A dual-targeting recombinant fusion protein and dual-targeting nanovesicle, and construction method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a double-targeting recombinant fusion protein and double-targeting nanovesicle as well as a construction method and application thereof. The structure of the double-targeting recombinant fusion protein comprises alphaCD16scFv, alphaHER2scFv and Lamp2b transmembrane protein connected in sequence. The double-targeting recombinant fusion protein takes the NK cell activation receptor CD16 molecule and the HER2 molecule on the surface of gastric cancer cells as targets, can guide the NK cell to recognize the HER2 positive gastric cancer cell, and activates the NK cell through the CD16 molecule to further target and kill the gastric cancer cell, so as to achieve the purpose of treating gastric cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a dual-targeting recombinant fusion protein and dual-targeting nanovesicles, their construction methods, and applications. Background Technology

[0002] Gastric cancer is a common digestive tract tumor. Because early symptoms are often subtle, it is usually diagnosed at an advanced stage or has metastasized. Traditional chemotherapy drugs can improve the survival of patients with this type of gastric cancer, but the treatment effect is not ideal, with long disease courses and drug resistance. NK cell adoptive therapy is an emerging cancer treatment method that has shown promise in clinical trials for hematological malignancies. However, the tumor suppressor microenvironment in solid tumors severely affects the efficacy of NK cells in solid tumors. Therefore, new methods are needed to activate and guide NK cells to enhance their anti-tumor effects. Summary of the Invention

[0003] The purpose of this invention is to provide a dual-targeting recombinant fusion protein and dual-targeting nanovesicles, their construction methods and applications. The dual-targeting recombinant fusion protein can guide NK cells to recognize cancer cells and activate NK cells through CD16 molecules to target and kill cancer cells, thereby achieving an anti-tumor effect.

[0004] The present invention provides a dual-targeting recombinant fusion protein, the structure of which includes αCD16 scFv, αHER2 scFv and Lamp2b transmembrane protein connected in sequence.

[0005] As a preferred embodiment, the dual-targeting recombinant fusion protein is composed of signal peptide SP, HAtag, αCD16scFv, αHER2 scFv and Lamp2b transmembrane protein linked sequentially.

[0006] As a preferred embodiment, the heavy chain amino acid sequence of the αCD16 scFv is shown in SEQ ID NO.1, and the light chain amino acid sequence of the αCD16 scFv is shown in SEQ ID NO.2.

[0007] As a preferred embodiment, the heavy chain amino acid sequence of the αHER2 scFv is shown in SEQ ID NO.3, and the light chain amino acid sequence of the αHER2 scFv is shown in SEQ ID NO.4.

[0008] As a preferred embodiment, the amino acid sequence of the Lamp2b transmembrane protein is shown in SEQ ID NO.5.

[0009] This invention provides a dual-targeting nanovesicle expressing the above-mentioned dual-targeting recombinant fusion protein.

[0010] As a preferred embodiment, the nanovesicles are extracted from HEK293 cells.

[0011] The present invention provides a method for constructing the above-mentioned dual-targeting nanovesicles, comprising the following steps: infecting eukaryotic cells with a lentivirus containing the above-mentioned dual-targeting recombinant fusion protein, and then extracting the dual-targeting nanovesicles.

[0012] This invention provides the application of the above-mentioned dual-targeting recombinant fusion protein or the above-mentioned dual-targeting nanovesicles in the preparation of drugs for inhibiting tumors.

[0013] As a preferred embodiment, the tumor includes: gastric cancer and / or rectal cancer.

[0014] Beneficial Effects: This invention provides a dual-targeting recombinant fusion protein, the structure of which comprises sequentially linked αCD16 scFv, αHER2 scFv, and Lamp2b transmembrane protein. The dual-targeting recombinant fusion protein of this invention targets both the NK cell activating receptor CD16 molecule and the HER2 molecule on the surface of gastric cancer cells. It can guide NK cells to recognize HER2-positive gastric cancer cells, and activate NK cells through CD16 molecules to target and kill gastric cancer cells. It expresses a dual-targeting protein, achieving the goal of treating gastric cancer.

[0015] This invention provides a dual-targeting nanovesicle containing the above-mentioned dual-targeting recombinant fusion protein. The dual-targeting nanovesicle can act on the HER2-highly expressed colorectal cancer cell line SW480, and has the effect of inhibiting the activity of various HER2-positive tumor cell lines, thus exhibiting a broad-spectrum anti-tumor effect.

[0016] This invention provides a method for constructing the aforementioned dual-targeting nanovesicles. Utilizing the advantages of nanovesicles, such as their natural material transport properties, small molecular size, and excellent biocompatibility, they serve as excellent carriers for therapeutic molecules. The constructed dual-targeting nanovesicles target the NK cell activating receptor CD16 and the HER2 molecule on the surface of tumor cells. This dual-targeting fusion protein is expressed on the outer surface of the nanovesicle membrane and can be used to activate and guide NK cells to target and kill HER2-positive malignant tumors, effectively avoiding problems such as short blood half-life and gene manipulation at the NK cell level. Attached Figure Description

[0017] Figure 1 The preparation process for αCD16 / αHER2 NV;

[0018] Figure 2 Characterization diagrams of αCD16 / αHER2 NVs; where A: Nanoparticle analyzer detection diagram, B: Gel electrophoresis detection diagram, and C: Flow cytometry detection diagram;

[0019] Figure 3 Figure 1 shows the effect of Blank NV and αCD16 / αHER2 NV on the activity of gastric cancer cells BGC823 and HGC27 as observed by CCK-8 assay; A: different concentrations with the same effector-to-target ratio, B: different effector-to-target ratios with the same concentration. Detailed Implementation

[0020] The purpose of this invention is to provide a dual-targeting recombinant fusion protein and dual-targeting nanovesicles, their construction methods, and applications. The dual-targeting recombinant fusion protein of this invention can guide NK cells to recognize HER2-positive gastric cancer cells and activate NK cells through CD16 molecules, thereby targeting and killing gastric cancer cells to achieve the purpose of cancer treatment.

[0021] This invention provides a dual-targeting recombinant fusion protein, the structure of which comprises sequentially linked αCD16 scFv, αHER2 scFv, and Lamp2b transmembrane protein. As one embodiment, the single-stranded variable region nucleic acid sequences (scFv) of CD16 and HER2 antibodies were determined by consulting the literature; the nucleic acid sequences were then sequentially assembled into the target fragment according to the order of signal peptide (SP), HAtag, αCD16 scFv, αHER2 scFv, and Lamp2b transmembrane protein sequence; these fragments are all linked by the same linker, the amino acid sequence of which is shown in SEQ ID NO.6: GGGGSGGGGSGGGGS, and the transmembrane sequence is derived from human. The heavy chain and parental chain portions are linked by the linker, with the relative positions being αCD16scFv light chain - linker - αCD16 scFv heavy chain - linker - αHER2 scFv heavy chain - linker - αHER2 scFv light chain.

[0022] As one embodiment, the heavy chain amino acid sequence of the αCD16 scFv is shown in SEQ ID NO.1: EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLE WVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCA RGRSLLFDYWGQGTLVTVSR; the light chain amino acid sequence of the αCD16 scFv is shown in SEQ ID NO.2: SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAP VLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNH VVFGGGTKLTVG.

[0023] As one embodiment, the heavy chain amino acid sequence of the αHER2 scFv is shown in SEQ ID NO.3: EVQLQQSGAEFVKPGASVKLSCTASGFNIKDTYIHWVKQRPEQGLEWI G RIDPANGNTKYDPNFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCACPTE EITLKYWYFDVWGAGTTVTASS, and the light chain amino acid sequence of the αHER2 scFv is shown in SEQ ID NO.4: DIKLTQSPAIMSASLGEEITLTCSASSSVSYMHWYQQKSGTSP KLLIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPT FGAGTKLELKRA.

[0024] As one embodiment, the amino acid sequence of the Lamp2b transmembrane protein is shown in SEQ ID NO.5: ILIPIIVGAGLSGLIIVIVIAYVI.

[0025] This invention provides a dual-targeting nanovesicle containing the aforementioned dual-targeting recombinant fusion protein. The dual-targeting nanovesicles provided by this invention can, on the one hand, increase the blood circulation half-life of bispecific antibodies; on the other hand, as a natural carrier, the nanovesicles can be modified to load chemotherapeutic drugs, nucleic acid drugs, etc., providing the possibility of achieving all-in-one combination therapy of bispecific antibodies and chemotherapeutic / nucleic acid drugs. In one embodiment, the nanovesicles are extracted from HEK293 cells.

[0026] This invention provides a method for constructing the aforementioned dual-targeting nanovesicles, comprising the following steps: infecting eukaryotic cells with a lentivirus containing the aforementioned dual-targeting recombinant fusion protein, and then extracting the aforementioned dual-targeting nanovesicles. As one embodiment, HEK293 cells are infected with a lentivirus containing the aforementioned dual-targeting recombinant fusion protein, and then cultured and expanded in a high-glucose medium containing 10% fetal bovine serum; after digestion of the cells, the cell pellet is washed with PBS, the cells are counted, resuspended in PBS, and the cell density is adjusted to 1×10⁻⁶. 6 The concentration of liposomes was increased to 1 / mL, and then the Avanti liposome extruder was used to push and pull the liposomes back and forth 10 times through a 400 nm polycarbonate membrane. The resulting mixture was centrifuged, the supernatant was discarded, PBS was added to dissolve the precipitate, and after filtration and sterilization, dual-targeting nanovesicles (αCD16 / αHER2NV) were obtained. The nanovesicles were used as carriers for the dual-targeting fusion protein, and the dual-targeting fusion protein was expressed on the outer surface of the nanovesicle membrane (see...). Figure 2 (C)

[0027] This invention provides the application of the above-mentioned dual-targeting recombinant fusion protein or the above-mentioned dual-targeting nanovesicles in the preparation of drugs for inhibiting tumors. As one embodiment, the tumor includes gastric cancer and / or rectal cancer. In one embodiment, the above-prepared dual-targeting nanovesicles were applied to the co-culture of gastric cancer cells and PBMCs, and it was found that they had the effect of targeting and activating NK cells to kill HER2-positive gastric cancer cells. As one embodiment, the above-prepared dual-targeting nanovesicles were applied to the HER2-highly expressed colorectal cancer cell line SW480, and it was found that they had the effect of inhibiting the activity of various HER2-positive tumor cell lines, exhibiting broad-spectrum anti-tumor activity.

[0028] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a dual-targeting recombinant fusion protein and dual-targeting nanovesicles, their construction methods, and applications, but these should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] 1. Preparation of HEK293 cells expressing dual-targeting fusion protein

[0031] (1) Construction and synthesis of the target fragment nucleotide sequence

[0032] Following the sequence of signal peptide (SP), HAtag, αCD16 scFv, αHER2 scFv, Lamp2b transmembrane region, and GFP, the nucleic acid sequences were sequentially assembled into the target fragment. The target fragment was synthesized by Nanjing GenScript Technology Co., Ltd., ligated into an overexpression plasmid, and packaged into a lentivirus.

[0033] (2) Screening of HEK293 cell lines infected with lentiviruses

[0034] HEK293 cells in logarithmic growth phase were digested, counted, and their density adjusted to 2 × 10⁻⁶. 5 Cells were cultured in 24-well plates for 5 hours. A 1:1 mixture of high-glucose DMEM and lentivirus supernatant was added to the 5-hour culture, followed by the addition of 10 g / mL polybrene. After 4 hours, the medium was replaced with 500 μL of complete culture medium, and the cells were cultured for another 24 hours. Cells were then digested and a portion was harvested for flow cytometry analysis to determine infection efficiency. The cells were then divided into 2 × 10⁶ cells / well. 5 The cells were seeded in 24-well plates and screened for 7 days with 1 g / mL puromycin to obtain positive cell lines.

[0035] 2. Preparation of αCD16 / αHER2 NV

[0036] The prepared positive cell lines were cultured and expanded in a high-glucose medium containing 10% fetal bovine serum. When the cell number reached 1 × 10⁶ cells / year... 6 After digesting the cells, centrifuge at 300g for 5 minutes, discard the supernatant, wash the cell pellet twice with PBS, count the cells, resuspend the cells in PBS, and adjust the cell density to 1×10⁻⁶. 6 The sample was extracted at a density of 1 / mL and then pushed and pulled back and forth 10 times through a 400 nm polycarbonate membrane using an Avanti liposome extruder. The resulting mixture was centrifuged at 800 g for 10 min at 4 °C. The supernatant was collected and centrifuged again at 10,000 g for 30 min at 4 °C. The supernatant was then centrifuged again at 100,000 g for 1.5 h at 4 °C. The supernatant was discarded, and an appropriate amount of PBS was added to dissolve the precipitate. After filtration through a 0.22 μm filter for sterilization, dual-targeted nanovesicles (αCD16 / αHER2 NV) were obtained. After aliquoting, the nanovesicles were stored at -80 °C for subsequent experiments and characterization.

[0037] The preparation process of dual-targeted nanovesicles αCD16 / αHER2 NV is as follows: Figure 1 As shown.

[0038] Compare with Example 1

[0039] The procedure was carried out in accordance with Example 1, except that: the lentivirus prepared in step 1 (1) was replaced with a blank lentivirus (without any gene fragments); step 1 (2) was performed to obtain blank HEK293 cells (control HEK293); and step 2 was performed to obtain blank nanovesicles (BlankNV).

[0040] Identification of αCD16 / αHER2 NV in Experiment 1

[0041] (1) Particle size analysis of αCD16 / αHER2 NV: αCD16 / αHER2 NV prepared in Example 1 or Blank NV prepared in Control Example 1 were diluted with purified water to an appropriate ratio of 1.0 × 10⁻⁶. 8 ~2.5×10 9 Particles / mL; 1 mL of the diluted sample was drawn using a 1 mL syringe and injected into the sample chamber of the nanoparticle tracing analyzer. The Brownian motion of NVs was analyzed and recorded using the software built into the Nanosight tracing analyzer, and the particle concentration and particle size were detected. The results are shown in [Figure number missing]. Figure 2 A.

[0042] (2) Identification of αCD16 / αHER2 NV markers: Fresh αCD16 / αHER2 NV suspension or Blank NV was mixed with an equal volume of RIPA lysis buffer (RIPA:PMSF volume ratio = 250:1), and shaken for 1 minute every 10 min, repeated 4 times; centrifuged at 12000g for 15 min at 4℃, and the supernatant was collected. BCA protein concentration was determined by quantitative analysis. The expression of αCD16 / αHER2 NV marker proteins CD9, HSP70, Alix, and Calenxin was detected by Western blotting. The expression of the dual-targeting fusion protein was detected using the tag protein HA, and the results are shown in […]. Figure 2 B.

[0043] (3) Identification of αCD16 / αHER2 NV outer surface fusion protein expression: Take 30 μg of αCD16 / αHERNV suspension or BlankNV and place it together with 5 μL of latex microbeads in a 1.5 mL EP tube. Adjust the volume to 20 μL with PBS and incubate at room temperature for 15 min. Add 1 mL of PBS and place on a shaker. Incubate at room temperature for 2 h. Add 20 μL of PBS, pipette and mix well. Block at room temperature for 30 min. Centrifuge at 3000g for 5 min, discard the supernatant, and a white precipitate will be visible. Add 1 mL of PBS, centrifuge at 3000g for 5 min, and wash once. Discard the supernatant, resuspend in 100 μL of PBS, add 5 μL of APC-anti-HA.11epitope tag antibody, pipette and mix well, and incubate at 4℃ in the dark for 30 min. Add 1 mL of PBS, centrifuge at 3000g for 5 min, wash once, and analyze the results. See below. Figure 2 C.

[0044] from Figure 2 As can be seen from the data, the hydrated particle size of Blank NV and αCD16 / αHER2 NV, as detected by the Nanosight nanoparticle analyzer, is mainly concentrated around 120 nm. Figure 2 (A). Western blot analysis of NV expression-related proteins showed that both NV types highly expressed CD9, Alix, HSP70, and Calenxin, but only the αCD16 / αHER2 NV expressed the tag protein HA. Figure 2 (See Figure B). This result indicates that αCD16 / αHER2NV can express the fusion protein. To further confirm this, the constructed fusion protein was expressed on the outer surface of the NV membrane, and flow cytometry was used to detect the expression of the tag protein HA on the cell membrane. The results showed that αCD16 / αHER2 NV could display the fusion protein on the outer surface of the NV membrane, with an expression efficiency of 15.8% (Figure B). Figure 2 (C)

[0045] Experimental Example 2: αCD16 / αHER2 NV inhibits the activity of HER2-positive gastric cancer cells.

[0046] Gastric cancer cells BGC823 (HER2-low expressing cells) and HGC27 (HER2-high expressing cells) were digested with trypsin, counted, and resuspended. 1×10⁻⁶ cells were then collected. 4 Cell suspensions of 100 cells / well were seeded into 96-well plates. To detect the inhibitory effect of different concentrations of NV on the activity of gastric cancer cells, after the gastric cancer cells adhered overnight, 1×10⁻⁶ cells / well were added to the cell culture medium. 5 Personal peripheral blood mononuclear cells (PBMCs) were used to achieve an effector-to-target ratio (E:T) of 10:1 for PBMCs and gastric cancer cells. Then, 10 μL of PBS and different concentrations of Blank NV (10 μL / L) were added to the PBMCs and gastric cancer cell co-culture system, respectively. -2 10 -1 10 0 10 1 10 2 10 3 ng / mL) or αCD16 / αHER2 NV (10 -2 10 -1 10 0 10 1 10 2 10 3 The cells were incubated with 1×10⁻⁶ ng / mL for 24 hours. To investigate the effect of different effector-to-target ratios on the inhibitory activity of NV on gastric cancer, after the gastric cancer cells adhered overnight, 1×10⁻⁶ ng / mL of the NV-containing medium was added to the cell culture medium. 4 2×10 4 5×10 4 1×10 5 1×10 6 PBMCs were selected to achieve effector-to-target ratios (E:T) of 1:1, 2:1, 5:1, 10:1, and 20:1 for gastric cancer cells. Then, PBS and 10 mg / L PBS were added to the co-culture system of PBMCs and gastric cancer cells, respectively. 2 ng / mL Blank NV and 10 2 Cells were incubated with ng / mL αCD16 / αHER2 NV for 24 hours. At the 24-hour detection time point, cells were harvested, the cell supernatant was discarded, and the cells were washed twice with PBS to remove suspended NK cells and dead gastric cancer cells. 100 μL / well of pre-prepared CCK-8 reagent was added to each well of adherent gastric cancer cells. After incubation at 37°C for 2 hours, the absorbance at 450 nm was measured using a microplate reader. The absorbance of PBS-treated cells served as a 100% cell viability control for each treatment group. Results are shown below. Figure 3 .

[0047] from Figure 3 As can be seen, at the same effector-to-target ratio, the activity of HER2-positive gastric cancer cells HGC27 in the αCD16 / αHER2 NV treatment group was lower than that in the BlankNV treatment group, and its activity decreased with increasing αCD16 / αHER2 NV concentration; however, the activity of HER2-negative gastric cancer cells BGC823 showed no difference between the αCD16 / αHER2 NV treatment group and the BlankNV treatment group. Figure 3 (A). When the same concentration of NV was added to culture systems with different effector-to-target ratios, it was found that the activity of HER2-positive gastric cancer cells HGC27 in the αCD16 / αHER2 NV treatment group was lower than that in the Blank NV group, and its activity decreased with increasing effector-to-target ratio; however, the activity of HER2-negative gastric cancer cells BGC823 showed no difference between the αCD16 / αHER2 NV treatment group and the Blank NV treatment group. Figure 3 (B) The results showed that αCD16 / αHER2 NV has the effect of targeting and activating NK cells to kill HER2-positive gastric cancer cells.

[0048] Experimental Example 3: Broad-spectrum antitumor effect of αCD16 / αHER2 NV in vitro

[0049] SW480 colorectal cancer cells (HER2-highly expressing cells) were digested with trypsin, counted, and resuspended. 1×10⁶ cells were collected. 4 Cell suspensions of 100 cells / well were seeded into 96-well plates. To detect the inhibitory effect of different concentrations of NV on the activity of colorectal cancer cells, after the colorectal cancer cells adhered overnight, 1×10⁻⁶ cells / well were added to the cell culture medium. 5 Personal peripheral blood mononuclear cells (PBMCs) were used to achieve an effector-to-target ratio (E:T) of 10:1 between PBMCs and colorectal cancer cells. Then, PBS and different concentrations of Blank NV (10⁻⁶) were added to the co-culture system of PBMCs and colorectal cancer cells, respectively. -2 10 -1 10 0 10 1 10 2 10 3 ng / mL) and αCD16 / αHER2 NV (10 -2 10 -1 10 0 10 1 10 2 10 3 The cells were incubated with 1×10⁻⁶ ng / mL for 24 hours. To investigate the effect of different effector-to-target ratios on the inhibitory activity of NV on colorectal cancer, after the colorectal cancer cells adhered overnight, 1×10⁻⁶ ng / mL of the NV was added to the cell culture medium. 4 2×10 4 5×104 1×10 5 1×10 6 PBMCs were selected to achieve effector-to-target ratios (E:T) of 1:1, 2:1, 5:1, 10:1, and 20:1 with colorectal cancer cells. Then, PBS and 10 mg / L PBS were added to the co-culture system of PBMCs and colorectal cancer cells, respectively. 2 ng / mL Blank NV and 10 2 Cells were incubated with ng / mL αCD16 / αHER2 NV for 24 hours. At the 24-hour detection time point, cells were harvested, the cell supernatant was discarded, and the cells were washed twice with PBS to remove suspended NK cells and dead gastric cancer cells. 100 μL / well of pre-prepared CCK-8 reagent was added to each well of adherent gastric cancer cells. After incubation at 37°C for 2 hours, the absorbance at 450 nm was measured using a microplate reader. The absorbance of PBS-treated cells served as a 100% cell viability control for each treatment group. Results are shown below. Figure 3 .

[0050] from Figure 3 As can be seen, under the same effector-to-target ratio, the activity of HER2-positive colorectal cancer cells SW480 in the αCD16 / αHER2 NV treatment group was lower than that in the Blank NV group, and its activity decreased with increasing αCD16 / αHER2 NV concentration. Figure 3 (A). When the same concentration of NV was added to culture systems with different effector-to-target ratios, it was found that the activity of HER2-positive colorectal cancer cells SW480 in the αCD16 / αHER2 NV treatment group was lower than that in the Blank NV group, and its activity decreased with increasing effector-to-target ratio. Figure 3 (B)

[0051] Therefore, the dual-targeting nanovesicles constructed in this invention can be used to activate and guide NK cells to target and kill HER2-positive malignant tumors by targeting the NK cell activation receptor CD16 and the HER2 molecule on the surface of tumor cells, effectively avoiding problems such as short blood half-life and gene manipulation at the NK cell level.

[0052] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A dual-targeting recombinant fusion protein, characterized in that, The dual-targeting recombinant fusion protein is composed of signal peptide SP, HAtag, αCD16 scFv, αHER2 scFv and Lamp2b transmembrane protein linked sequentially; these fragments are linked by the same linker, the amino acid sequence of which is shown in SEQ ID NO. 6; The heavy chain amino acid sequence of the αCD16 scFv is shown in SEQ ID NO. 1, and the light chain amino acid sequence of the αCD16 scFv is shown in SEQ ID NO. 2; The heavy chain amino acid sequence of the αHER2scFv is shown in SEQ ID NO. 3, and the light chain amino acid sequence of the αHER2 scFv is shown in SEQ ID NO. 4; The amino acid sequence of the Lamp2b transmembrane protein is shown in SEQ ID NO.

5.

2. A dual-targeting nanovesicle containing the dual-targeting recombinant fusion protein of claim 1.

3. The dual-targeting nanovesicles according to claim 2, characterized in that, The nanovesicles were extracted from HEK293 cells.

4. The method for constructing dual-targeted nanovesicles according to claim 2 or 3, characterized in that, Includes the following steps: The dual-targeting nanovesicles were obtained by infecting eukaryotic cells with a lentivirus containing the dual-targeting recombinant fusion protein of claim 1.

5. The use of the dual-targeting recombinant fusion protein of claim 1 or the dual-targeting nanovesicles of claim 2 or 3 in the preparation of a drug for inhibiting tumors; wherein the tumor is: HER2-positive gastric cancer and / or HER2-overexpressing colorectal cancer.

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