Exosome capable of simultaneously expressing antigen targeting antibody and VSVG as well as preparation method and application of exosome

Abstract

The invention belongs to the technical field of biological medicine, and discloses an exosome capable of simultaneously expressing an antigen-targeted antibody and VSVG (Vascular Scalable Vector Graphics) as well as a preparation method and application thereof, and the surface of the exosome expresses a fusion protein of the antigen-targeted antibody and VSVG. The exosome capable of simultaneously expressing the antigen targeting antibody and the VSVG is obtained, the targeting property of the exosome is realized, and the membrane fusion capability of the exosome is enhanced, so that the exosome uptake efficiency of target cells is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an exosome that simultaneously expresses an antigen-targeting antibody and a VSVG, its preparation method, and its application. Background Technology

[0002] Exosomes are cellularly secreted, spherical, lipid bilayer vesicles with a diameter of approximately 30-100 nm. As endogenous nanocarriers, exosomes play a crucial role in mediating intercellular communication. The exosome membrane is characterized by a phospholipid bilayer and abundant transmembrane tetraprotein CD9, which promotes direct fusion of the membrane with target cells and facilitates the cellular delivery of therapeutic agents. Another advantage of exosomes is that, due to their biologically derived membranes, which are inert to protein corona formation, endogenously derived exosomes are expected to exhibit significantly lower immunogenicity and higher biocompatibility compared to viruses and synthetic nanocarriers, which often exhibit high immunoreactivity. Given these unique and pharmacologically important properties, various methods have been developed for loading exosomes with various types of endogenous and exogenous drugs for therapeutic delivery.

[0003] However, natural exosomes lack tissue or organ targeting. To achieve site-specific drug delivery via exosomes, the exosome surface needs to be modified to attach targeting ligands and reach the intended site of action. Currently, exosome surface modification methods mainly include genetic engineering, covalent modification (chemical and metabolic engineering of the exosome parent cell), and non-covalent modification (multivalent electrostatic interactions, ligand-receptor interactions, hydrophobic interactions, aptamer-based modification, and CP05 peptide anchoring modification). Using chemical modifications other than genetic engineering to endow exosomes with targeting properties may have adverse effects due to the complexity of the exosome surface, such as reduced reaction efficiency, decreased control over specific sites, damage to the structure and function of the vector, or increased exosome toxicity.

[0004] Furthermore, vesicular stomatitis virus (VSV) is a model virus of the Rhabdoviridae family and is widely used in vaccine vectors and gene therapy. When VSV infects target cells, its G glycoprotein structure induces endocytosis, facilitating viral invasion. The G glycoprotein can mediate the fusion of the viral envelope with the cell membrane. The lipid bilayer membrane of exosomes can spontaneously fuse with other types of membrane structures. In one study, exosomes carrying the vasculostomy virus G protein (VSVG) directly delivered the membrane protein to target cells, thus providing a new tool for membrane protein therapy. .

[0005] Currently, there are no targeted exosomes designed to enhance the efficiency of exosome uptake. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a recombinant expression vector that simultaneously expresses an antigen-targeting antibody and a VSVG, and to further prepare exosomes that simultaneously express an antigen-targeting antibody and a VSVG and promote membrane fusion for application.

[0007] Therefore, in one aspect, the present invention provides a targeted exosome that simultaneously expresses an antigen-targeting antibody and a VSVG on its surface, i.e., a fusion protein expressing an antigen-targeting antibody and a VSVG. In embodiments of the present invention, the fusion protein of the antigen-targeting antibody and the VSVG comprises the following elements:

[0008] 1) Signal peptide region; 2) Histidine tag and Flag tag; 3) Single-chain antibody region targeting antigen; 4) Dimerization sequence 1; 5) Transmembrane region; 6) Fluorescent protein; 7) 2A sequence; 8) VSVG sequence; 9) Flexible linker; and 10) Dimerization sequence 2.

[0009] In embodiments of the present invention, the signal peptide can be a signal peptide of any mammalian cell surface protein, such as human albumin signal peptide, human insulin signal peptide, or mouse IgG kappa signal peptide. Preferably, the signal peptide is mouse IgG kappa signal peptide, and its amino acid sequence is shown in SEQ ID NO: 1.

[0010] In embodiments of the present invention, the positions of the histidine tag (His-tag) and the flag tag are interchangeable. In a specific embodiment, the amino acid sequences of the histidine tag (His-tag) and the flag tag are as shown in SEQ ID NO: 2.

[0011] In embodiments of the present invention, the single-chain antibody region targeting the antigen can be any single-chain antibody targeting a cell surface protein. In a specific embodiment, the single-chain antibody region targeting the antigen is an antibody targeting Her2 on the surface of breast cancer cells, and its amino acid sequence is shown in SEQ ID NO: 3.

[0012] In an embodiment of the present invention, the amino acid sequence of dimerization sequence 1 is shown in SEQ ID NO: 4; and the amino acid sequence of dimerization sequence 2 is shown in SEQ ID NO: 10.

[0013] In embodiments of the present invention, any fluorescent protein can be selected according to experimental needs. In a specific embodiment, the amino acid sequence of the fluorescent protein is shown in SEQ ID NO: 6.

[0014] In an embodiment of the present invention, the transmembrane region may be the transmembrane region of human CD8, human CD28, human IL-15R, or human PDGFR. Preferably, the transmembrane region is the transmembrane region of human PDGFR, and its amino acid sequence is shown in SEQ ID NO: 5.

[0015] In an embodiment of the present invention, the amino acid sequence of the 2A sequence is shown in SEQ ID NO: 7.

[0016] In an embodiment of the present invention, the amino acid sequence of the VSVG sequence is shown in SEQ ID NO: 8.

[0017] In an embodiment of the present invention, the amino acid sequence of the flexible linker is shown in SEQ ID NO: 9.

[0018] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned fusion protein, the fusion protein comprising an antigen-targeting antibody and a VSVG.

[0019] In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule encoding the aforementioned fusion protein.

[0020] In a fourth aspect, the present invention provides a recombinant lentivirus comprising the nucleic acid molecule of the second aspect or prepared by an expression vector of the third aspect.

[0021] In a fifth aspect, the present invention provides a method for preparing the recombinant lentivirus described in the fourth aspect, comprising co-transfecting mammalian host cells with the expression vector and helper plasmid of the third aspect to obtain the recombinant lentivirus.

[0022] In the implementation of the fifth aspect, the helper plasmid can be pMDLG-pRRE-Kana, pMD2G-Kana, or pRSV-REV-Kana.

[0023] In the fifth aspect of the implementation scheme, the mammalian host cell can be, but is not limited to, HEK293 cells, human PER.C6 cells, human HeLa cells, and mouse CHO cells, with HEK293 cells being preferred.

[0024] In a sixth aspect, the present invention provides cells capable of secreting the aforementioned targeted exosomes.

[0025] In a seventh aspect, the present invention provides a method for preparing the cells described in the sixth aspect, comprising transfecting host cells with a recombinant lentivirus as described in the fifth aspect.

[0026] In an embodiment of the seventh aspect of the present invention, the host cell may be HEK293, T cells, mesenchymal stem cells, etc.

[0027] In a specific implementation of the seventh aspect, the method for preparing the cells described in the sixth aspect specifically includes the following steps:

[0028] (1) Construct a recombinant lentiviral plasmid carrying a nucleic acid molecule encoding the second aspect;

[0029] (2) Use recombinant lentiviral plasmids and helper plasmids to transfect host cells and prepare recombinant lentiviruses that can infect cells;

[0030] (3) Transfect the recombinant lentivirus obtained in step (2) into cells to produce cells that can secrete exosomes that simultaneously express antigen-targeting antibodies and VSVG.

[0031] In specific implementation schemes, the helper plasmids are pMDLG-pRRE-Kana, pMD2G-Kana, and pRSV-REV-Kana.

[0032] In an eighth aspect, the present invention provides a method for preparing the targeted exosomes described in the first aspect, comprising the following steps:

[0033] (1) Culturing and isolating the cells described in aspect six;

[0034] (2) Collect the culture supernatant of cells that can secrete the above-mentioned targeted exosomes, and centrifuge and filter to remove impurities;

[0035] (3) The exosomes expressing both antigen-targeting antibody and VSVG were purified and concentrated using a hollow fiber column.

[0036] In a ninth aspect, the present invention provides a drug delivery tool comprising the targeted exosomes described in the first aspect, the nucleic acid molecules described in the second aspect, the expression vector described in the third aspect, and the cells described in the sixth aspect.

[0037] In a tenth aspect, the present invention provides the above-described targeted exosomes, nucleic acid molecules, expression vectors, recombinant lentiviruses, or their use as delivery tools in the preparation of therapeutic drugs for diseases.

[0038] In embodiments of the present invention, a disease refers to a disease characterized by high expression of Her2 protein on the cell surface. In specific embodiments, the disease can be breast cancer, liver cancer, etc.

[0039] In embodiments of the present invention, Her2-scFv-VSVG represents a fusion protein of Her2 targeting antibody and VSVG, Her2-scFv-VSVG-293F cell represents a cell that can secrete exosomes that simultaneously express Her2 targeting antibody and VSVG, and Her2-scFv-VSVG exosome represents an exosome that simultaneously expresses Her2 targeting antibody and VSVG.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The inventors of this invention designed a transmembrane protein vector that simultaneously expresses an antigen-targeting antibody and a VSVG (Vacuum-Induced Static Vosome). This vector is then introduced into cells to produce targeted exosomes with enhanced uptake efficiency, thereby improving the application of exosomes as drug delivery vehicles. This invention offers high flexibility; by introducing an antigen-targeting antibody and a VSVG into cells via a transmembrane protein expression vector, exosomes simultaneously expressing both antigen-targeting antibodies and VSVG can be obtained, achieving targeted exosome delivery, enhancing exosome membrane fusion capabilities, and improving the uptake efficiency of exosomes by target cells. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the carrier structure constructed in this implementation plan.

[0043] Figure 2 This is a schematic diagram of exosomes that simultaneously express antigen-targeting antibodies and VSVG in this implementation scheme.

[0044] Figure 3 This is a graph showing the number and particle size of Her2-scFv-VSVG exosomes in this implementation scheme.

[0045] Figure 4 This is a comparison diagram of SKBR3 cells taking up different exosomes in this implementation plan. Detailed Implementation

[0046] The preferred embodiments of the present invention will now be described in detail so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0047] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2012), or as recommended by the manufacturer's instructions.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0049] Example 1: Design, Construction and Preparation of Antigen-Targeting Antibodies and VSVG Plasmids

[0050] In this embodiment, the plasmid encoding the antigen-targeting antibody and VSVG was synthesized by Nanjing Baode Biotechnology Co., Ltd. The antigen-targeting antibody is an antibody targeting HER2 on the surface of breast cancer cells (Her2-scFv). The plasmid contains, from the 5' to the 3' direction of the gene structure, nucleic acid sequences encoding the following elements: signal peptide (SEQ ID NO: 1), histidine tag (His-tag) and Flag tag (SEQ ID NO: 2), single-chain antibody region targeting the antigen (SEQ ID NO: 3), dimerization sequence 1 (SEQ ID NO: 4), transmembrane region (SEQ ID NO: 5), fluorescent protein (SEQ ID NO: 6), 2A sequence (SEQ ID NO: 7), VSVG sequence (SEQ ID NO: 8), flexible linker (SEQ ID NO: 9), and dimerization sequence 2 (SEQ ID NO: 10). The plasmid was cloned into the lentiviral master plasmid pMSGV-IRES (Shanghai Newp Biotechnology Co., Ltd., catalog number: V000092) to obtain a plasmid expressing Her2-scFv-VSVG, named pMSGV-Her2-scFv-VSVG, which serves as the master plasmid in lentiviral packaging. The structure of pMSGV-Her2-scFv-VSVG is as follows. Figure 1 As shown.

[0051] To verify the differences in exosome membrane fusion capabilities, we modified the pMSGV-Her2-scFv-VSVG plasmid to obtain the pMSGV-Her2-scFv and pMSGV-NC plasmids, both of which were synthesized by Nanjing Bayode Biotechnology Co., Ltd.

[0052] The pMSGV-Her2-scFv plasmid contains, from 5' to 3', nucleic acid sequences encoding the following elements: signal peptide (SEQ ID NO: 1), histidine tag (His-tag) and flag tag (SEQ ID NO: 2), single-chain antibody region targeting the antigen (SEQ ID NO: 3), transmembrane region (SEQ ID NO: 5), and fluorescent protein (SEQ ID NO: 6). This plasmid was cloned into the lentiviral master plasmid pMSGV-IRES to obtain a plasmid expressing Her2-scFv, named pMSGV-Her2-scFv.

[0053] The pMSGV-NC plasmid contains, from 5' to 3' in the gene structure, nucleic acid sequences encoding the following elements: signal peptide (SEQ ID NO: 1), histidine tag (His-tag) and flag tag (SEQ ID NO: 2), transmembrane region (SEQ ID NO: 5), and fluorescent protein (SEQ ID NO: 6). This plasmid was cloned into the lentiviral master plasmid pMSGV-IRES to obtain a plasmid expressing only the His-tag, flag tag, and fluorescent protein; this plasmid is named pMSGV-NC.

[0054] Example 2: Preparation of recombinant lentivirus

[0055] The methods for preparing recombinant lentiviruses using pMSGV-Her2-scFv-VSVG, pMSGV-Her2-scFv, or pMSGV-NC are exactly the same. The following text uses pMSGV-Her2-scFv-VSVG as an example to illustrate the process of preparing recombinant lentiviruses.

[0056] Recombinant lentivirus capable of infecting host cells can be prepared by co-transfecting the WayneLVPro HEK293 cell line (Cell Bank of Type Culture Collection, Chinese Academy of Sciences, catalog number: SCSP-5207, hereinafter referred to as 293F cells) with the master plasmid pMSGV-Her2-scFv-VSVG and three helper plasmids pMDLG-pRRE-Kana (Changsha Aibiwei Biotechnology Co., Ltd., catalog number: HG-VMA0374), pMD2G-Kana (Changsha Aibiwei Biotechnology Co., Ltd., catalog number: HG-VMA0648), and pRSV-REV-Kana (Changsha Aibiwei Biotechnology Co., Ltd., catalog number: HG-VMA0370). The specific operation steps are as follows:

[0057] 1. Adjust the density of 293F cells to 0.5 × 10⁻⁶. 6 Cells / mL were cultured in SMM 293-TII Expression Medium (Science & Technology Co., Ltd., catalog number: M293TII) at 37 ℃, 5% CO2, and 160 rpm for 48 h, and counted at 4 × 10⁶ cells / mL. 6 per mL.

[0058] 2. Take 144 µL of PEI (Merck Biotech Ltd., 937762) with a concentration of 1 mg / mL, add it to 1 mL of opti-MEM (Thermo Fisher Scientific Ltd., catalog number: 11058021), mix well, and let stand at room temperature for 20 min.

[0059] 3. Add 16 µg of main plasmid pMSGV-Her2-scFv-VSVG and 12 µg of pMDLG-pRRE-Kana, 4 µg of pMD2G-Kana and 4 µg of pRSV-REV-Kana auxiliary plasmids to 1 mL of SMM 293-TII Expression Medium and mix well.

[0060] 4. Add the mixed solution of PEI and 1 mL opti-MEM from step 2 to the solution from step 3, mix well, and let stand at room temperature for 20 min.

[0061] 5. Take the mixture prepared in step 4 and add it to the cells prepared in step 1, then mix well.

[0062] 6. After 6-8 hours, add 2% LV Feed 04 (Zhongshan Kangsheng Biotechnology Co., Ltd., product number: QRD003) to replenish the feed.

[0063] 7. After continuous culture for 48 hours, collect the supernatant containing lentivirus from the culture dish, filter it through a 0.22 μm filter cup, incubate at 4℃ for 2 hours, transfer it to a centrifuge bottle, balance it, and centrifuge at 20000 g at 4℃ for 2 hours. After centrifugation, carefully aspirate the liquid from the centrifuge tube in a biosafety cabinet, add 120 μL of PBS buffer to resuspend the precipitate, and obtain the recombinant lentivirus. Store it at -80℃.

[0064] Example 3: Lentiviral titer detection

[0065] Jurkat cells (ATCC, catalog number: MD130) that have been revived and cultured for 2 passages were centrifuged at 300 g for 10 min and resuspended in RPMI 1640 medium (Stenofan Biotechnology (Hangzhou) Co., Ltd., catalog number: SH30809-01) to a concentration of 2×10⁻⁶. 6 Cells / mL were seeded into 48-well plates, with 0.1 mL seeded per well. 10 μL of the lentivirus obtained in Example 2 was diluted with 100 μL of RPMI 1640 medium. 10 μL of the diluted lentivirus was added to the seeded cells and mixed thoroughly. After 6 h, 800 µL of RPMI 1640 + 10% FBS was added for complete culture. The cells were cultured in a 37°C, 5% CO2 cell culture incubator. Flow cytometry was performed after 48 h.

[0066] Example 4: Preparation of Her2-scFv-VSVG-293F cells

[0067] HEK-293 cell-derived exosomes exhibit extremely low immunogenicity and demonstrate excellent preclinical safety in both in vitro and in vivo tests. 293F cells are derived from human embryonic kidney cells HEK-293, therefore 293F cells were used as the host cells.

[0068] 1. Calculate the required viral load based on MOI=3. The formula is as follows: Required viral load (mL) = (MOI * number of cells) / viral titer.

[0069] 2. Virus particles were transfected into 293F cells (Cell Bank of the Chinese Academy of Sciences: Catalog No.: SCSP-5207) to obtain Her2-scFv-VSVG-293F cells.

[0070] 3. Take 1×10 6 Her2-scFv-VSVG-293F cells were incubated once for 30 min with anti-Flag antibody (Beyotime Biotechnology Co., Ltd., catalog number: AG8050); then incubated a second time for 20 min with FITC-labeled goat anti-rabbit IgG (H+L) (Beyotime Biotechnology Co., Ltd., catalog number: A0562); the cells were then ready for detection.

[0071] 4. Take 1×10 6 Her2-scFv-VSVG-293F cells were incubated for 20 min with FITC-labeled anti-His antibody (Wuhan Sanying Biotechnology Co., Ltd., catalog number: FITC-66008) and then tested.

[0072] 5. The positivity rates of Flag and His in Her2-scFv-VSVG-293F cells were detected by flow cytometry to measure the positivity rates of Her2-scFv and VSVG proteins.

[0073] Flow cytometry results showed that 99% of Her2-scFv-VSVG-293F cells expressed Her2-scFv-VSVG.

[0074] Example 5: Pretreatment of Her2-scFv-VSVG exosomes

[0075] 1. Her2-scFv-VSVG-293F cells were suspended in serum-free medium, specifically a 1:1 mixture of SMM 293-TII Expression Medium (Science & Technology Co., Ltd., catalog number: M293TII) and OPM-293 CD05 Medium (Shanghai Aopomai Biotechnology Co., Ltd., catalog number: 81075-001). The cells were cultured at 37°C and 5% CO2 with shaking at 160 rpm. The serum-free medium was replaced every 1-2 days. The replaced medium, containing Her2-scFv-VSVG exosomes, was collected.

[0076] 2. Centrifuge 12000 g of the culture medium collected in step 1 at 4℃ for 30 min, take the supernatant, and filter the supernatant through a 0.22 μm filter; it can be stored at 4℃, -20℃, or -80℃ as needed.

[0077] Example 6: Purification and concentration of Her2-scFv-VSVG exosomes.

[0078] In this embodiment, Her2-scFv-CD9-VSVG exosomes were prepared, purified, and concentrated using a hollow fiber column tangential flow filtration system. The hollow fiber pore size in the hollow fiber column is 300 kD. The hollow fiber module generates less shear force, which helps protect the integrity of the exosomes. Secondly, the hollow fiber column can efficiently concentrate large volumes of fermentation supernatant, facilitating subsequent steps such as anion exchange chromatography. Furthermore, the pore size of the hollow fiber column effectively filters out most impurities, such as cell debris, proteins, and nucleic acids, while retaining the exosomes, achieving efficient separation and concentration of exosomes. More importantly, compared to other concentration techniques, the hollow fiber column is simple to operate, easy to control and maintain, providing convenience for experimental operations.

[0079] 1. Assemble the hollow fiber column and its inlet and return pipes (Hangzhou Kebaite Filter Material Co., Ltd., item number: HFEMI03000530P), flush the entire circuit with water for injection for 3-5 minutes, and keep the pressure at the inlet end below 5 psi.

[0080] 2. Rinse the circuit with 0.5 M NaOH for 30 min, adjusting the flow rate to maintain a shear force of 6000 s. -1 Maintain the pressure at the reflux and injection ends below 5 psi; adjust the pressure to make the flow rates at the permeate and reflux ends basically the same; after rinsing, drain the NaOH from the system.

[0081] 3. Flush the entire circuit with water for injection until the pH is neutral, and keep the injection pressure below 5 psi.

[0082] 4. Take 4-6 times the dead volume of PBS for rinsing, and drain the PBS from the system;

[0083] 5. Close the permeation end, place the injection tube and reflux tube into the injection bottle, and add 1L of the culture supernatant containing Her2-scFv-VSVG exosomes obtained in Example 4 to the injection bottle; adjust the injection end pressure to less than 5 psi and adjust the flow rate to maintain a shear force of less than 6000 s. -1 Record the flow rate at the permeate end; after concentrating to 15 mL, close the permeate end and circulate the sample back into the system for 3-5 min to degelatinate; empty the system and connect the concentrated sample to the reflux tube.

[0084] 6. Close the permeate end and wash the tubing with 1-2 dead volumes of PBS for 3-5 min; drain the system, connect the reflux end to the sample, and mix with the concentrated sample from step 5 to obtain Her2-scFv-VSVG exosomes (Her2-scFv-VSVG exo). A schematic diagram of exosomes simultaneously expressing the antibody targeting the antibody and VSVG is shown below. Figure 2 As shown.

[0085] Example 7: Targeting detection of Her2-scFv-VSVG exosomes

[0086] 100 μL of Her2-scFv-VSVG exosomes were sent to Guangzhou Ruibei Medical Technology Co., Ltd. for analysis of the number and particle size of Her2-scFv-CD9-VSVG exosomes using nanoflow cytometry. Figure 3 .

[0087] Figure 3 The results showed that Her2-scFv-VSVG exosomes secreted by 293F cells accounted for only 6.4% of the total vesicles, and the particle size was between 50 nm and 100 nm.

[0088] To verify the differences in exosome membrane fusion capabilities, we prepared Her2-scFv exosomes (Her2-scFv exo) and NC exosomes (NC exo) based on pMSGV-Her2-scFv and pMSGV-NC from Example 1, following the specific operating steps in Examples 2-6. The Her2-scFv exosome membrane was modified with Her2-scFv, His-tag, Flag tag, and fluorescent protein; the NC exosome membrane was modified only with His-tag, Flag tag, and fluorescent protein.

[0089] Take 10 each 8 Particles Flag positive exosomes, namely Her2-scFv-VSVG exosomes, Her2-scFv exosomes, and NC exosomes, 10 each.8 Particles were added to SKBR3 cells (Cell Bank of the Chinese Academy of Sciences, catalog number: TCHU225) and incubated for 2 hours. Then, the cells were incubated with Flag-FTIC antibody (Wuhan Sanying Biotechnology Co., Ltd., catalog number: FITC-66008) for 30 minutes. The cells were then observed under a fluorescence microscope, and the number of positive cells (green fluorescence) was counted. See [link to table]. Figure 4 .

[0090] Figure 4 The results showed that Her2-scFv-VSVG exosomes were taken up by SKBR3 cells faster than NC exosomes and Her2-scFv exosomes. After co-incubation for 2 hours, the number of targeted exosome-positive cells in the field of view increased significantly, indicating that Her2-scFv-VSVG exosomes exhibited stronger Her2-positive cell targeting and membrane fusion ability in vitro.

[0091] Although the proportion of Her2-scFv-VSVG exosomes among total vesicles was relatively low, this may be related to the molecular weight of the fusion protein, leading to a decrease in the efficiency of Her2-scFv-VSVG encoding in 293F cells. However, with the addition of the same amount of exosomes, more Her2-scFv-VSVG exosomes were detected in Her2-positive cells, indicating that Her2-scFv-VSVG exosomes have a stronger membrane fusion capacity.

[0092] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

[0093] References

[0094] LIAO, W., DU, Y., ZHANG, C., PAN, F., YAO, Y., ZHANG, T. & PENG, Q.(2019), "Exosomes: The next generation of endogenous nanomaterials for advanced drug delivery and therapy.", Acta biomaterialia, Vol. 861-14.

[0095] MENTKOWSKI, K. I., SNITZER, J. D., RUSNAK, S. & LANG, J. K. (2018), "Therapeutic Potential of Engineered Extracellular Vesicles.", The AAPSjournal, Vol. 20 No. 3, pp. 50.

[0096] SALUNKHE, S., DHEERAJ, BASAK, M., CHITKARA, D. & MITTAL, A. (2020), "Surface functionalization of exosomes for target-specific delivery and invivo imaging & tracking: Strategies and significance.", Journal of controlledrelease : official journal of the Controlled Release Society, Vol. 326599-614.

[0097] TEMCHURA, V. V., TENBUSCH, M., NCHINDA, G., NABI, G., TIPPLER, B.,ZELENYUK, M., WILDNER, O., UBERLA, K. & KUATE, S. (2008), "Enhancement ofimmunostimulatory properties of exosomal vaccines by incorporation of fusion-competent G protein of vesicular stomatitis virus.", Vaccine, Vol. 26 No. 29-30, pp. 3662-72.

[0098] YANG, Y., HONG, Y., NAM, G., CHUNG, J. H., KOH, E. & KIM, I. (2017),"Virus-Mimetic Fusogenic Exosomes for Direct Delivery of Integral MembraneProteins to Target Cell Membranes.", Advanced materials (Deerfield Beach,Fla.), Vol. 29 No. 13, pp.

[0099] ZHU, X., BADAWI, M., POMEROY, S., SUTARIA, D. S., XIE, Z., BAEK, A.,JIANG, J., ELGAMAL, O. A., MO, X., PERLE, K. L., CHALMERS, J., SCHMITTGEN, T.D. & PHELPS, M. A. (2017), "Comprehensive toxicity and immunogenicity studiesreveal minimal effects in mice following sustained dosing of extracellularvesicles derived from HEK293T cells.", Journal of extracellular vesicles,Vol. 6 No. 1, pp. 1324730.

Claims

1. A targeted exosome, characterized in that, The exosomes express a fusion protein of an antigen-targeting antibody and VSVG on their surface.

2. The targeted exosomes according to claim 1, characterized in that... The fusion protein comprises the following elements: 1) Signal peptide; 2) Histidine tag and Flag tag; 3) Single-chain antibody regions targeting antigens; 4) Dimerization sequence 1; 5) Transmembrane region; 6) Fluorescent proteins; 7) 2A sequence; 8) VSVG sequences; 9) Flexible connectors; and 10) Dimerization sequence 2, The amino acid sequence of the single-chain antibody region targeting the antigen is shown in SEQ ID NO:

3.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fusion protein of claim 2 that simultaneously expresses an antigen-targeting antibody and VSVG.

4. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 3.

5. Recombinant lentivirus, characterized in that... The recombinant lentivirus comprises a nucleic acid molecule as described in claim 3 or an expression vector as described in claim 4.

6. The method for preparing the recombinant lentivirus of claim 5, characterized in that, The procedure includes the following steps: co-transfecting mammalian host cells with the vector and helper plasmid as described in claim 4.

7. A cell, characterized in that, The cells express the fusion protein of claim 2 and secrete the targeted exosomes of claim 1.

8. The method for preparing the cells of claim 7, characterized in that, The method includes the following steps: infecting host cells with the recombinant lentivirus according to claim 5.

9. The method for preparing the targeted exosomes according to claim 1, characterized in that, The method includes the following steps: (1) Culturing and isolating the cells of claim 7; (2) Collect the culture supernatant of cells that can secrete exosomes that simultaneously express antigen-targeting antibodies and VSVG, and centrifuge and filter to remove impurities; (3) The supernatant was purified and concentrated using a hollow fiber column to obtain exosomes that simultaneously express antigen-targeting antibodies and VSVG.

10. A drug delivery vehicle, characterized in that, The delivery tool comprises the targeted exosome of claim 1, the nucleic acid molecule of claim 3, the expression vector of claim 4, or the cell of claim 7.

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