A biomimetic nanovesicle expressing a SLAMF7-GPC3 bispecific antibody and its preparation method and application

Abstract

The application belongs to the technical field of tumor immunotherapy, and particularly relates to a SLAMF7-GPC3 bispecific antibody-expressed biomimetic nanovesicle and a preparation method and application thereof. The SLAMF7-GPC3 bispecific antibody-expressed biomimetic nanovesicle is composed of biological cells, and the cell surface expresses the SLAMF7-GPC3 bispecific antibody. The gene sequence of the bispecific antibody is shown as SEQ ID NO. 7. The biomimetic nanovesicle can improve the half-life of the bispecific antibody, retain good tissue permeability and targeting thereof, simultaneously recognize SLAMF7 antigens on lymphocytes and GPC3 antigens on liver cancer cells, activate various immune cells by using the expression of SLAMF7 on the immune cells, has a synergistic killing ability on tumor cells expressing GPC3 target proteins, and is expected to remodel the tumor microenvironment through SLAMF7 signals, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor immunotherapy, and specifically relates to a biomimetic nanovesicle expressing a SLAMF7-GPC3 bispecific antibody, and a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common histological subtype of liver cancer. According to statistics, only 15%-20% of HCC cases are diagnosed in the early stages, and most advanced patients are diagnosed with unresectable advanced HCC, macroscopically visible vascular invasion, and extrahepatic metastasis, so there are considerable challenges in actual clinical treatment. The current standard treatment with sorafenib, lenvatinib, and regorafenib is still unsatisfactory in advanced HCC. Recently, two PD-1 inhibitors, pembrolizumab and nivolumab, have been approved for the treatment of HCC patients who have progressed after sorafenib, providing promising clinical benefits for a variety of malignancies including HCC. However, only about 20% of HCC patients show a clear and lasting response to PD-1 blockade, so there is an urgent need to find more effective and novel target immunotherapy strategies.

[0003] The transmembrane receptor signaling lymphocyte activation molecule (SLAM) family is a group of type I transmembrane glycoproteins widely expressed on hematopoietic cells and belongs to the CD2 subgroup of the immunoglobulin superfamily. SLAMF7, also known as CRACC, CS1, and CD319, is the seventh member of the SLAM family. It is consistently expressed at low levels on various human immune cells, including NK cells, CD4+ T cells, CD8+ T cells, some B cells, macrophages, and dendritic cells, but is absent in other tissues. SLAMF7 is consistently expressed at high levels in multiple myeloma (MM) and its malignant plasma cells, and has become a novel biomarker for the diagnosis of MM. SLAMF7 is a "self-ligand," capable of recognizing the same receptor molecule on another cell as a ligand through its IgV domain. Furthermore, the monoclonal antibody targeting SLAMF7, elotuzumab, binds to the IgC2 domain. Together, these two functions trigger the intracellular ITSM to recruit the SLAM adaptor protein, mediating activation signals in human leukocytes. Several studies have reported that SLAMF7 can regulate immune cell function in various immune cells through the binding of SLAMF7-specific antibodies or self-ligands, enhancing the cytotoxic effects of NK cells and CD8+ T cells. Therefore, SLAMF7 plays an important role in tumor immune responses and is a novel target for tumor immunotherapy.

[0004] Currently, the immunotherapy drug targeting SLAMF7 as a target is mainly elotuzumab, which is used for the treatment of relapsed or refractory MM. However, elotuzumab has not been reported in solid tumors, and the main reason is that solid tumor cells do not express SLAMF7, and a single antibody cannot effectively target tumor cells and immune cells at the same time. In recent years, bispecific antibodies further developed based on antibody structure are one of the most potential cancer immunotherapy drugs at present. Bispecific antibodies (BsAbs) are antibodies that can specifically bind to two antigens or two different epitopes of the same antigen, mainly used for the treatment of tumors and autoimmune diseases. In the treatment of tumors, BsAbs are antibodies that can specifically recognize tumor antigens and immune cell receptors (such as CD3, CD16) in two directions, promote the interaction between endogenous effector T cells or NK cells and tumor cells, and then kill tumor cells. BsAbs have small molecular weight, are easy to manufacture, and have enhanced tissue penetration, but their half-life is short, which can lead to rapid blood clearance and poor retention time at the target site. Although researchers have made a lot of improvements in the format of BsAbs based on the above shortcomings, there is still no clear preferred best mode. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide a kind of SLAMF7-GPC3 bispecific antibody expression biomimetic nanovesicle and its preparation method and application, introduce biomimetic nanovesicle as the carrier of double specific antibody, solve the defects of the existing double specific antibody format.

[0006] The primary purpose of the present application is to provide a kind of SLAMF7-GPC3 bispecific antibody expression biomimetic nanovesicle.

[0007] The present application realizes the above-mentioned purpose by the following technical solutions:

[0008] A kind of SLAMF7-GPC3 bispecific antibody expression biomimetic nanovesicle is composed of biological cells, and the cell surface expresses SLAMF7-GPC3 bispecific antibody.

[0009] Preferably, the cell is any one of HEK293T, HepG2, Huh-7, Hepa1-6, SK-Hep-1, lymphocyte.

[0010] More preferably, the cell is HEK293T cell.

[0011] Preferably, the genetic sequence of the bispecific antibody is as shown in SEQ ID NO.7.

[0012] The second purpose of the present application is to provide a preparation method of the above-mentioned SLAMF7-GPC3 bispecific antibody expression biomimetic nanovesicle.

[0013] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0014] A method for preparing biomimetic nanovesicles expressing a SLAMF7-GPC3 bispecific antibody comprises the following steps:

[0015] S1. Sequentially assemble the signal peptide, single-chain antibody targeting SLAMF7, (G4S)2 connecting peptide, single-chain antibody targeting GPC3, CD8α hinge region, CD8α transmembrane region, and Myc tag genes, ligate them to a vector after enzyme digestion, and transform them into competent cells. After culture, plasmid extraction and sequencing are performed to obtain a plasmid containing the SLAMF7-GPC3 bispecific antibody.

[0016] S2. The plasmid obtained in step S1 is packaged into a lentiviral vector, and the lentiviral vector system is transfected into cells. After culture, the cells are screened for resistance to obtain stably transfected cells that stably express the SLAMF7-GPC3 bispecific antibody;

[0017] S3. Expand the culture of the stably transfected cells obtained in step S2, collect the cells, perform lysis, grinding, gradient centrifugation, resuspend the precipitate, and then squeeze to form biomimetic nanovesicles.

[0018] Preferably, the nucleotide sequence of the signal peptide in step S1 is shown as SEQ ID NO.1, the nucleotide sequence of the single-chain antibody targeting SLAMF7 is shown as SEQ ID NO.2, the nucleotide sequence of the (G4S)2 connecting peptide is shown as SEQ ID NO.3, the nucleotide sequence of the single-chain antibody targeting GPC3 is shown as SEQ ID NO.4, the nucleotide sequences of the CD8α hinge region and transmembrane region are shown as SEQ ID NO.5, and the nucleotide sequence of the Myc tag is shown as SEQ ID NO.6.

[0019] Preferably, the lentiviral packaging vectors in step S2 are psPAX2 and pMD2G plasmids.

[0020] The third object of the present invention is to provide the use of the biomimetic nanovesicles expressing the SLAMF7-GPC3 bispecific antibody in the preparation of anti-tumor drugs.

[0021] Preferably, the tumor includes liver cancer, lung cancer, esophageal cancer, gastric cancer, melanoma, colorectal cancer, bladder cancer, pancreatic cancer, ovarian cancer, breast cancer and neuroblastoma.

[0022] More preferably, the tumor is liver cancer.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The application provides a kind of expression SLAMF7-GPC3 bispecific antibody biomimetic nanovesicle.One aspect, SLAMF7 is a kind of activation receptor expressed on a variety of immune cells, and the activation signal based on SLAMF7 can enhance the anti-tumor ability of a plurality of immune cells, and it is a new target of tumor immunotherapy.GPC3 antigen is specifically highly expressed in HCC, but there is no expression in normal liver tissue, kidney and gastric gland, and it is a specific target antigen of HCC.Therefore, the SLAMF7-GPC3 bispecific antibody provided by the application can activate NK cells, T cells and other immune cells, and has good killing ability to tumor cells expressing GPC3 target protein, and can exert stronger anti-tumor effect.On the other hand, the bispecific antigen is expressed on the biomimetic cell vesicle, which solves the original format defect of the bispecific antibody, prolongs the half-life of the bispecific antibody, increases the tumor permeability, activates more immune cells in the tumor microenvironment, inhibits the growth rate of tumor, reduces the volume of tumor, so that the bispecific antibody has stronger anti-solid tumor activity, and shows good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a structural schematic diagram of SLAMF7-GPC3 bispecific antibody.

[0026] Figure 2 It is a nanometer particle size diagram of SLAMF7@GPC3 NVs.

[0027] Figure 3 It is a SDS-PAGE identification map of SLAMF7@GPC3 NVs.

[0028] Figure 4 It is a result graph of in vitro cytotoxicity test of SLAMF7@GPC3 NVs and PBMC on HepG2 cells.

[0029] Figure 5 It is a result graph of in vitro cytotoxicity test of SLAMF7@GPC3 NVs and PBMC on Bel-7402 cells.

[0030] Figure 6 It is a result graph of in vivo animal test of SLAMF7@GPC3 NVs. DETAILED DESCRIPTION

[0031] The technical solutions of the application will be described clearly and completely in combination with the embodiments of the application.It is obvious that the described embodiments are only a part of the embodiments of the application, not all the embodiments.Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor belong to the protection scope of the application.

[0032] Unless otherwise specified, the experimental methods used in the examples of the present invention are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0033] Example 1. Preparation of SLAMF7@GPC3 NVs

[0034] 1. Synthesis of SLAMF7-GPC3 bispecific antibody gene

[0035] The present invention provides a bispecific antibody that simultaneously targets SLAMF7 and GPC3, named SLAMF7@GPC3 BsAbs. The bispecific antibody comprises: a signal peptide, a single-chain antibody targeting SLAMF7, a (G4S)2 connecting peptide, a single-chain antibody targeting GPC3, a CD8α hinge region, a CD8α transmembrane region, and a Myc tag connected in sequence in series. The codons are optimized to ensure that the encoded amino acid sequence remains unchanged and is more suitable for expression in 293T cells. The structure is as follows: Figure 1 As shown. The single-chain variable fragment is a heavy chain and light chain pair against SLAMF7, and the variable region nucleotide sequence refers to the sequence of the monoclonal antibody huLuc63 (reference WO 2019 / 241358 A2 sequence number 56), including anti-SLAMF7VH, VL and hinge region domains; the variable region nucleotide sequence of the single-chain antibody sequence targeting GPC3 refers to the sequence of the monoclonal antibody GC33 (reference WO 2021 / 186395 A1 Table 2), including anti-GPC3VH, VL and hinge region domains. In order for SLAMF7@GPC3 BsAbs to be expressed on the cell surface, the CD8α signal peptide was selected as the membrane expression signal peptide to guide its expression on the membrane, and the CD8α transmembrane region further supports its stable presence on the membrane. The signal peptide is directly connected to the N-terminus of the antibody variable region, and the protein tag Myc is connected to the C-terminus of the sequence as a detection indicator for protein expression. The nucleotide sequences of each domain and signal peptide are as follows:

[0036] The nucleotide sequence of the signal peptide is shown in SEQ ID NO.1 in the sequence listing, the nucleotide sequence of the single-chain antibody targeting SLAMF7 is shown in SEQ ID NO.2 in the sequence listing, the nucleotide sequence of the (G4S)2 connecting peptide is shown in SEQ ID NO.3 in the sequence listing, the nucleotide sequence of the single-chain antibody targeting GPC3 is shown in SEQ ID NO.4 in the sequence listing, the nucleotide sequences of the CD8α hinge region and transmembrane region are shown in SEQ ID NO.5 in the sequence listing, and the nucleotide sequence of the Myc tag is shown in SEQ ID NO.6 in the sequence listing.

[0037] 2. Construction of SLAMF7-GPC3 bispecific antibody plasmid

[0038] Overlap PCR and other molecular cloning techniques were used to complete the splicing of the genes in Example 1. After digestion with EcoRI / BamHI, the genes were ligated into the pCDH-CMV-MCS-EF1-GFP+Puro (CD513B-1) cloning vector and transformed into Stbl3 competent cells. After culturing for 16-18 hours, single colonies were picked and cultured, and then plasmids were extracted and sequenced to obtain the target gene plasmid.

[0039] 3. Transfection of 293T cells with lentiviral three-plasmid system

[0040] The specific implementation process is as follows:

[0041] (1) Culture 293T cells in good growth condition into a 6-well culture plate. The optimal confluence is when the cells reach about 70%;

[0042] (2) The target plasmid: psPAX2: pMD2G = 4:3:1 ratio was transfected into 293T cells using PEI and then cultured in an incubator;

[0043] (3) 12 hours after transfection, the medium was replaced with 10% FBS, and the cells were passaged after 24 hours of culture.

[0044] (4) After the cell state improves, 3 μg / mL puromycin is added to the culture medium for screening. The cells are maintained in a 37°C, 5% CO2 incubator, passaged every 2-3 days, and supplemented with antibiotics to finally obtain a polyclonal stably transfected cell line that stably expresses the SLAMF7-GPC3 bispecific antibody.

[0045] 4. Preparation of SLAMF7@GPC3 NVs

[0046] Stably transfected polyclonal cells were expanded and cultured, then harvested, lysed, ground, and subjected to ultrahigh-speed centrifugation before being extruded through an extruder to form biomimetic nanovesicles. The main process was as follows: After harvesting the cells, they were washed three times with PBS. The cell pellet was dispersed in separation buffer and incubated at 4°C overnight. The next day, the mixture was loaded into a Dounce homogenizer and repeatedly ground to disrupt the cells. The mixture was spun at 800×g for 5 minutes to remove large debris. The supernatant was collected and centrifuged again at 10,000×g for 25 minutes. The supernatant was collected again and finally centrifuged at 100,000×g for 60 minutes. The precipitate was collected and dispersed in PBS. The mixture was extruded through an extruder containing 800nm ​​and 200nm polycarbonate membranes, respectively, to obtain nanovesicles.

[0047] 5. Characterization and analysis of SLAMF7@GPC3 NVs

[0048] (1) Nanoparticle size diagram of SLAMF7@GPC3 NVs

[0049] According to the above method, biomimetic nanovesicles expressing bispecific antibodies were prepared by hypotonic lysis, mechanical disruption, differential centrifugation, and finally artificial extrusion. The particle size of the vesicles was analyzed by dynamic light scattering (DLS). Figure 2 As shown, the hydrated particle size of SLAMF7@GPC3 NVs is ∼176 nm.

[0050] (2) Characterization and analysis of SLAMF7@GPC3 NVs

[0051] Western blot was used to analyze the expression of NVs protein tag Myc to detect the expression of the target protein. Figure 3 As shown, compared with untransfected HEK293T NVs, the expression of protein tag Myc can be detected in transfected SLAMF7@GPC3 NVs, and the molecular weight is consistent with expectations.

[0052] The experimental results showed that a biomimetic nanovesicle (SLAMF7@GPC3 NVs) expressing SLAMF7-GPC3 bispecific antibody was successfully constructed.

[0053] Example 2: Detection of SLAMF7@GPC3 NVs Effectively Mediating PBMC Killing of GPC3-Positive Tumor Cells

[0054] Flow cytometry was used to evaluate whether SLAMF7@GPC3 NVs could enhance the cytotoxicity of PBMCs against tumor cells. HepG2 cells expressing the GPC3 antigen were stained with CFSE and plated into 96-well cell culture plates. After adherence, the medium was aspirated and PBMCs were added at a target cell:effector cell ratio of 1:5. Then, various concentrations of SLAMF7@GPC3 NVs or Free293TNVs as a control group were added to each well. After 14 hours of mixed culture, cells were harvested and stained for viability. Flow cytometry was used to assess cytotoxicity.

[0055] Result Analysis

[0056] The results of in vitro cytotoxicity tests are shown in Figure 4 Compared with the control group and Free293TNVs group, the addition of different concentrations of SLAMF7@GPC3 NVs can enhance the killing effect of PBMC on HepG2 cells in a concentration-dependent manner, suggesting that SLAMF7@GPC3 NVs can activate a variety of immune cells, thereby having an effective killing effect on liver cancer cells.

[0057] Example 3: SLAMF7@GPC3 NVs cannot mediate PBMC killing of GPC3-negative tumor cells

[0058] To further verify the specific killing effect of SLAMF7@GPC3 NVs, the human liver cancer cell line Bel-7402 that does not express GPC3 was used as the target cell, and the killing effect of SLAMF7@GPC3 NVs on Bel-7402 cells was evaluated using the experimental method described in Example 2.

[0059] The flow cytometry results are shown in Figure 5 The addition of different concentrations of Free293TNVs and SLAMF7@GPC3 NVs could not enhance the killing effect of PBMC on Bel-7402 cells, and no obvious toxic effect was observed with the increase of concentration. There was no significant difference with the Control group. It can be seen that the SLAMF7@GPC3 NVs targeting HCC provided by the embodiment of the present invention has good specific anti-tumor effect.

[0060] Example 4. Preparation of mouse SLAMF7@GPC3 NVs

[0061] To further analyze the antitumor activity of SLAMF7@GPC3 NVs in mice, mouse-derived SLAMF7@GPC3 NVs were prepared.

[0062] 1. Plasmid construction

[0063] The gene sequences of mouse CD8α signal peptide (SEQ ID NO.8), single-chain antibody targeting mouse SLAMF7 (SEQ ID NO.9), (G4S)2 connecting peptide (SEQ ID NO.3), single-chain antibody targeting GPC3 (SEQ ID NO.4), mouse CD8α hinge region and transmembrane region (SEQ ID NO.10) and Myc tag (SEQ ID NO.6) were connected in sequence, wherein the single-chain variable fragment was a heavy chain and light chain pair of anti-mouse SLAMF7, and the amino acid sequence of the variable region was based on the sequence of the monoclonal antibody Murine Luc90 (reference WO2019 / 241358 A2 sequence number 47). The mouse SLAMF7@GPC3 BsAbs gene fragment (SEQ ID NO.11), digested with EcoRI / BamHI, and ligated to the pCDH-CMV-MCS-EF1-GFP+Puro (CD513B-1) cloning vector, transformed into Stbl3 competent cells, cultured for 16-18 h, and single clones were picked for culture. Plasmids were then extracted and sequenced to obtain the target gene plasmid.

[0064] 2. Preparation of SLAMF7@GPC3 NVs

[0065] The specific implementation process is as described in Example 1, which is briefly as follows: the target plasmid: psPAX2:pMD2G = 4:3:1 ratio is transfected into 293T cells with PEI, and 3 μg / mL puromycin is used for screening after 24-48 hours. After one week of continuous screening with puromycin, a polyclonal stably transfected cell line stably expressing the mouse SLAMF7-GPC3 bispecific antibody is obtained. After the cell line is expanded and cultured, nanovesicles are finally obtained by lysis, repeated grinding, ultracentrifugation, and extrusion.

[0066] Example 5. Detection of the efficacy of SLAMF7@GPC3 NVs in killing subcutaneous transplanted tumors in mice

[0067] It is planned to establish a mouse subcutaneous liver cancer model by subcutaneous injection of Hepa1-6-huGPC3 cells, and to inject mouse-derived SLAMF7@GPC3 NVs into the tail vein to explore its in vivo anti-tumor effect. 6 Hepa1-6-huGPC3 cells were injected into the right groin of C57BL / 6 mice. After about 10 days, when the tumor volume reached 100 mm, 3 The mice were randomly divided into groups at about 10 days and injected with drugs via tail vein. (1) Negative control group, injected with PBS only; (2) Control group, injected with 200 μg Free 293T NVs; (3) Experimental group, injected with 200 μg SLAMF7@GPC3 NVs. The day of administration was the 10th day, and the mice were injected with tail vein every 3 days without changing the dose. The size of the Hepa1-6-huGPC3 subcutaneous transplanted tumor was measured with a vernier caliper, and the changes in tumor volume and body weight of each group of mice were recorded. The volume was calculated using the formula of 1 / 2×length×width×width (mm 3 ).

[0068] Result Analysis

[0069] The results are as follows Figure 6 As shown in Figure A, in a wild-type mouse subcutaneous liver cancer model implanted with Hepa1-6-huGPC3 cells, treatment with SLAMF7@GPC3 NVs significantly inhibited tumor growth, showing significant differences compared to the control and negative control groups. Treatment with Free 293T NVs failed to inhibit tumor growth, with the tumors gradually increasing in size over time, showing no significant difference from the negative control group. This suggests that biomimetic nanovesicles expressing the SLAMF7 / GPC3 bispecific antibody can specifically bind to tumor-specific antibodies and activate immune cells to kill them, demonstrating a modest anti-tumor effect in wild-type tumor-bearing mice.

[0070] exist Figure 6In Figure B, Hepa1-6-huGPC3 cells were inoculated subcutaneously into SLAMF7 knockout mice (SLAMF7 KO) to establish a mouse subcutaneous liver cancer model. Different nanovesicles were injected four times to compare their inhibitory effects on mouse subcutaneous liver cancer. The results showed that treatment with SLAMF7@GPC3 NVs failed to inhibit tumor growth. The tumor gradually increased over time and showed no significant difference from the control group and the negative control group. This suggests that after knocking out SLAMF7 on immune cells, SLAMF7@GPC3 NVs lost their binding receptors and were unable to effectively activate immune cells. It also further suggests that nanovesicles expressing the GPC3 / SLAMF7 bispecific antibody activate immune cells by binding to SLAMF7 on immune cells, thereby killing tumor cells.

[0071] The present invention provides a bispecific antibody biomimetic nanovesicle targeting GPC3. GPC3 is a member of the heparan sulfate proteoglycan family and is involved in cell proliferation, differentiation, migration, and apoptosis. Studies have shown that GPC3 is expressed in 70-80% of HCCs, but rarely expressed in normal tissues, making it a very promising target for liver cancer immunotherapy. Therefore, the bispecific antibody provided by the present invention uses SLAMF7 as an activating receptor and the GPC3 antigen as a liver cancer target, so that SLAMF7@GPC3 NVs can specifically recognize the GPC3 antigen, thereby having good killing ability against GPC3 target cells and exerting a stronger anti-tumor effect. At the same time, the bispecific antibody biomimetic nanovesicle targeting GPC3 only shows killing ability against liver cancer cells expressing GPC3, demonstrating its specific anti-tumor activity, and has good safety and application prospects.

[0072] Obviously, the specific implementation scheme described above is only a further detailed description of the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above is only a specific example of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A biomimetic nanovesicle expressing a SLAMF7-GPC3 bispecific antibody, characterized in that: It is composed of biological cells, and the cell surface expresses the SLAMF7-GPC3 bispecific antibody. The gene sequence of the SLAMF7-GPC3 bispecific antibody is shown in SEQ ID NO. 7 or SEQ ID NO.

11.

2. The biomimetic nanovesicle expressing the SLAMF7-GPC3 bispecific antibody according to claim 1, characterized in that: The cell is any one of HEK293T, HepG2, Huh-7, Hepa1-6, SK-Hep-1, and lymphocytes.

3. The biomimetic nanovesicle expressing the SLAMF7-GPC3 bispecific antibody according to claim 1, characterized in that: The cells are HEK293T.

4. The method for preparing the biomimetic nanovesicles expressing the SLAMF7-GPC3 bispecific antibody according to any one of claims 1 to 3, characterized in that: The steps include: S1. Sequentially assemble the signal peptide, single-chain antibody targeting SLAMF7, (G4S)2 connecting peptide, single-chain antibody targeting GPC3, CD8α hinge region, CD8α transmembrane region, and Myc tag genes, ligate them to a vector after enzyme digestion, and transform them into competent cells. After culture, plasmid extraction and sequencing are performed to obtain a plasmid containing the SLAMF7-GPC3 bispecific antibody. S2. The plasmid obtained in step S1 is packaged into a lentiviral vector, and the lentiviral vector system is transfected into cells. After culture, the cells are screened for resistance to obtain stably transfected cells that stably express the SLAMF7-GPC3 bispecific antibody; S3. Expand the culture of the stably transfected cells obtained in step S2, collect the cells, perform lysis, grinding, gradient centrifugation, resuspend the precipitate, and then squeeze to form biomimetic nanovesicles.

5. The preparation method according to claim 4, characterized in that The nucleotide sequence of the signal peptide in step S1 is shown in SEQ ID NO. 1, the nucleotide sequence of the single-chain antibody targeting SLAMF7 is shown in SEQ ID NO. 2, the nucleotide sequence of the (G4S)2 connecting peptide is shown in SEQ ID NO. 3, the nucleotide sequence of the single-chain antibody targeting GPC3 is shown in SEQ ID NO. 4, the nucleotide sequences of the CD8α hinge region and transmembrane region are shown in SEQ ID NO. 5, and the nucleotide sequence of the Myc tag is shown in SEQ ID NO. 6; Alternatively, the nucleotide sequence of the signal peptide in step S1 is shown as SEQ ID NO.8, the nucleotide sequence of the single-chain antibody targeting SLAMF7 is shown as SEQ ID NO.9, the nucleotide sequence of the (G4S)2 connecting peptide is shown as SEQ ID NO.3, the nucleotide sequence of the single-chain antibody targeting GPC3 is shown as SEQ ID NO.4, the nucleotide sequences of the CD8α hinge region and transmembrane region are shown as SEQ ID NO.10, and the nucleotide sequence of the Myc tag is shown as SEQ ID NO.

6.

6. The preparation method according to claim 4, characterized in that The lentiviral vectors in step S2 are psPAX2 and pMD2G plasmids.

7. Use of the biomimetic nanovesicle expressing the SLAMF7-GPC3 bispecific antibody according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The tumor is liver cancer.

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