Dual-targeting recombinant fusion protein and dual-targeting nano vesicle as well as construction method and application of dual-targeting recombinant fusion protein and dual-targeting nano vesicle

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a double-targeting recombinant fusion protein and a double-targeting nano vesicle as well as a construction method and application thereof, and the structure of the double-targeting recombinant fusion protein comprises alpha CD16scFv, alpha HER2scFv and Lamp2b transmembrane protein which are sequentially connected. The dual-targeting recombinant fusion protein disclosed by the invention takes NK cell activated receptor CD16 molecules and gastric cancer cell surface HER2 molecules as targets, can guide NK cells to recognize HER2 positive gastric cancer cells, and activates the NK cells through the CD16 molecules so as to kill the gastric cancer cells in a targeting manner, so that the aim of treating gastric cancer is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a dual-targeting recombinant fusion protein and a dual-targeting nanovesicle, and a construction method and application thereof. Background Art

[0002] Gastric cancer is a common digestive tract tumor. Since the early symptoms are not obvious, it is usually in the late stage or metastatic when it is discovered. Traditional chemotherapy drugs can improve the survival of patients with this type of gastric cancer, but the treatment effect is poor, and there are phenomena such as long course of disease and drug resistance. NK cell adoptive therapy is an emerging tumor treatment method that has achieved results in clinical trials for the treatment of hematological tumors, but the tumor suppressive microenvironment in solid tumors has seriously affected the efficacy of NK cells in solid tumors. Therefore, new methods are needed to activate and guide NK cells and enhance the anti-tumor effect of NK cells. Summary of the invention

[0003] The purpose of the present invention is to provide a dual-targeting recombinant fusion protein and a dual-targeting nanovesicle and a construction method and application thereof. The dual-targeting recombinant fusion protein can guide NK cells to recognize cancer cells, activate NK cells through CD16 molecules, and then 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 the dual-targeting recombinant fusion protein comprises αCD16 scFv, αHER2 scFv and Lamp2b transmembrane protein which are connected in sequence.

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

[0006] As a preferred embodiment, the heavy chain amino acid sequence of the αCD16 scFv is shown as SEQ ID NO.1, and the light chain amino acid sequence of the αCD16 scFv is shown as 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] The present invention provides a dual-targeting nanovesicle expressing the dual-targeting recombinant fusion protein.

[0010] As a preferred embodiment, the extraction source of the nanovesicles includes HEK293 cells.

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

[0012] The present invention provides the use of the dual-targeting recombinant fusion protein or the dual-targeting nanovesicle in the preparation of a drug for inhibiting tumors.

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

[0014] Beneficial effects: The present invention provides a dual-targeted recombinant fusion protein, the structure of which includes αCD16 scFv, αHER2 scFv and Lamp2b transmembrane protein connected in sequence. The dual-targeted recombinant fusion protein of the present invention targets the NK cell activating receptor CD16 molecule and the HER2 molecule on the surface of gastric cancer cells, can guide NK cells to recognize HER2-positive gastric cancer cells, activate NK cells through CD16 molecules and then target and kill gastric cancer cells, can express dual-targeted proteins, and achieve the purpose of treating gastric cancer.

[0015] The present invention provides a dual-targeted nanovesicle containing the dual-targeted recombinant fusion protein. The dual-targeted nanovesicle can act on the colorectal cancer cell line SW480 with high HER2 expression, has the effect of inhibiting the activity of multiple HER2-positive tumor cell lines, and has a broad-spectrum anti-tumor effect.

[0016] The present invention provides a method for constructing the above-mentioned dual-targeted nanovesicles, which utilizes the advantages of nanovesicles such as natural material transport properties, small molecular size and excellent biocompatibility as good carriers of therapeutic molecules. The constructed dual-targeted nanovesicles target NK cell activating receptor CD16 and HER2 molecules on the surface of tumor cells. The dual-targeted 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 genetic manipulation at the NK cell level. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 αCD16 / αHER2 NV characterization diagram; A: nanoparticle analyzer detection diagram, B: gel electrophoresis detection diagram, C: flow cytometry detection diagram;

[0019] Figure 3 This is a graph showing the effects of Blank NV and αCD16 / αHER2 NV on the activity of gastric cancer cells BGC823 and HGC27 observed by the CCK-8 method; A: same effector-target ratio but different concentrations, B: same concentration but different effector-target ratios. DETAILED DESCRIPTION

[0020] The purpose of the present invention is to provide a dual-targeted recombinant fusion protein and a dual-targeted nanovesicle and a construction method and application thereof. The dual-targeted recombinant fusion protein of the present invention 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, thereby achieving the purpose of treating cancer.

[0021] 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. As an embodiment, the literature is consulted to determine the single-chain variable region nucleic acid sequence (scFv) of CD16 and HER2 antibodies; the nucleic acid sequence is sequentially spliced ​​into target fragments in 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, and the amino acid sequence of linker is shown in SEQ ID NO.6: GGGGSGGGGSGGGGS, and the transmembrane sequence is derived from humans. Among them, the heavy chain and the affinity chain are connected by linker, and the relative position is 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 GRIDPANGNTKYDPNFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCACPTE 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 as shown in SEQ ID NO.5: ILIPIIVGAGLSGLIIVIVIAYVI.

[0025] The present invention provides a dual-targeted nanovesicle containing the above-mentioned dual-targeted recombinant fusion protein. The dual-targeted nanovesicle provided by the present invention can, on the one hand, increase the blood circulation half-life of the bispecific antibody; on the other hand, the nanovesicle, as a natural carrier, can be loaded with chemotherapeutic drugs, nucleic acid drugs, etc. through modification, providing the possibility of realizing all-in-one combined treatment of bispecific antibodies and chemotherapeutic drugs / nucleic acid drugs. In one embodiment, the extraction source of the nanovesicle includes HEK293 cells.

[0026] The present invention provides a method for constructing the above-mentioned dual-targeted nanovesicles, comprising the following steps: extracting and obtaining the dual-targeted nanovesicles after infecting eukaryotic cells with a lentivirus containing the above-mentioned dual-targeted recombinant fusion protein. As an embodiment, HEK293 cells are infected with a lentivirus containing the above-mentioned dual-targeted recombinant fusion protein, and then cultured and amplified in a high-glucose medium containing 10% fetal bovine serum; after the cells are digested, the cell pellet is washed with PBS, the cells are counted, and the cells are resuspended with PBS and the cell density is adjusted to 1×10 6 / mL, and then use Avanti liposome extruder to push and pull back and forth through a 400nm polycarbonate membrane 10 times; the resulting mixed solution is centrifuged and the supernatant is discarded, PBS is added to dissolve the precipitate, and after filtering and sterilization, the dual-targeted nanovesicles (αCD16 / αHER2NV) can be obtained. The nanovesicles are used as carriers of dual-targeted fusion proteins, and the dual-targeted fusion proteins are expressed on the outer surface of the nanovesicle membrane (see Figure 2 Middle C).

[0027] The present invention provides the use of the above-mentioned dual-targeted recombinant fusion protein or the above-mentioned dual-targeted nanovesicle in the preparation of a drug for inhibiting tumors. As an embodiment, the tumor includes: gastric cancer and / or rectal cancer. In one embodiment, the dual-targeted nanovesicle prepared above is applied to the co-culture of gastric cancer cells and PBMCs, and it is found that it has the effect of targeted activation of NK cells to kill HER2-positive gastric cancer cells. As an embodiment, the dual-targeted nanovesicle prepared above can be applied to the colorectal cancer cell line SW480 with high expression of HER2, and it is found that it has the effect of inhibiting the activity of multiple HER2-positive tumor cell lines, and has a broad-spectrum anti-tumor effect.

[0028] In order to further illustrate the present invention, a dual-targeting recombinant fusion protein and a dual-targeting nanovesicle provided by the present invention, as well as a construction method and application thereof are described in detail below in conjunction with the embodiments, but they 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 proteins

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

[0032] The nucleic acid sequences were spliced ​​into the target fragment in the order of signal peptide (SP), HAtag, αCD16 scFv, αHER2 scFv, Lamp2b transmembrane region, and GFP; the target fragment was commissioned to Nanjing GenScript Technology Co., Ltd. for synthesis, ligation to the overexpression plasmid, and packaging into lentivirus.

[0033] (2) Screening of HEK293 cell lines positive for lentivirus infection

[0034] HEK293 cells in logarithmic growth phase were digested and counted, and the cell density was adjusted to 2×10 5 100 μL / well of the culture medium was added to the cells cultured for 5 h. The volume ratio of high-glucose DMEM medium and lentiviral supernatant was 1:1. After remixing, it was added to the cells cultured for 5 h. Polybrene was added at a final concentration of 10 g / mL. After 4 h, it was replaced with 500 L of complete medium. After further culture for 24 h, some cells were digested and removed. The infection efficiency was detected by flow cytometry. The cells were divided into 2×10 5 / well were seeded in a 24-well plate, and 1 g / mL puromycin was added for 7 days to obtain positive cell lines.

[0035] 2. Preparation of αCD16 / αHER2 NV

[0036] The positive cell lines prepared above were cultured and expanded in a high-glucose medium containing 10% fetal bovine serum. When the cell number reached 1×10 6 After digestion, the cells were centrifuged at 300 g for 5 min, the supernatant was removed, the cell pellet was washed twice with PBS, the cells were counted, and the cells were resuspended with PBS and the cell density was adjusted to 1×10 6 / mL, and then use an Avanti liposome extruder to push and pull back and forth 10 times through a 400nm polycarbonate membrane; the resulting mixture is centrifuged at 4°C, 800g for 10 minutes, the supernatant is taken and centrifuged at 4°C, 10,000g for 30 minutes, the supernatant is taken again and centrifuged at 4°C, 100,000g for 1.5 hours, the supernatant is discarded, an appropriate amount of PBS is added to dissolve the precipitate, and after filtering and sterilization with a 0.22μm filter, dual-targeted nanovesicles (αCD16 / αHER2 NV) can be obtained, which are packaged and stored in a -80°C refrigerator for subsequent experiments and characterization and identification.

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

[0038] Comparative Example 1

[0039] The method of Example 1 is followed, except that: the lentivirus prepared in step 1(1) is replaced with a blank lentivirus (without any gene fragment); step 1(2) is performed to obtain blank HEK293 cells (control HEK293); and step 2 is performed to obtain blank nanovesicles (BlankNV).

[0040] Experimental Example 1 Identification of αCD16 / αHER2 NV

[0041] (1) αCD16 / αHER2 NV particle size analysis: The αCD16 / αHER2 NV prepared in Example 1 or the Blank NV prepared in Control Example 1 was diluted with purified water to an appropriate multiple to 1.0×10 8 ~2.5×10 9 / mL; use a 1mL syringe to draw 1mL of the diluted sample and inject it into the sample chamber of the nanoparticle tracking analyzer. Use the software provided by the Nanosight tracing analyzer to analyze and record the Brownian motion of the NV, detect the particle concentration and particle size, and the results are shown in Figure 2 Middle 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 (the volume ratio of RIPA:PMSF in the lysis buffer was 250:1), and the mixture was shaken once every 10 minutes for 1 minute each time, and repeated 4 times; centrifuged at 4°C and 12,000 g for 15 minutes, and the supernatant was collected. The protein concentration was determined by BCA protein quantification. 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. The results are shown in Figure 2 Middle B.

[0043] (3) Identification of αCD16 / αHER2 NV outer surface fusion protein expression: Take 30 μg of αCD16 / αHER2 NV suspension or BlankNV, place it together with 5 μL of latex beads in a 1.5 mL EP tube, dilute to 20 μL with PBS, and incubate at room temperature for 15 min; continue to add 1 mL of PBS, place on a shaker, and incubate at room temperature for 2 h; continue to add 20 μL of FBS, mix by pipetting, and block at room temperature for 30 min; centrifuge at 3000 g for 5 min, discard the supernatant, and white precipitate can be seen; add 1 mL of PBS, centrifuge at 3000 g for 5 min, wash once; discard the supernatant, resuspend in 100 μL of PBS, add 5 μL of APC-anti-HA.11 epitope tag antibody, mix by pipetting, and incubate at 4°C in the dark for 30 min; add 1 mL of PBS, centrifuge at 3000 g for 5 min, wash once, and detect on a machine. The results are shown in the table. Figure 2 Middle C.

[0044] from Figure 2 As can be seen from the results, the hydrated particle sizes of BlankNV and αCD16 / αHER2 NV were mainly concentrated around 120 nm using the Nanosight nanoparticle analyzer. Figure 2 (A). Western blot analysis of NV expression-related proteins showed that both NVs highly expressed CD9, Alix, HSP70, and Calenxin, but only αCD16 / αHER2 NVs expressed the tag protein HA ( Figure 2 (B). The results showed that αCD16 / αHER2NV was able to express the fusion protein. To further confirm that the constructed fusion protein was expressed on the outer surface of the NV membrane, flow cytometry was used to detect the expression of the tag protein HA on the cell membrane. The results showed that αCD16 / αHER2NV was able to display the fusion protein on the outer surface of the NV membrane, and the expression efficiency reached 15.8% ( Figure 2 Middle C).

[0045] Experimental Example 2: Activity of αCD16 / αHER2 NV in inhibiting 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, and 1×10 4 To detect the inhibitory effect of different concentrations of NV on the activity of gastric cancer cells, 1×10 5 Peripheral blood mononuclear cells (PBMCs) of individuals were cultured to make the effector-target ratio of PBMCs to gastric cancer cells E:T = 10:1; then, 10 μL PBS, different concentrations of BlankNV (10 -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 ng / mL) and incubated for 24 hours. To detect the effect of different effector-target ratios on the inhibitory activity of NV on gastric cancer, 1×10 4 , 2×10 4 , 5×10 4 , 1×10 5 , 1×10 6 PBMCs were cultured so that the effector-target ratio of PBMCs to gastric cancer cells was 1:1, 2:1, 5:1, 10:1, and 20:1. Then, PBS, 10 2 ng / mL Blank NV and 10 2 ng / mLαCD16 / αHER2 NV, co-incubated for 24 hours. At 24h of the detection time point, the cells were removed, the cell supernatant was discarded, and washed twice with PBS to wash away the suspended NK cells and dead gastric cancer cells; 100μL / well of the pre-prepared CCK-8 reagent was added to the adherent gastric cancer cells; after incubation at 37℃ for 2 hours, the absorbance value at 450nm was detected with an enzyme reader. The absorbance of cells treated with PBS was used as the 100% cell activity control for each treatment group. The results are shown in Figure 3 .

[0047] from Figure 3 It can be seen that at the same effector-target ratio, the activity of HER2-positive gastric cancer cell HGC27 in the αCD16 / αHER2 NV treatment group was lower than that in the Blank NV group, and its activity decreased with the increase of αCD16 / αHER2 NV concentration; however, the activity of HER2-negative gastric cancer cell BGC823 showed no difference between the αCD16 / αHER2 NV treatment group and the Blank NV treatment group ( Figure 3 The same concentration of NV was added to the culture system with different effector-target ratios. It was found that the activity of HER2-positive gastric cancer cell HGC27 in the αCD16 / αHER2 NV treatment group was lower than that in the Blank NV group, and its activity decreased with the increase of the effector-target ratio; however, the activity of HER2-negative gastric cancer cell 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 targeted activation of NK cells to kill HER2-positive gastric cancer cells.

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

[0049] Colorectal cancer SW480 cells (HER2 high-expressing cells) were digested with trypsin, counted and resuspended, and 1×10 4 To detect the inhibitory effect of different concentrations of NV on the activity of colorectal cancer cells, 1×10 5 Peripheral blood mononuclear cells (PBMCs) of individuals were cultured to make the effector-target ratio of PBMCs to colorectal cancer cells E:T = 10:1; then, PBS, different concentrations of BlankNV (10 -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 ng / mL) and incubated for 24 hours. To detect the effect of different effector-target ratios on the inhibitory activity of NV on colorectal cancer, after the colorectal cancer cells were attached overnight, 1×10 4 , 2×10 4 , 5×104 , 1×10 5 , 1×10 6 PBMCs were cultured so that the effector-target ratio of PBMCs to colorectal cancer cells was 1:1, 2:1, 5:1, 10:1, and 20:1. Then, PBS, 10 2 ng / mL Blank NV and 10 2 ng / mLαCD16 / αHER2 NV, co-incubated for 24 hours. At 24h of the detection time point, the cells were removed, the cell supernatant was discarded, and washed twice with PBS to wash away the suspended NK cells and dead gastric cancer cells; 100μL / well of the pre-prepared CCK-8 reagent was added to the adherent gastric cancer cells; after incubation at 37℃ for 2 hours, the absorbance value at 450nm was detected with an enzyme reader. The absorbance of cells treated with PBS was used as the 100% cell activity control for each treatment group. The results are shown in Figure 3 .

[0050] from Figure 3 It can be seen that at the same effector-target ratio, the activity of HER2-positive colorectal cancer cell SW480 in the αCD16 / αHER2 NV treatment group was lower than that in the Blank NV group, and its activity decreased with the increase of αCD16 / αHER2 NV concentration ( Figure 3 The same concentration of NV was added to the culture system with different effector-target ratios. It was found that the activity of HER2-positive colorectal cancer cell SW480 in the αCD16 / αHER2 NV treatment group was lower than that in the Blank NV group, and its activity decreased with the increase of the effector-target ratio ( Figure 3 (middle B).

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

[0052] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, 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 structure of the dual-targeting recombinant fusion protein includes αCD16 scFv, αHER2 scFv and Lamp2b transmembrane protein connected in sequence.

2. The dual-targeting recombinant fusion protein according to claim 1, characterized in that: The dual-targeting recombinant fusion protein is formed by sequentially connecting a signal peptide SP, a HAtag, an αCD16 scFv, an αHER2 scFv and a Lamp2b transmembrane protein.

3. The dual-targeting recombinant fusion protein according to claim 1 or 2, characterized in that: 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.

4. The dual-targeting recombinant fusion protein according to claim 1 or 2, characterized in that: 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.

5. The dual-targeting recombinant fusion protein according to claim 1 or 2, characterized in that: The amino acid sequence of the Lamp2b transmembrane protein is shown in SEQ ID NO.

5.

6. A dual-targeting nanovesicle containing the dual-targeting recombinant fusion protein according to any one of claims 1 to 5.

7. The dual-targeting nanovesicle according to claim 6, characterized in that: The extraction sources of the nanovesicles include HEK293 cells.

8. The method for constructing the dual-targeting nanovesicles according to claim 6 or 7, characterized in that: The following steps are involved: After infecting eukaryotic cells with a lentivirus comprising the dual-targeting recombinant fusion protein according to any one of claims 1 to 5, the dual-targeting nanovesicles are extracted.

9. Use of the dual-targeting recombinant fusion protein according to any one of claims 1 to 5 or the dual-targeting nanovesicle according to claim 6 or 7 in the preparation of a drug for inhibiting tumors.

10. The use according to claim 9, characterized in that: The tumors include: gastric cancer and / or colorectal cancer.

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