Bacterial outer membrane vesicle containing CD47 antibody as well as preparation and application of bacterial outer membrane vesicle

Abstract

The invention relates to preparation of engineered bacterium outer membrane vesicles and application of the engineered bacterium outer membrane vesicles in postoperative tumor recurrence resistance, and belongs to the technical field of tumor immune checkpoint blocking preparations. Escherichia coli is subjected to gene recombination, an engineering strain capable of efficiently expressing the CD47 antibody is constructed, and an outer membrane vesicle (OC47) loaded with the CD47 antibody is extracted from the engineering strain. Furthermore, a photosensitizer Ce6 is loaded into the OC47. The OC47 can be combined with CD47 on tumor cells and Toll-like receptors on tumor-related macrophages at the same time, so that the tumor-related macrophages are promoted to enter an M1 polarization state, a'eating-my 'signal on the tumor cells is blocked at the same time, and the tumor-related macrophages are stimulated to enter an'eating-my' state to effectively swallow the tumor cells. The synergistic effect obviously enhances the phagocytosis and removal capabilities of macrophages on tumor cells. Under laser irradiation, Ce6 generates reactive oxygen species (ROS), and the effect of killing tumor cells is further enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor immune checkpoint blockade agents, and specifically, to the preparation of engineered bacterial outer membrane vesicles and their application in anti-tumor recurrence after surgery. Background Art

[0002] Surgical resection, as the core means for the treatment of solid tumors, although continuously innovating in the directions of precision and minimally invasive, the residual and metastatic tumor cells after surgery remain a fatal bottleneck leading to treatment failure. In essence, traditional surgery has two major biological limitations: First, physical resection is difficult to completely remove infiltrating tumor cells. Infiltrating tumor cells have strong invasiveness and migratory ability, can penetrate into the surrounding tissues, resulting in the existence of microscopic residual lesions. These cells are not completely removed during the operation and are difficult to detect by conventional imaging examinations such as CT, MRI, and ultrasound. Existing detection methods mainly target larger lesions, and the sensitivity for detecting microscopic lesions is low, so the signals of early recurrence are often missed. Second, the postoperative inflammatory environment will attract a large number of immunosuppressive macrophages, which form an immunosuppressive microenvironment by secreting anti-inflammatory factors (such as IL-10), further promoting the escape of residual tumor cells and accelerating their metastasis and recurrence. Therefore, although surgical resection can effectively remove part of the tumor, due to the influence of the above biological factors, the risk of tumor recurrence still exists, and there is an urgent need to develop more efficient treatment strategies to reduce the occurrence of recurrence and improve the long-term survival rate of patients. Summary of the Invention

[0003] In view of the above problems existing in postoperative recurrence and metastasis, the present invention provides a method for preventing tumor recurrence and metastasis after surgery by using engineered bacterial outer membrane vesicles (OC47) to transform tumor-associated macrophages from immunosuppressive type to anti-tumor M1 type macrophages and immune checkpoint inhibition of CD47 antibody. Further, taking OC47 as a carrier to load photosensitizer Ce6 (OC47-Ce6), which generates reactive oxygen species (ROS) under laser irradiation to further kill tumor cells. Finally, a method of synergistic action of macrophage phenotype reprogramming - immune checkpoint blockade - in-situ phototherapy is formed.

[0004] According to the first aspect of the present invention, there is provided a method for preparing a bacterial outer membrane vesicle containing a CD47 antibody, comprising the following steps:

[0005] (1) Insert the DNA fragment of the CD47 antibody into an expression plasmid, and then transfect the plasmid into Escherichia coli to obtain an engineered bacterium; the sequence of the DNA fragment is as shown in SEQ NO:1;

[0006] (2) Culture the engineered bacteria obtained in step (1), then inoculate single colonies for culture. Centrifuge the single colony culture to remove bacteria. Filter the supernatant through an ultrafiltration tube, then concentrate the filtrate. Centrifuge the filtrate, resuspend the precipitate to obtain bacterial outer membrane vesicles containing CD47 antibody.

[0007] Preferably, mix the bacterial outer membrane vesicles containing CD47 antibody with the photosensitizer Ce6 so that the bacterial outer membrane vesicles are loaded with Ce6.

[0008] According to another aspect of the present invention, there is provided the bacterial outer membrane vesicles containing CD47 antibody prepared by the above method.

[0009] According to another aspect of the present invention, there is provided the use of the bacterial outer membrane vesicles containing CD47 antibody in the preparation of a drug for inhibiting tumor recurrence and / or inhibiting tumor metastasis.

[0010] Preferably, the bacterial outer membrane vesicles are used to inhibit tumor recurrence and metastasis; and by activating the immune system, enhancing the immune clearance of tumor cells, thereby reducing the occurrence of tumor recurrence and metastasis.

[0011] Preferably, the bacterial outer membrane vesicles are used to interact with macrophages and induce M1 polarization of macrophages; and the bacterial outer membrane vesicles block the CD47-SIRPα pathway on the surface of tumor cells through the carried CD47 antibody.

[0012] Preferably, the bacterial outer membrane vesicles enhance the local concentration of the photosensitizer by delivering Ce6; under laser irradiation, the photosensitizer Ce6 generates a large amount of reactive oxygen species, causing strong oxidative damage to tumor cells, thereby leading to the destruction of cell membranes and DNA, and further inducing apoptosis of tumor cells.

[0013] Generally speaking, compared with the prior art by the above technical solutions conceived by the present invention, the main technical advantages are as follows:

[0014] (1) Aiming at the difficult problems of tumor recurrence and metastasis caused by residual microtumors after surgical resection and the immunosuppressive microenvironment, the present invention recombinantly modifies the genes of Escherichia coli by synthetic biology means to construct an engineered strain capable of highly expressing CD47 antibody, and extracts outer membrane vesicles (OC47) loaded with CD47 antibody therefrom. Further, the present invention also loads the small molecule photosensitizer Ce6 into OC47 to form an OC47-Ce6 complex. OC47 can simultaneously bind to CD47 on tumor cells and Toll-like receptors on tumor-associated macrophages, thereby promoting tumor-associated macrophages to enter the M1 polarization state, while blocking the "don't eat me" signal on tumor cells, stimulating tumor-associated macrophages to enter the "eat me" state and effectively phagocytosing tumor cells. This synergistic effect reverses the immunosuppressive microenvironment and significantly enhances the phagocytosis and clearance ability of macrophages against tumor cells. At the same time, under laser irradiation, Ce6 generates reactive oxygen species (ROS), further enhancing the effect of killing tumor cells. Combining the immune activation effect of OC47 with the photodynamic therapy of Ce6 can not only enhance the direct killing effect on tumor cells, but also activate the anti-tumor immune response by remodeling the tumor microenvironment, thereby improving the overall treatment effect and effectively inhibiting postoperative tumor recurrence and metastasis.

[0015] (2) The preparation method of the present invention is simple and efficient: the present invention efficiently prepares outer membrane vesicles of CD47 antibody through an Escherichia coli expression system, avoiding the complex steps in traditional antibody production and improving production efficiency and cost-effectiveness.

[0016] (3) The present invention enhances the immune regulation function: the present invention can effectively induce macrophages to polarize into pro-inflammatory M1 type, enhancing the immune response, and has stronger immune regulation ability compared with the prior art.

[0017] (4) The present invention has a significant anti-tumor effect: the present invention significantly inhibits tumor growth and enhances immune memory by activating CD8 + T cells and promoting macrophage immune polarization, and the effect is better than that of traditional treatment methods.

[0018] (5) The present invention avoids the safety risks brought by live bacteria: since bacterial outer membrane vesicles rather than live bacteria are used as carriers, the present invention avoids the safety problems that may be brought by using live bacteria and ensures the safety of treatment.

[0019] (6) The present invention can enhance the immune response by transforming tumor-associated macrophages from an immunosuppressive phenotype into anti-tumor M1 macrophages and combining immune checkpoint inhibition. Bacterial outer membrane vesicles (OMV) as natural immune regulators can activate tumor-associated macrophages and enhance their phagocytic function, contributing to the clearance of residual tumor cells. By engineering bacteria to secrete OMV containing CD47 antibody, postoperative tumor recurrence and metastasis can be effectively prevented.

[0020] (7) In the present invention, the photosensitizer Ce6 is loaded on OC47 as a carrier, combining the function of OC47 with photodynamic therapy, enhancing the direct killing effect on tumor cells, activating the anti-tumor immune response, and reducing the risk of tumor recurrence and metastasis after surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the TEM image of OC47 of the present invention.

[0022] Figure 2 It is the fluorescence intensity of CD47 antibody detected by flow cytometry for OC47 of the present invention.

[0023] Figure 3 It is the content of CD47 antibody in each milligram of OMV and OC47 of the present invention.

[0024] Figure 4 It is the ultraviolet-visible absorption spectrum of OC47-Ce6 and Ce6 of the present invention.

[0025] Figure 5 It is the fluorescence spectrum of OC47-Ce6 and Ce6 of the present invention.

[0026] Figure 6 It is the ultraviolet-visible absorption spectrum of OC47-Ce6 decomposing DPBF of the present invention.

[0027] Figure 7 It is the proportion of CD86 + cells in RAW 246.7 at different times after treatment with OC47 of the present invention.

[0028] Figure 8 It is the mRNA levels of IL-6, IL-12 and TNF-α in RAW 246.7 after different treatments with OC47 of the present invention.

[0029] Figure 9 It is the confocal microscopy image of RAW 246.7 cells expressing matrix metalloproteinase 9 (MMP9) after treatment with OC47 of the present invention.

[0030] Figure 10 It is the CD86 in the tumor tissue after treating mice with OC47-Ce6 of the present invention. + F4 / 80 + and CD206 + F4 / 80 + Flow cytometry of cells.

[0031] Figure 11 It is the CD8 in the tumor, lymph nodes and spleen after treating mice with OC47-Ce6 of the present invention. + CD3 +Flow cytometry of T cells.

[0032] Figure 12 This shows the growth of distal tumors in mice after treatment with OC47-Ce6 of the present invention.

[0033] Figure 13 This shows CD8 in lymph nodes and spleen in the distal model after treatment with OC47-Ce6 of the present invention. + CD3 + Flow cytometry of T cells.

[0034] Figure 14 This shows the changes in blood routine and blood biochemical related indicators of mice after treatment with OC47-Ce6.

[0035] Figure 15 This shows the anti-tumor mechanism of OC47-Ce6. Detailed implementation manners

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0037] The present invention prepared a bacterial outer membrane vesicle (OC47) containing a CD47 antibody, and further loaded the photosensitizer Ce6 (OC47-Ce6). The OC47-Ce6 solution was injected in situ at the local site after tumor resection surgery. OC47-Ce6 can bind to CD47 on the surface of tumor cells and Toll-like receptors on the surface of tumor-associated macrophages at the same time, promote the polarization of tumor-associated macrophages to the M1 type, and at the same time block the "don't eat me" signal expressed on tumor cells, thereby significantly enhancing the phagocytic function of tumor-associated macrophages, and further effectively clearing tumor cells. And under laser irradiation, it can activate Ce6 to generate reactive oxygen species (ROS) to further kill tumor cells. Combining the immune activation effect of OC47 with the photodynamic therapy of Ce6 can not only enhance the direct killing effect on tumor cells, but also reshape the immunosuppressive tumor microenvironment, thereby stimulating an anti-tumor immune response. This combined strategy can not only improve the treatment response rate, but also reduce the probability of postoperative recurrence and metastasis through the immune memory effect.

[0038] The core content of the present invention can be summarized as the following aspects:

[0039] (1) Expression of CD47 antibody and preparation of outer membrane vesicles:

[0040] The present invention constructs a system capable of expressing CD47 antibody by using an Escherichia coli expression system and recombinant Escherichia coli. The CD47 antibody gene fragment is inserted into an appropriate expression vector and transferred into Escherichia coli for expression. Then, outer membrane vesicles (OC47) containing CD47 antibody are obtained by extraction. This method purifies the outer membrane vesicles through steps such as high-speed centrifugation and filtration, making the preparation process simple and efficient.

[0041] (2) Loading photosensitizer Ce6 onto OC47

[0042] The present invention uses OC47 as a drug carrier to deliver photosensitizer Ce6, enhancing the local concentration of the photosensitizer. Under laser irradiation, Ce6 can generate a large amount of reactive oxygen species (ROS), causing strong oxidative damage to tumor cells, leading to the destruction of cell membranes and DNA, and then inducing apoptosis of tumor cells.

[0043] (3) Role of OC47-Ce6 in immunomodulation:

[0044] Studies have shown that OC47-Ce6 can interact with macrophages and enhance the pro-inflammatory response of macrophages by inducing the polarization transformation of macrophages. OC47-Ce6 can effectively regulate the function of macrophages, increase the secretion of inflammatory factors, and promote local immune responses. This process has important significance for exerting immunosuppressive effects on tumor cells with immune escape.

[0045] (4) Application of OC47-Ce6 in anti-tumor immunity:

[0046] The present invention further studies the role of OC47-Ce6 in anti-tumor immunity. By regulating the polarization of macrophages, OC47-Ce6 can effectively induce pro-inflammatory immune responses of immune cells in the tumor microenvironment, inhibit tumor growth, and enhance the immune activity of T cells. OC47-Ce6 effectively enhances the anti-tumor immune response by increasing the proportion of CD8 + T cells in the tumor microenvironment. In addition, OC47-Ce6 can also activate the immune memory function and play a positive role in preventing tumor recurrence.

[0047] (5) Activation of immune memory:

[0048] The present invention further verifies the activation effect of OC47-Ce6 on immune memory through experiments. OC47-Ce6 can improve the ability of the immune system to recognize and eliminate tumor cells, enhance the reactivity and memory of T cells, and thus effectively prevent tumor recurrence. This activation mechanism of immune memory provides new ideas for cancer immunotherapy and has potential clinical application value.

[0049] The present invention provides a method for extracting bacterial outer membrane vesicles containing anti-CD47 antibodies, comprising the following steps:

[0050] (1) Construction of Escherichia coli expressing anti-CD47 antibodies

[0051] First, the DNA sequence of the anti-CD47 antibody is inserted into the expression plasmid pBV220, and then the plasmid is transfected into Escherichia coli MG1655.

[0052] (2) Extraction of outer membrane vesicles OMV and outer membrane vesicles OC47

[0053] Escherichia coli MG1655 expressing anti-CD47 antibodies is cultured on a solid LB agar plate at 37 °C for 12 hours. Then, a single colony is inoculated into LB liquid medium and cultured in a shaking incubator at 37 °C until the optical density (OD) reaches 1.0. 1 L of the bacterial culture is centrifuged at 8000 g for 20 minutes to remove bacteria, and the supernatant is filtered through a 0.45 μm vacuum filter. The filtrate is concentrated using a centrifugal filter with a molecular weight cut-off of 100 kDa. Then, the concentrated medium is centrifuged at 200000 g at 4 °C for 3 hours. The OC47 precipitate is resuspended in PBS and filtered through a 0.22 μm vacuum filter to prevent bacterial contamination. The method for extracting vesicles from the original Escherichia coli (MG1655) is the same, and outer membrane vesicles OMV are obtained.

[0054] Furthermore, it includes loading the photosensitizer Ce6 with OC47 as a carrier, specifically: OC47 and Ce6 are mixed, stirred in the dark at 4 °C for 2 hours, and then centrifuged and washed 3 times with an ultrafiltration tube. OC47-Ce6 is obtained.

[0055] Figure 1 This is the TEM image of the outer membrane vesicles OC47 prepared in this example. The figure shows that the size of OC47 is 27.27 ± 3.74 nm.

[0056] Figure 2 This is the flow cytometry of the outer membrane vesicles OC47 prepared in this example. The figure verifies the expression of anti-CD47 antibodies in OC47.

[0057] Figure 3 This is the ELISA detection of the outer membrane vesicles OC47 prepared in this example. Through detection with an ELISA kit, the precise content of anti-CD47 antibodies in OC47 is 90 μg / mg.

[0058] OC47 and Ce6 are mixed, stirred in the dark at 4 °C for 2 hours, and then centrifuged and washed 3 times with an ultrafiltration tube. OC47-Ce6 is obtained.

[0059] Figure 4The UV-Vis absorption spectra of OC47-Ce6 and Ce6 prepared in this example are shown. The results indicate that the UV-Vis spectrum of OC47-Ce6 contains the characteristic absorption peaks of Ce6.

[0060] Figure 5 The fluorescence emission spectra of OC47-Ce6 and Ce6 prepared in this example are shown. The results indicate that the emission spectrum of OC47-Ce6 contains the fluorescence emission peaks of Ce6.

[0061] Figure 6 The UV-Vis absorption spectra of OC47-Ce6 prepared in this example for the decomposition of DPBF (1,3-diphenylisobenzofuran) are shown. OC47-Ce6 was added to the DPBF solution, and the mixed solution was irradiated with a 606 nm laser. The irradiation was stopped at different time points (2 minutes, 5 minutes, 10 minutes, and 15 minutes), and the absorption spectra were immediately measured. DPBF reacts with singlet oxygen to produce colorless products, resulting in a decrease in absorbance. As the irradiation time increases, the absorbance of the DPBF and OC47-Ce6 mixed solution at 410 nm gradually decreases. This indicates that OC47-Ce6 generates ROS under light irradiation, leading to the bleaching of DPBF, thus demonstrating the photodynamic activity of OC47-Ce6.

[0062] The present invention further studied the role of OC47 in macrophage polarization, including the following content:

[0063] Figure 7 The flow cytometry analysis of OC47 prepared in this example after incubation with macrophages is shown. OC47 with a total protein of 5 μg / mL was incubated with M0 macrophages for 0 h, 6 h, 24 h, and 48 h respectively. After collecting the cells and labeling them with FITC-anti-mouse CD86, the cells were analyzed by flow cytometry to detect the proportion of CD86-positive cells. The figure shows that as the incubation time of extracellular vesicle OC47 with M0 macrophages prolongs, the proportion of CD86-positive cells gradually increases.

[0064] Figure 8 The qPCR results of inflammatory factors of extracellular vesicle OC47 prepared in this example after incubation with macrophages are shown. Extracellular vesicle OC47 was co-incubated with M0 macrophages for 48 h. After the incubation, the cells were collected and RNA was extracted. RNA was transcribed into cDNA using reverse transcriptase, and the expression levels of inflammatory factors such as IL-6, IL-12, and TNF-α were detected by quantitative PCR technology. The figure shows that after OC47 acts on M0 macrophages, the release of IL-6, IL-12, and TNF-α increases.

[0065] Figure 9The expression of MMP9 on the surface of macrophages after incubation with the extracellular membrane vesicles OC47 prepared in this example. The extracellular membrane vesicles OC47 were co-incubated with M0 macrophages for 48 h. After the incubation, the macrophages were labeled with anti-MMP9 antibody, and detected by combining with a fluorescence-labeled secondary antibody. Subsequently, a confocal microscope was used to observe the expression of MMP9. The figure shows that the expression of MMP9 on the surface of macrophages treated with OC47 was enhanced, indicating that OC47 can regulate the function of macrophages and enhance their pro-inflammatory response.

[0066] To evaluate the anti-tumor efficacy of OC47-Ce6, 90% of the tumor in tumor-bearing mice was resected to mimic the environment of residual tumors after surgical resection, and the inhibition of tumor recurrence after surgery was studied. To simulate the situation of incomplete tumor resection clinically, tumor cells were inoculated into the back of mice. When the tumor volume reached 120 mm 3 ³, 90% of the tumor tissue was surgically resected, and then a solution of extracellular membrane vesicles OC47-Ce6 was injected at the wound site and then sutured.

[0067] The present invention further studied the role of OC47-Ce6 in activating immunity in mice with a surgical resection model, including the following:

[0068] Figure 10 The polarization state of tumor-associated macrophages of OC47-Ce6 prepared in this example in the surgical resection tumor model. After the surgical resection tumor model was established, each group of mice received different treatments (each mouse was administered 50 μg total protein of OMV or 50 μg total protein of OC47). Three days after administration, the tumor tissue was removed and made into a single-cell suspension. Macrophages were labeled with surface markers F4 / 80, CD206 and CD86, and then the polarization state of tumor-associated macrophages in the tumor microenvironment was analyzed by flow cytometry. Flow cytometry analysis showed that the proportion of M2-type macrophage markers (F4 / 80 + CD206 + cells) in the OC47 group was significantly reduced, and the proportion of M1-type macrophage markers (F4 / 80 + CD86 + cells) was significantly increased compared with the PBS group. It is shown that the treatment with OC47-Ce6 can effectively induce macrophages to polarize into pro-inflammatory M1 type and inhibit the generation of anti-inflammatory M2 type. This change in the polarization state not only enhances the local immune response, but also contributes to the remodeling of the tumor microenvironment.

[0069] Figure 11 CD8 in the tumor, spleen and lymph nodes of mice in the surgical resection tumor model of OC47-Ce6 prepared in this example +Changes in T cells. After the establishment of the tumor resection model, each group of mice received different treatments (each mouse was administered 50 μg of total protein of OMV, 50 μg of total protein of OC47, or 5 μg of Ce6). Seven days after administration, the spleen, lymph nodes, and tumor tissues were removed and made into single-cell suspensions. T cells were labeled with surface markers CD3, CD8, and CD4, and then analyzed by flow cytometry. The figure shows that the percentages of CD8 + T cells in the spleen, lymph nodes, and tumor of the OC47 group were 12.3%, 15.1%, and 20.4% higher than those in the PBS group, respectively.

[0070] The present invention further studied the activation effect of OC47-Ce6 on immune memory, including the following content:

[0071] To verify the activation effect of OC47-Ce6 on immune memory, tumor cells were subcutaneously injected into the contralateral side on the 20th day of treatment.

[0072] Figure 12 This shows the growth of the contralateral tumor after 20 days of treatment with the OC47-Ce6 prepared in this example. Twenty days after the treatment of the mice in the surgical resection model, tumor cells were subcutaneously inoculated into the contralateral side, and the growth of the contralateral tumor in the mice was regularly monitored, and the tumor volume was measured. The figure shows that the subcutaneous tumor growth in the mice treated with OC47-Ce6 was slow, and there was a significant difference compared with the blank group.

[0073] Figure 13 This shows the flow cytometry analysis of the lymph nodes and spleen of the mice after 20 days of treatment with the OC47-Ce6 prepared in this example. Twenty days after the treatment of the mice in the surgical resection model, tumor cells were subcutaneously inoculated into the contralateral side, and the growth of the contralateral tumor in the mice was regularly monitored. After the monitoring was completed, the spleen and lymph node tissues of the mice were collected and made into single-cell suspensions. Flow cytometry was used to analyze the proportion of CD8 + T cells in the spleen and lymph nodes. The figure shows that the proportions of CD8 + T cells in the spleen and lymph nodes of the OC47-Ce6 group were 12.9% and 17.1% higher than those in the PBS group, respectively.

[0074] Figure 14Changes in the blood routine and blood biochemical related indicators of mice treated with OC47-Ce6 prepared in this example. The mice were divided into a PBS group, an OC47 group, and an OC47-Ce6 group, and corresponding treatments were carried out (each mouse was administered 50 μg of total protein of OC47). After the treatment was completed, blood samples of the mice were collected for blood routine and blood biochemical analysis. By detecting relevant indicators in the blood, such as white blood cell count, red blood cell count, platelet count, liver and kidney functions, etc., the effects of OC47-Ce6 treatment on the hematological and biochemical indicators of mice were evaluated. The results showed that OC47-Ce6 treatment did not cause significant changes in hematological or biochemical indicators, proving its high safety.

[0075] Figure 15 Mechanism diagram of OC47-Ce6 prepared in this example for jointly inhibiting postoperative tumor recurrence through polarization of macrophage phenotype, immune checkpoint blockade, and photodynamic therapy. In the present invention, an engineered strain highly expressing CD47 antibody was constructed by recombinant Escherichia coli, and outer membrane vesicles (OC47) loaded with CD47 antibody were extracted. Subsequently, the photosensitizer Ce6 was loaded into OC47. OC47 can simultaneously bind to CD47 on tumor cells and Toll-like receptors on tumor-associated macrophages, promoting the polarization of macrophages to M1, while blocking the "don't eat me" signal of tumor cells and activating the "eat me" signal of macrophages, thereby enhancing their ability to phagocytose tumor cells. Under laser irradiation, the reactive oxygen species (ROS) generated by Ce6 further enhance the tumor cell killing effect.

[0076] It is easy for those skilled in the art to understand that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a bacterial outer membrane vesicle containing a CD47 antibody, characterized in that, Comprising the following steps: (1) Insert the DNA fragment of the CD47 antibody into an expression plasmid, and then transfect the plasmid into Escherichia coli to obtain an engineered bacterium; the sequence of the DNA fragment is as shown in SEQ NO:1; (2) Culture the engineered bacterium obtained in step (1), then inoculate single colonies, centrifuge the single colony culture to remove bacteria, filter the supernatant through an ultrafiltration tube, then concentrate the filtrate, centrifuge the filtrate, and resuspend the precipitate to obtain bacterial outer membrane vesicles containing the CD47 antibody.

2. The preparation method of the bacterial outer membrane vesicles containing the CD47 antibody according to claim 1, characterized in that, Mix the bacterial outer membrane vesicles containing the CD47 antibody with the photosensitizer Ce6 so that the bacterial outer membrane vesicles are loaded with Ce6.

3. Bacterial outer membrane vesicles containing the CD47 antibody prepared by the method according to claim 1 or 2.

4. Use of the bacterial outer membrane vesicles containing the CD47 antibody according to claim 3 in the preparation of a drug for inhibiting tumor recurrence and / or inhibiting tumor metastasis.

5. The application according to claim 4, characterized in that The bacterial outer membrane vesicles are used for inhibiting tumor recurrence and metastasis; and by activating the immune system, enhancing the immune clearance of tumor cells, thereby reducing the occurrence of tumor recurrence and / or metastasis.

6. The application according to claim 4 or 5, characterized in that, The bacterial outer membrane vesicles are used for interacting with macrophages to induce M1 polarization of macrophages; and the bacterial outer membrane vesicles block the CD47-SIRPα pathway on the surface of tumor cells through the carried CD47 antibody.

7. The application according to claim 6, wherein The bacterial outer membrane vesicles enhance the local concentration of the photosensitizer by delivering the photosensitizer Ce6; under laser irradiation, the photosensitizer Ce6 generates reactive oxygen species, causing oxidative damage to tumor cells, thereby leading to the destruction of cell membranes and DNA, and further inducing apoptosis of tumor cells.

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