the rate of cancer cell invasion into surrounding organs, or a reduction in tumor metastasis or tumor growth rate). The treatment method that effectively treats the patient may vary depending on various factors (e.g., the patient's disease state, age, weight, and the treatment method that stimulates the subject's anti-cancer response capacity).

[0073] As those skilled in the art will understand, the appropriate dose level used for treatment varies in part depending on the molecule being delivered, the indication, the route of administration, and the patient's condition (body weight, body surface or organ size) and / or circumstances (age and general health). In some embodiments, the clinician may titrate the dose or change the route of administration to obtain the optimal therapeutic effect. [Effects of the Invention]

[0074] This approach utilizes a mechanism that links endocytosis and endosomal / lysosome escape to modify the vector to incorporate a primary molecule capable of binding to the endocytosis receptor of target cells, thereby obtaining a targeting vector and exhibiting the following beneficial effects.

[0075] Based on highly efficient endocytosis receptors specific to different cell types, it is possible to rapidly develop targeted vectors for different cell types without blind screening or trial and error. By designing the first molecule based on the endocytosis receptor of the target cell, it is possible to target different types of cells, especially cells that do not express or express low levels of viral envelope protein receptors (e.g., LDL-R), which are already widely used, thus having a wide range of applications. By introducing rational mutations into the vector, it is possible to avoid infecting cells that do not require targeting, thereby significantly improving accuracy.

[0076] All publications, documents and patents referenced herein are incorporated as a whole, particularly in a manner of reference, and are the same as each individual publication, document or patent that is not specifically and independently indicated to be incorporated as a whole in a manner of reference. In the event of any conflict, this application (including any definitions herein) shall prevail. However, any references, documents, publications, patents, patent publications and patent applications referenced herein should not be considered endorsements or suggestions of any form, but rather constitute valid prior art or form part of the common public knowledge of any country worldwide.

[0077] The section headings used herein are for organizational purposes only and are not intended to be interpreted as limiting the themes described. [Brief explanation of the drawing]

[0078] [Figure 1] This is a schematic diagram of the targeting vector. [Figure 2] This is a schematic diagram of the pGClenti-GFP lentiviral vector skeleton. [Figure 3] This is a flowchart illustrating viral infection of peripheral blood NK cells in Example 1. [Figure 4] This is a schematic diagram of the VSVG mutant from Example 2. [Figure 5] This is a detection map of the P24 capsid protein of 15 pairs of lentivirals constructed using different VSVG mutants to form envelope glycoproteins. [Figure 6] This is a titer detection map of 15 pairs of lentiviruses constructed using different VSVG mutants to create envelope glycoproteins. [Figure 7] This is a flowchart illustrating how the virus from Example 3 infects CD7+ cells. [Figure 8] This is a flowchart illustrating the infection of Jurkat cells, Nalm6 cells, and THP1 cells using the targeted lentivirus A33-VSV-G-1. [Figure 9]This is a flowchart illustrating the infection of Jurkat cells and THP1 cells using the targeted lentivirus A33-VSV-G-2. [Figure 10] This is a flowchart illustrating the infection of Jurkat cells and Nalm6 cells using the targeted lentivirus A19-VSV-G-2. [Figure 11] This is a detection chart of the titers of the targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2. [Figure 12] This is a flowchart illustrating the infection of Jurkat cells, HuH-7 cells, and Hep-G2 cells using the targeted lentivirus A-ASGPR-VSV-G-2. [Figure 13] This is a flowchart illustrating the infection of Jurkat cells and MCF7 cells using the targeted lentivirus A-HER-2-VSV-G-2. [Figure 14] This is a flowchart illustrating the infection of Jurkat cells and Jurkat-MSN cells using the targeted lentivirus A-Jurkat-MSN-VSV-G-2. [Figure 15] This is a flowchart illustrating the infection of Jurkat cells, Nalm6 cells, and PBMCs using the targeted lentivirus A8-VSV-G-1. [Figure 16] This is a flowchart illustrating the infection of Jurkat cells and PBMCs using the targeted lentivirus A8-VSV-G-2. [Figure 17] These are flowcharts illustrating the infection of Jurkat cells using lentiviruses dK47-VSV-G-A7, dR354-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7, respectively. [Figure 18] These are flowcharts illustrating the infection of Nalm6 cells using lentiviruses dK47-VSV-G-A7, dR354-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7, respectively. [Modes for carrying out the invention]

[0079] Example 1 Construction of a lentivirus that targets NK cells using wild-type VSVG. Because NK cells have low expression of the VSVG receptor LDL-R, ordinary VSVG lentiviruses do not easily infect NK cells. Therefore, the inventors constructed a single membrane-expressed CD7 antibody on the lentiviral envelope and bound it to CD7 on NK cells to induce lentiviral endocytosis. Subsequently, VSVG mediates the fusion of the viral envelope and the endosomal / lysosomal membrane, causing lysosomal escape and further inducing the expression of GFP supported by the vector in the cells. The structure of the lentiviral targeting vector is shown in Figure 1, where 1 is the first molecule, the CD7 antibody, which can target the endocytosis receptor, and 2 is the second molecule, VSVG, which mediates endosomal / lysosomal escape.

[0080] 1. Design of membrane-expressed CD7 antibodies The membrane-bound CD7 antibody sequence contains, in order from the 5' to the 3' end, the CD8 signal peptide, TH-69 heavy chain (VH), GS linker peptide, TH-69 light chain (VL), CD8 hinge region, and CD8 transmembrane region, where the CD8 signal peptide amino acid sequence is SEQ ID NO:1. MALPVTALLLPLALLLHAARP

[0081] The TH-69 heavy chain (VH) amino acid sequence is SEQ ID NO:2: EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSS

[0082] The amino acid sequence of the GS linker is SEQ ID NO:3: GGGGSGGGGSGGGGS

[0083] The TH-69 light chain (VL) amino acid sequence is SEQ ID NO: 4: AAYKDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKR

[0084] The amino acid sequence of the CD8 hinge region is SEQ ID NO: 5: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0085] The amino acid sequence of the CD8 transmembrane region is SEQ ID NO:6: IYIWAPLAGTCGVLLLSLVITLYC

[0086] 2. Building a Wild-Type VSVG The VSVG wild-type extracellular domain contains the amino acid sequence shown in SEQ ID NO:7: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0087] 3. Encapsulation of lentivirus Lentiviruses expressing pGClenti-GFP (GFP fluorescent protein), as shown in the skeleton in Figure 2, were encapsulated in plasmids carrying membrane-expressed CD7 antibodies, psPAX2, and the pMD2.G (VSVG wild-type) packaging plasmid. Specifically, the four plasmids described above were mixed, transfected into 293T cells by PEI, and the supernatant was collected after 48 hours. Centrifugation yielded lentiviruses capable of targeting NK cells. When the lentiviruses were added to an NK cell culture system, they could infect the NK cells. As shown in Figure 3, the left figure shows NK cells infected with VSVG lentivirus expressing GFP, and the right figure shows NK cells infected with VSVG lentivirus carrying membrane-type CD7 antibodies expressing GFP. While normal VSVG lentiviruses do not easily infect NK cells, VSVG lentiviruses carrying membrane-type CD7 antibodies can effectively target and infect NK target cells. This is because NK cells do not express LDL-R and highly express CD7.

[0088] Example 2 Screening for VSVG mutants that lack receptor binding ability but retain endosomal / lysosome escape ability. Based on the binding site of VSVG to LDL-R, the inventors designed a series of VSVG mutants, as shown in Figure 4, where Mut represents a mutant and Δ represents a base deletion, and tested their ability to bind to the receptor and escape from endosomes / lysosomes.

[0089] The viral envelope proteins were constructed using 15 sets of VSV-G mutants shown in Figure 4, and the 15 sets of lentiviruses, each containing a membrane-expressed anti-CD7 antibody in its viral envelope, were packaged according to the lentivirus packaging method described in Example 1.

[0090] The capsid protein P24 of 15 lentiviruses obtained by packaging was tested, and the results are shown in Figure 5. The method for detecting capsid protein P24 is known to those skilled in the art.

[0091] Fifteen sets of lentiviruses obtained by packaging were used to infect CD7+ Jurkat cells, and the infectivity of each lentivirus set was tested. The results are shown in Figure 6.

[0092] As can be seen in Figure 5, all 15 pairs of lentiviruses obtained through packaging showed detection of the lentiviral capsid protein P24, proving that each pair successfully packaged a lentivirus. However, as can be seen in Figure 6, there were relatively large differences in the biological activity and titer of the 15 pairs of lentiviruses obtained through packaging.

[0093] As can be seen in Figure 6, the titer of lentiviruses that construct the viral envelope glycoprotein using the K47 deletion VSV-G mutant was significantly higher than that of lentiviruses that construct the viral envelope glycoprotein using the R354Q mutation VSV-G mutant and the K47Q mutation VSV-G mutant. In contrast, the titer of lentiviruses that construct the viral envelope glycoprotein using the R354 deletion VSV-G mutant approached 0 and had almost no infectivity.

[0094] Example 3 Construction of a lentivirus that targets Jurkat cells using mutant VSVG. Using a mutant VSVG, we were able to retain its ability to escape lysosomes, lose its ability to bind to LDL-R, and specifically target cells expressing CD7. Cells that did not express CD7 but expressed LDL-R could not be infected by the lentivirus.

[0095] 1. Design of CD7 antibodies This is the same as Example 1.

[0096] 2. Construction of a mutated VSVG The extracellular region of the VSVG mutant (where amino acid 354 is mutated to Q) contains the amino acids shown in SEQ ID NO:8: KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEQELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0097] 3. Encapsulation of lentivirus Plasmids carrying membrane-expressed CD7 antibodies, psPAX2, and VSVG mutant packaging plasmids were used to encapsulate targeted lentiviruses expressing pGClenti-GFP (GFP fluorescent protein). Specifically, the four plasmids were mixed, transfected into 293T cells by PEI, and the supernatant was collected after 48 hours. Centrifugation yielded lentiviruses capable of targeting CD7+ cells, including Jurkat cells, CD7+NK cells, and CD7+T cells. When the lentiviruses were added to the culture system of these cells, they could infect the corresponding target cells.

[0098] As shown in Figure 7, the results show that VSVG lentivirus and targeted lentivirus infected Raji cells (which do not express CD7) and Jurkat cells (which express CD7), respectively. The left column shows the results of infection with the untargeted lentiviral vector, and the right column shows the results of infection with the targeted lentiviral vector. The control group consisted of Raji cells, which do not express CD7, while the experimental group consisted of CD7+ Jurkat cells. The untargeted lentiviral vector was capable of infecting both Raji and Jurkat cells, while the targeted lentiviral vector was capable of infecting only Jurkat cells and not Raji cells. The reason why the CD7-targeting lentivirus in this embodiment cannot effectively infect Raji cells but can effectively infect only Jurkat cells is that the mutant VSVG used in the targeted lentivirus has no ability to infect cells, its targeting depends on the CD7 antibody, and can target only cells that express CD7, while the wild-type VSVG carried by the untargeted lentivirus can bind to cells that express LDL-R, regardless of whether these cells express CD7 or not.

[0099] Example 4 Construction of a lentivirus that targets CD33+ cells 1. Design of membrane-type CD33 antibody The membrane-bound CD33 antibody sequence contains, in order from the 5' to the 3' end, the CD8 signal peptide, Gemtuzumab light chain (VL), GS linker peptide, Gemtuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region.

[0100] The amino acid sequence of the Gemtuzumab light chain (VL) is SEQ ID NO:9: DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVE VKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0101] The amino acid sequence of the Gemtuzumab heavy chain (VH) is SEQ ID NO: 10: EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0102] 2. Construction of mutant VSV-G, referring to Example 3. Referring to Example 3, a mutant VSV-G was constructed, and the extracellular region of the VSV-G mutant contained the amino acid sequence shown in SEQ ID NO:8 (mutation of the 354th amino acid arginine to glutamine Q, mutant VSV-G-1) or the amino acid sequence shown in SEQ ID NO:21 (deletion of the 47th amino acid lysine K, mutant VSV-G-2).

[0103] 3. Encapsulation of lentivirus Referring to Example 3, the targeted lentiviruses A33-VSV-G-1 and A33-VSV-G-2 were packaged and used to infect CD33+ and CD33- cells, respectively.

[0104] Each is 0.5 × 10 6 Jurkat cells, Nalm6 cells, and THP1 cells were taken and resuspended in 200 μL of medium each. The medium consisted of 1640 medium (brand: Elgbio, product number: EH80809) and 10% FBS (brand: EXCELL, product number: FSP500). Lentivirus A33-VSV-G-1, which targets the CD33+ cells, was added to each cell culture system according to MOI=1. The cells were mixed uniformly and cultured in an incubator at 37°C. On Day 2, the expression status of GFP in Jurkat cells, Nalm6 cells, and THP1 cells was detected. The results are shown in Figure 8.

[0105] As can be seen in Figure 8, the targeted lentivirus A33-VSV-G-1 expresses LDL-R but was unable to effectively infect Jurkat cells and Nalm6 cells that do not express CD33, but it was able to effectively infect THP1 cells that express CD33.

[0106] Refer to the method by which the targeted lentivirus A33-VSV-G-1 infects Jurkat cells and THP1 cells, respectively, 1 × 10⁻⁶ 5 Individual Jurkat cells and THP1 cells were taken, and the targeted lentivirus A33-VSV-G-2 was added to the Jurkat cell culture system and the THP1 cell culture system, respectively, according to MOI=1. On Day 2, the expression status of GFP in each culture system was detected, and the results are shown in Figure 9.

[0107] As can be seen in Figure 9, the targeted lentivirus A33-VSV-G-2 expressed LDL-R but was unable to effectively infect Jurkat cells that did not express CD33, but it was able to effectively infect THP1 cells that expressed CD33.

[0108] In this example, the viral envelopes of the targeted lentiviruses A33-VSV-G-1 and A33-VSV-G-2, constructed using the mutant VSV-G-1 or VSV-G-2, contain membrane-expressed anti-CD33 antibodies and can specifically bind to the endocytosis receptor CD33 on the surface of CD33+ cells. These antibodies enter and infect CD33+ cells via endocytosis. Furthermore, because the viral envelope glycoprotein is constructed using the mutant VSV-G-1 or VSV-G-2, the ability of the targeted lentiviruses A33-VSV-G-1 and A33-VSV-G-2 to specifically bind to LDL-R is weakened or lost, preventing them from effectively infecting cells expressing LDL-R. This effectively improves the targetability of the targeted lentiviruses A33-VSV-G-1 and A33-VSV-G-2 in infecting CD33+ cells.

[0109] Flow cytometry antibody used for CD33 detection by flow cytometry: Product name: APC-CD33, Brand: Biolegend, Product number: #366606.

[0110] Example 5 Construction of lentiviruses that target CD19+ cells 1. Design of membrane-expressed CD19 antibodies The membrane-bound CD19 antibody sequence contains, in order from the 5' to the 3' end, the CD8 signal peptide, FMC-63 heavy chain (VH), GS linker peptide, FMC-63 light chain (VL), CD8 hinge region, and CD8 transmembrane region.

[0111] The FMC-63 heavy chain (VH) amino acid sequence is shown in SEQ ID NO:11.

[0112] The amino acid sequence of the FMC-63 light chain (VL) is shown in SEQ ID NO:12.

[0113] 2. Construction of mutant VSV-G, referring to Example 3. Referring to Example 3, a mutant VSV-G was constructed, and the extracellular region of the VSV-G mutant contained the amino acid sequence shown in SEQ ID NO:8 (mutation of the 354th amino acid arginine to glutamine Q, mutant VSV-G-1) or the amino acid sequence shown in SEQ ID NO:21 (deletion of the 47th amino acid lysine K, mutant VSV-G-2).

[0114] 3. Encapsulation of lentivirus Referring to Example 3, the target lentiviruses A19-VSV-G-1 and A19-VSV-G-2 were constructed using the mutant VSV-G-1 and mutant VSV-G-2.

[0115] Each is 1 x 10 5 Individual CD19- Jurkat cells and CD19+ Nalm6 cells were taken and resuspended in 200 μL each of medium containing 1640 (brand: Elgbio, product number: EH80809) medium and 10% FBS (brand: EXCELL, product number: FSP500), and the targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2 were added to each cell culture system according to MOI=1, targeting CD19+ cells. The cells were mixed uniformly and cultured in an incubator at 37°C for 2 days. In step 2, the expression status of GFP in the Jurkat cells and Nalm6 cells was detected. The result of the targeted lentivirus A19-VSV-G-2 infecting Jurkat cells and Nalm6 cells is shown in Figure 10, and the titer detection results for the targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2 are shown in Figure 11.

[0116] As can be seen in Figure 10, the targeted lentivirus A19-VSV-G-2 was unable to effectively infect Jurkat cells that do not express CD19, but it was able to effectively infect Nalm6 cells that express CD19.

[0117] The targeted lentiviruses A19-VSV-G-1 and A19-VSV-G-2 specifically bind to the endocytosis receptor CD19 on the surface of Nalm6 cells via anti-CD19 antibodies contained in their viral envelopes, and then infect CD19+ Nalm6 cells through endocytosis.

[0118] As can be seen from Figure 11, the titer of the targeted lentivirus A19-VSV-G-2, whose envelope glycoprotein is constructed using the mutant VSV-G-2 (K47 deletion), was significantly increased compared to the targeted lentivirus A19-VSV-G-1, whose envelope glycoprotein is constructed using the mutant VSV-G-1 (R354Q mutation).

[0119] Flow cytometry antibody used for CD19 detection by flow cytometry: Product name: APC-CD19, Brand: Sianchi, Product number: #S0098.

[0120] Example 6 Construction of a lentivirus that targets asialoglycoprotein receptor-positive (ASGPR+) hepatocytes

[0121] The targeted asialoglycoprotein receptor (ASGPR) may be N-acetylgalactosamine (GalNAc) or an ASGPR antibody. GalNAc can be added to the lentiviral envelope surface by modification, or a lentivirus that targets hepatocytes can be produced by membrane expression of an ASGPR antibody.

[0122] 1. Design of membrane-expressed ASGPR antibodies The membrane-bound CD33 antibody sequence contains, in order from the 5' to the 3' end, the CD8 signal peptide, ASGPR light chain B11 (VL), GS linker peptide, ASGPR heavy chain (VH), CD8 hinge region, and CD8 transmembrane region.

[0123] The amino acid sequence of the ASGPR antibody light chain (VL) is SEQ ID NO: 13: DIVLTQPPSASGTPGQRVTISCTGSSSGIGNAYVSWYQQLPGKAPKLLIYKNGQRPSGVSDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKVTVL The amino acid sequence of the ASGPR antibody heavy chain (VH) is SEQ ID NO: 14: MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSAITTGGGSPNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRTAGYFDYWGQGALVTVSSGSA 2. Construction of mutant VSV-G, referring to Example 3. Referencing Example 3, a mutant VSV-G-2 was constructed, and the extracellular region of the mutant VSV-G-2 contained the amino acid sequence shown in SEQ ID NO:21.

[0124] 3. Encapsulation of lentivirus Referring to Example 3, a lentivirus A-ASGPR-VSV-G-2 that is targeted using the mutant VSV-G-2 was constructed.

[0125] Each is 1 x 10 5Individual ASGPR- Jurkat cells and ASGPR+ HuH-7 and Hep-G2 cells were taken and resuspended in 200 μL of culture medium, which consisted of DMEM medium and 10% FBS (DMEM, brand: Gibco, product number: #C12430500BT; FBS, brand: Ikesai, product number: #FSP500). The targeted lentivirus A-ASGPR-VSV-G-2, which targets ASGPR+ cells, was added to each cell culture system according to MOI=1, mixed uniformly, and cultured in an incubator at 37°C. On Day 2, the expression status of GFP in Jurkat cells, HuH-7 cells, and Hep-G2 cells was detected, and the results are shown in Figure 12.

[0126] As can be seen in Figure 12, the targeted lentivirus A-ASGPR-VSV-G-2 was unable to effectively infect Jurkat cells that do not express ASGPR, but it was able to effectively infect HuH-7 cells and Hep-G2 cells that express ASGPR.

[0127] The targeted lentivirus A-ASGPR-VSV-G-2 specifically binds to the endocytosis receptor ASGPR on the surface of ASGPR+ cells via anti-ASGPR antibodies contained in its viral envelope, and then infects ASGPR+ HuH-7 and Hep-G2 cells through endocytosis.

[0128] Flow cytometry antibody used for ASGPR detection by flow cytometry: Product name: PE-ASGPR1, Brand: BD, Product number: #563655.

[0129] Example 7 Construction of a lentivirus that targets HER2+ cells 1. Design of membrane-expressed HER2 antibodies The membrane-bound HER2+ antibody sequence contains, in order from the 5' to the 3' end, the CD8 signal peptide, Pertuzumab light chain (VL), GS linker peptide, Pertuzumab heavy chain (VH), CD8 hinge region, and CD8 transmembrane region.

[0130] The amino acid sequence of the pertuzumab light chain (VL) is SEQ ID NO: 15: DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0131] The amino acid sequence of the Pertuzumab heavy chain (VH) is SEQ ID NO:16: EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHNHYTQKSLSLSPG

[0132] 2. Construction of mutant VSV-G, referring to Example 3. Referencing Example 3, a mutant VSV-G-2 was constructed, and the extracellular region of the mutant VSV-G-2 contained the amino acid sequence shown in SEQ ID NO:21.

[0133] 3. Encapsulation of lentivirus Referring to Example 3, a lentivirus A-HER-2-VSV-G-2 that is targeted using the mutant VSV-G-2 was constructed.

[0134] Each is 1 x 10 5 Individual HER-2- Jurkat cells and HER-2+ MCF7 cells were taken and resuspended in 200 μL of medium each, which consisted of DMEM medium and 10% FBS (DMEM, brand: Gibco, product number: #C12430500BT; FBS, brand: Ikesai, product number: #FSP500). The targeted lentivirus A-HER-2-VSV-G-2, which targets HER-2+ cells, was added to each cell culture system according to MOI=1, mixed uniformly, and cultured in an incubator at 37°C. On Day 2, the expression status of GFP in the Jurkat cells and MCF7 cells was detected, and the results are shown in Figure 13.

[0135] As can be seen in Figure 13, the targeted lentivirus A-HER-2-VSV-G-2 was unable to effectively infect Jurkat cells that do not express HER-2, but it was able to effectively infect MCF7 cells that express HER-2.

[0136] The targeted lentivirus A-HER-2-VSV-G-2 specifically binds to the HER-2 endocytosis receptor on the surface of MCF7 cells via anti-HER-2 antibodies contained in its viral envelope, and then infects HER-2+ MCF7 cells through endocytosis.

[0137] Flow cytometry antibody used for HER-2 detection by flow cytometry: Product name: APC-CD340(HER-2), Brand: Biolegend, Product number: #324407.

[0138] Example 8 Construction of a lentivirus that targets MESOTHELIN+ cells 1. Design of membrane-expressed mesothelin antibodies The membrane-expressed MESOTHELIN+(MSN) antibody sequence contains, in order from the 5' to the 3' end, a CD8 signal peptide, a PE38 heavy chain (VH), a GS linker peptide, a PE38 light chain (VL), a CD8 hinge region, and a CD8 transmembrane region.

[0139] The PE38 heavy chain (VH) amino acid sequence is SEQ ID NO: 17: MQVQLQQSGPELEKPGASVKISKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSGV

[0140] The PE38 light chain (VL) amino acid sequence is SEQ ID NO: 18: DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIK

[0141] 2. Construction of mutant VSV-G, referring to Example 3. Referencing Example 3, a mutant VSV-G-2 was constructed, and the extracellular region of the mutant VSV-G-2 contained the amino acid sequence shown in SEQ ID NO:21.

[0142] 3. Encapsulation of lentivirus Referring to Example 3, a lentivirus A-MSN-VSV-G-2 was constructed that is targeted using the mutant VSV-G-2.

[0143] 4. Construction of a Jurkat-MSN overexpression cell line A method for constructing a Jurkat-MSN cell line that overexpresses MSN is known to those skilled in the art, which involves using a normal lentivirus whose envelope glycoprotein is wild-type VSV-G to carry nucleic acid encoding MSN, infecting a Jurkat cell line with it, and constructing a Jurkat-MSN cell line that overexpresses MSN.

[0144] Each is 1 x 10 5 Jurkat-MSN cells (one MSN- Jurkat cell and one MSN+ cell) were taken and resuspended in 200 μL of medium each, which contained 1640 + 10% FBS (1640, brand: Elgbio, product number: #EH80809; FBS, brand: Ikesai, product number: #FSP500). The targeted lentivirus A-MSN-VSV-G-2, which targets MSN+ cells, was added to each cell culture system according to MOI=1, mixed uniformly, and cultured in an incubator at 37°C. On Day 2, the expression status of GFP in Jurkat cells and Jurkat-MSN cells was detected, and the results are shown in Figure 14.

[0145] As can be seen in Figure 14, the targeted lentivirus A-Jurkat-MSN-VSV-G-2 was unable to effectively infect Jurkat cells that do not express MSN, but it was able to effectively infect Jurkat-MSN cells that express MSN.

[0146] The targeted lentivirus A-MSN-VSV-G-2 specifically binds to the endocytosis receptor MSN on the surface of Jurkat-MSN cells via anti-MSN antibodies contained in its viral envelope, and then infects MSN+ Jurkat-MSN cells through endocytosis.

[0147] Flow cytometry antibody used for MSN detection by flow cytometry: Product name: APC-Mesothelin, Brand: R&D, Product number: #FAB32652A.

[0148] Example 9 Construction of a lentivirus that targets CD8+ cells based on a non-endocytosis receptor CD8 antibody. 1. Design of membrane-expressed CD8 antibodies The membrane-bound CD8+ antibody sequence contains, in order from the 5' to the 3' end, the CD8 signal peptide, the CD8 antibody heavy chain (VH), the GS linker peptide, the CD8 antibody light chain (VL), the CD8 hinge region, and the CD8 transmembrane region.

[0149] The amino acid sequence of the CD8 antibody heavy chain (VH) is SEQ ID NO: 19: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHFVRQAPGKGLEWIGRIDPANDNTLYASKFQGKATISADTSKNTAYLQMNSLRAEDTAVYYCGRGYGYYVFDHWGQGTLVTVSS

[0150] The amino acid sequence of the CD8 antibody light chain (VL) is SEQ ID NO: 20: DVQITQSPSSLSASVGDRVTITCRTSRSISQYLAWYQQKPGKVPKLLIYSGSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQQHNENPLTFGGGTKVEIK

[0151] 2. Construction of mutant VSV-G, referring to Example 3. Referring to Example 3, a mutant VSV-G was constructed, and the extracellular region of the VSV-G mutant contained the amino acid sequence shown in SEQ ID NO:8 (mutation of amino acid 354 to Q, mutant VSV-G-1) or the amino acid sequence shown in SEQ ID NO:21 (deletion of amino acid lysine K at position 47, mutant VSV-G-2).

[0152] 3. Encapsulation of lentivirus Referring to Example 3, the mutant VSV-G-1 or mutant VSV-G-2 was used to package a targetable lentivirus A8-VSV-G-1 or A8-VSV-G-2 in which the viral envelope contains a membrane-expressed anti-CD8 antibody.

[0153] Each is 1 x 10 5 Individual Jurkat cells, Nalm6 cells, and PBMCs were isolated and resuspended in 200 μL of culture medium. The medium for resuspending the Jurkat cells and Nalm6 cells consists of 1640 medium (brand: Elgbio, product number: EH80809) and 10% FBS (brand: EXCELL, product number: FSP500), and the medium for resuspending the PBMCs consists of XVT medium (product name PRIME-XV T cell CDM, brand: IRVINE (FUJIFILM), product number: 91154), IL-7 (product name: IL-7 Protein, Human, Recombinant, brand: Yiqiao Shenzhou, product number: 11821-HNAE) with a final concentration of 20 ng / mL and IL-15 (product name: IL-15 Protein, Human, Recombinant (His Tag), brand: Yiqiao Shenzhou, product number: 10360-H07E) with a final concentration of 20 ng / mL.

[0154] According to MOI=1, the targeted lentivirus A8-VSV-G-1 was added to the Jukart cells, Nalm6 cells, and PBMCs cell culture systems, respectively, and mixed uniformly. The cultures were then incubated at 37°C, and on Day 2, the expression status of GFP in the Jukart cells, Nalm6 cells, and PBMCs was detected. The results are shown in Figure 15.

[0155] As can be seen in Figure 15, the targeted lentivirus A8-VSV-G-1, constructed using the mutant VSV-G-1, expresses LDL-R but was unable to effectively infect Jurkat cells and Nalm6 cells that do not express CD8, nor was it able to effectively infect PBMCs that express CD8.

[0156] Each is 1 x 10 5 Individual Jurkat cells and PBMCs were taken, and referring to the method described above for uniformly mixing the targeted lentivirus A8-VSV-G-1 with each set of cells and culturing them, the targeted lentivirus A8-VSV-G-2 was added to the Jurkat cell culture system and the PBMC culture system, respectively, according to MOI=1. On Day 2, the expression status of GFP in the Jurkat cells and PBMCs was detected, and the results are shown in Figure 16.

[0157] As can be seen from Figure 16, the targeted lentivirus A8-VSV-G-2 expresses LDL-R but was unable to effectively infect Jurakt cells that do not express CD8, nor was it able to effectively infect PBMCs that express CD8.

[0158] After CD8+ cells came into contact with the targeted lentivirus A8-VSV-G-1 or A8-VSV-G-2, the membrane-expressed anti-CD8 antibody contained in the viral envelope of the targeted lentivirus A8-VSV-G-1 or A8-VSV-G-2 specifically bound to the CD8 receptor on the surface of CD8+ cells in PBMCs. However, because CD8 is a non-endocytosis receptor and cannot efficiently mediate endocytosis, the targeted lentivirus A8-VSV-G-1 or A8-VSV-G-2 was unable to effectively enter and infect the CD8+ cells.

[0159] The results showed that after contact between CD8+ cells and lentivirus, the CD8 receptor was able to bind to the antibody, but effective endocytosis was not possible, and the membrane-expressed CD8 antibody-based lentiviral vector could not effectively mediate infection of CD8+ cells. Therefore, receptors lacking efficient endocytosis capability were not suitable as the first molecule for constructing the targeting vector of the present invention.

[0160] Flow cytometry antibody used for CD8 detection by flow cytometry: APC-CD8, Brand: BD, Product Number: 566852.

[0161] Example 10 Comparison of lentiviral infection efficiency in VSV-G cells containing K47 deletion or R354 deletion in the extracellular domain.

[0162] An envelope plasmid (K47 deletion-A7 envelope plasmid) carrying the nucleic acid encoding the membrane-expressed anti-CD7 antibody described in Example 1 and the nucleic acid encoding VSV-G with a K47 deletion in the extracellular region, a pMDLg / pRRE packaging plasmid, a pRSV-REV packaging plasmid, and a lentiviral GFP plasmid were prepared. The K47 deletion-A7 envelope plasmid was synthesized by a standard molecular cloning method. Lentivirus dK47-VSV-G-A7 is packaged and manufactured by referring to the packaging method for manufacturing lentiviruses described in Example 3. Of the four plasmids mentioned above, the envelope plasmid containing a K47 deletion in the VSV-G extracellular region is replaced with an envelope plasmid containing an R354 deletion, K47Q, or R354Q mutation in the VSV-G extracellular region. The lentivirus dR354-VSV-G-A7, sK47Q-VSV-G-A7, or sR354Q-VSV-G-A7, containing an R354 deletion, K47Q, or R354Q mutation in the VSV-G extracellular region, is then packaged according to the packaging method for the lentiviral vector dK47-VSV-G-A7. The VSV-G extracellular region containing the K47 deletion in the extracellular region includes the amino acid sequence shown in SEQ ID NO:21. The VSV-G extracellular region containing the R354 deletion in the extracellular region includes the amino acid sequence shown in SEQ ID NO:22. The VSV-G extracellular region containing the K47Q mutation in the extracellular region includes the amino acid sequence shown in SEQ ID NO:23. The VSV-G extracellular region containing the R354Q mutation in the extracellular region includes the amino acid sequence shown in SEQ ID NO:8.

[0163] According to MOI=1, each of the four types of lentiviruses was added to four sets of CD7+ Jurkat cells, mixed uniformly, and infected at room temperature for 10 minutes. Then, 10 mL of DPBS buffer was added and mixed uniformly, followed by centrifugation at 500 g for 3 minutes, the supernatant was aspirated and discarded, and 1 mL of 1640 medium containing 10% FBS (brand: ELGBIO, product number: #EH80809, FBS serum: brand: EXCELL, product number: #FSP500) was added. Each of the four sets of CD7+ Jurkat cells was resuspended, and in vitro culture was performed in an incubator at 37°C with a CO2 concentration of 5%. GFP expression was detected on Day 2, and the results are shown in Figure 17.

[0164] As can be seen from Figure 17, lentiviruses dK47-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7, which contain a K47 deletion or R354Q or K47Q mutation in the VSV-G extracellular domain, can specifically bind to the endocytosis receptor CD7 expressed by Jurkat cells and can effectively infect CD7+ Jurkat cells through endocytosis. On the other hand, lentivirus dR354-VSV-G-A7, which contains an R354 deletion in the VSV-G extracellular domain, is less likely to infect CD7+ Jurkat cells through endocytosis. This indicates that, unlike the K47 deletion, an unpredictable and unknown change occurred in VSV-G function after the R354 deletion in the extracellular domain, and even though its viral envelope contained membrane-expressed anti-CD7 antibodies, the lentivirus dR354-VSV-G-A7 still lost the ability to infect CD7+ cells.

[0165] Referring to the above-described virus infection and in vitro culture methods, the four types of lentiviruses dK47-VSV-G-A7, sK47Q-VSV-G-A7, sR354Q-VSV-G-A7, and dR354-VSV-G-A7 were each added to four sets of CD7- Nalm6 cells according to MOI=1. In vitro cell culture was performed in an incubator at 37°C with a CO2 concentration of 5%, and the expression status of GFP was detected on Day 2. The results are shown in Figure 18.

[0166] As can be seen from Figure 18, none of the lentiviruses dK47-VSV-G-A7, dR354-VSV-G-A7, sK47Q-VSV-G-A7, and sR354Q-VSV-G-A7 were able to infect CD7- Nalm6 cells. This indicates that the inclusion of a K47 deletion in the VSV-G extracellular domain does not affect the targeting ability of the lentivirus dK47-VSV-G-A7 to infect CD7+ cells.

[0167] As described above, these are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any modification or substitution that any person skilled in the art can easily conceive of within the scope of the art revealed by the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the claims.

Claims

1. It is a targeting vector, It comprises a first molecule that binds to the endocytosis receptor of the target cell and a second molecule that promotes the release of the substance carried by the targeting vector into the cytoplasm. The targeting vector is a lentiviral vector. The first molecule comprises a transmembrane peptide stretch, an antibody or ligand that binds to the endocytosis receptor of a target cell, The second molecule is a viral envelope protein, which is selected from variants of the bullous stomatitis virus envelope glycoprotein VSVG, and the variants of the bullous stomatitis virus envelope glycoprotein VSVG include one or more combinations of mutations at the K47 deletion, amino acid deletions between positions 321 and 374, amino acid deletions between positions 331 and 364, and amino acid deletions between positions 344 and 354, based on SEQ ID NO: 7, as a targeting vector.

2. The targeting vector according to claim 1, characterized in that the second molecule promotes the occurrence of endosomal escape or lysosomal escape in the targeting vector.

3. The targeting vector according to claim 1, characterized in that the first molecule further comprises an extracellular hinge region.

4. The endocytosis receptors mentioned above include HER2, CD20, CD19, CD79A, CD79B, CD56, CD22, CD138, CD37, CD98, CD309, CD33, CD163, CD163B, CD5, CD7, 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, endotherin receptors, ETB, TN-C, and Collagen. The targeting vector according to claim 1, characterized in that it is selected from 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, and ASGPR.

5. The targeting vector according to claim 1, characterized in that the antibody that binds to the endocytosis receptor of the target cell is a CD7 antibody, CD19 antibody, CD33 antibody, ASGRP antibody or ligand, Mesothelin antibody, or HER2 antibody.

6. The targeting vector according to claim 1, characterized in that the substance is at least one of a small molecule compound, a protein, a polypeptide, RNA, and DNA.

7. The targeting vector according to claim 1, characterized in that the target cells are lymphocytes, bone marrow cells, hematopoietic stem cells / hematopoietic progenitor cells, or non-blood cells.

8. The targeting vector according to claim 1, characterized in that the endocytosis receptor is not a lymphocyte-specific protein.

9. A method for manufacturing a targeting vector according to any one of claims 1 to 8, The first step is to design the molecule based on the endocytosis receptor of the target cell, The step of selecting a second molecule, The process includes the step of assembling a first molecule, a second molecule, and a substance to be supported on a vector to produce a targeting vector, The targeting vector is a lentiviral vector. The first molecule comprises a transmembrane peptide stretch, an antibody or ligand that binds to the endocytosis receptor of a target cell, The method wherein the second molecule is a viral envelope protein, the second molecule is selected from a variant of the bullous stomatitis virus envelope glycoprotein VSVG, the variant of the bullous stomatitis virus envelope glycoprotein VSVG comprising one or more combinations of mutations at the K47 deletion, amino acid deletions between positions 321 and 374, amino acid deletions between positions 331 and 364, and amino acid deletions between positions 344 and 354, based on SEQ ID NO:

7.

10. Use of the targeting vector according to any one of claims 1 to 8 in the manufacture of a drug or vaccine.

11. A targeting vector according to any one of claims 1 to 8, used for the delivery of small molecule compounds, proteins, polypeptides, RNA, or DNA.

12. A composition comprising a targeting vector according to any one of claims 1 to 8.

13. The composition according to claim 12, which is used as a drug.

14. The composition according to claim 13, used in gene therapy.

15. The composition according to claim 13, used in immunotherapy.

16. The composition according to claim 13, used in cell therapy.

17. The composition according to claim 13, used for the treatment of a gene deficiency disorder.

18. The composition according to claim 13, used for the treatment of an autoimmune disease.

19. The composition according to claim 13, used for the treatment of an infectious disease.

20. The composition according to claim 13, used for the treatment of cancer, wherein the cancer includes blood cancer and solid tumors.

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