Genetically engineered bacterium outer membrane vesicle as well as preparation and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a genetically engineered bacterial outer membrane vesicle as well as preparation and application thereof, and the genetically engineered bacterial outer membrane vesicle is a bacterial outer membrane vesicle of a CLEC9A-scFv / CD3-scFv bispecific antibody through gene engineering overexpression. The invention relates to an application of a bacterial outer membrane vesicle of a gene engineering overexpression CLEC9A-scFv / CD3-scFv bispecific antibody in promoting the interaction of cDC1 and CD8 + T lymphocytes. The invention relates to an application of a bacterial outer membrane vesicle of a gene engineering overexpressed CLEC9A-scFv / CD3-scFv bispecific antibody in preparation of a tumor drug for treating hepatocellular carcinoma. The genetically engineered bacterial outer membrane vesicle disclosed by the invention can couple cDC1 and CD8 + T cells, so that the interaction between the cDC1 and the CD8 + T cells is enhanced, and effective activation of the cDC1 on the CD8 + T cells is realized; when the genetically engineered bacterium outer membrane vesicle is applied to the treatment process of tumors, the tumor immune microenvironment of hepatocellular carcinoma can be remodeled, the immunosuppression of hepatocellular carcinoma is relieved, and finally efficient killing of tumors is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a genetically engineered bacterial outer membrane vesicle and its preparation and application. Background Art

[0002] Primary liver cancer mainly includes three different pathological types: hepatocellular carcinoma, intrahepatic bile duct carcinoma, and mixed hepatocellular carcinoma-cholangiocarcinoma, of which hepatocellular carcinoma accounts for 85% to 90%. The main causes include chronic hepatitis B / C infection, cirrhosis, and aflatoxin exposure. Primary liver cancer is currently the fourth most common malignant tumor and the second leading cause of cancer-related death in my country, posing a serious threat to the lives and health of the Chinese people. Data from the World Health Organization (WHO) show that the five-year survival rate for patients with intermediate and late-stage hepatocellular carcinoma in my country is less than 10%. Most patients with hepatocellular carcinoma in my country are already in the intermediate and late stages when they are discovered, and systemic treatment is essential for these patients. As a first-line treatment option, immunotherapy is crucial in the systemic treatment of intermediate and late-stage hepatocellular carcinoma. However, due to the low objective response rate and minimal clinical benefit to patients, finding new therapeutic targets and more effective treatment strategies has important clinical application value.

[0003] Tumor microenvironment (TME) immunosuppression is one of the core mechanisms by which tumors evade immune surveillance and resist immunotherapy. It weakens anti-tumor immune responses through multiple cellular and molecular pathways, significantly reducing the efficacy of immunotherapy (such as PD-1 / PD-L1 inhibitors and CAR-T cell therapy). The microenvironment of hepatocellular carcinoma is highly immunosuppressive, and the development of strategies that can effectively reduce or reverse immunosuppression in the tumor microenvironment is of great significance for immunotherapy of hepatocellular carcinoma.

[0004] CD8+T cells refer to T lymphocytes that express CD8 molecules, also known as cytotoxic T lymphocytes, and are the most important effector cells for tumor killing. Type 1 conventional dendritic cells (cDC1) are key to anti-cancer immunity. cDC1 is particularly critical in presenting antigens, activating CD8+T cells, and initiating specific anti-tumor immune responses. Studies have shown that the tumor microenvironment hinders the interaction between cDC1 and CD8+T cells. If the microenvironmental barriers can be overcome, it is expected to promote the interaction between cDC1 and CD8+T cells, effectively activate CD8+T cells, and enhance anti-tumor immune responses.

[0005] Bacterial outer membrane vesicles (BMVs) are biomembrane vesicles naturally secreted during bacterial growth. They are approximately 20-250 nm in size and naturally carry pathogen-associated molecular patterns (PAMs) from the parent bacteria. Studies have shown that these PAMs can act as immune adjuvants, recruiting large numbers of immune cells to infiltrate tumor sites and activating the immune microenvironment. Furthermore, BMVs have high biocompatibility, good stability, and a long half-life, making them suitable as drug carriers and easily crossing biological barriers. These nanoparticles can also carry antibodies, receptors, or ligands while maintaining their original structure and function, enabling them to perform their biological functions.

[0006] The technical problem solved by the present invention is: how to improve the problem of immunosuppression in the tumor microenvironment of hepatocellular carcinoma, how to overcome the microenvironmental barriers that hinder the interaction between cDC1 and CD8+ T cells in hepatocellular carcinoma, synergistically enhance the anti-tumor immune response, and thus improve the effect of tumor immunotherapy.

[0007] To this end, a genetically engineered bacterial outer membrane vesicle and its preparation and application are provided to solve the above problems. Summary of the Invention

[0008] The present invention provides a genetically engineered bacterial outer membrane vesicle and its preparation and application, aiming to improve the problem of immunosuppression in the tumor microenvironment of hepatocellular carcinoma, overcome the microenvironmental barriers that hinder the interaction between cDC1 and CD8+ T cells in hepatocellular carcinoma, synergistically enhance the anti-tumor immune response, and improve the effect of tumor immunotherapy.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A genetically engineered bacterial outer membrane vesicle is a bacterial outer membrane vesicle that overexpresses a CLEC9A-scFv / CD3-scFv bispecific antibody through genetic engineering.

[0011] The method for preparing genetically engineered bacterial outer membrane vesicles comprises the following steps:

[0012] Step 1: Transform the plasmid of the CLEC9A-scFv / CD3-scFv bispecific antibody into competent E. coli W3110 (ΔmsbB), and pick monoclonal colonies using LB plates with corresponding resistance;

[0013] Step 2: Take the monoclonal colony obtained in step 1 and transfer it to LB liquid medium of corresponding resistance, shake and culture at 180 rpm and 37°C until its OD value reaches 0.6;

[0014] Step 3: Take the culture medium obtained in step 2, add 1 mM IPTG inducer, and culture with shaking at 180 rpm and 16°C for 16 hours;

[0015] Step 4: Centrifuge the culture medium obtained in step 3 at 4000 rpm for 15 min at 4°C, collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to obtain a filtered supernatant;

[0016] Step 5: The filtered supernatant obtained in step 4 was ultracentrifuged at 150,000 g for 2 h, the supernatant was discarded, the precipitate was resuspended with PBS, and the resuspended solution was collected to obtain bacterial outer membrane vesicles overexpressing the CLEC9A-scFv / CD3-scFv bispecific antibody.

[0017] Application of genetically engineered bacterial outer membrane vesicles overexpressing CLEC9A-scFv / CD3-scFv bispecific antibodies in promoting the interaction between cDC1 and CD8+ T lymphocytes.

[0018] Application of genetically engineered bacterial outer membrane vesicles overexpressing CLEC9A-scFv / CD3-scFv bispecific antibodies in the preparation of tumor drugs for the treatment of hepatocellular carcinoma.

[0019] The present invention has the following beneficial effects:

[0020] The genetically engineered bacterial outer membrane vesicles described in the present invention can couple cDC1 with CD8+ T cells, thereby enhancing the interaction between the two and achieving effective activation of CD8+ T cells by cDC1;

[0021] The genetically engineered bacterial outer membrane vesicles, when used in the treatment of tumors, can reshape the immune microenvironment of hepatocellular carcinoma tumors, reduce their immunosuppression, and ultimately achieve efficient tumor killing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the structure of the plasmid in step 1 of the method for preparing genetically engineered bacterial outer membrane vesicles of the present invention;

[0023] Figure 2 This is a WB characterization result diagram of the genetically engineered bacterial outer membrane vesicles of the present invention;

[0024] Figure 3 This is an electron microscopic image of the outer membrane vesicles of the genetically engineered bacteria of the present invention;

[0025] Figure 4 This is a diagram showing the NTA characterization results of the genetically engineered bacterial outer membrane vesicles of the present invention;

[0026] Figure 5 This is a graph showing the ELISA results of the binding of the CLEC9A-scFv / CD3-scFv bispecific antibody carried on the surface of the genetically engineered bacterial outer membrane vesicles of the present invention to the corresponding antigen;

[0027] Figure 6 This is a representative flow cytometry image of the genetically engineered bacterial outer membrane vesicles enhancing the interaction between cDC1 and T cells according to the present invention;

[0028] Figure 7 This is a curve diagram of the changes in tumor volume of mouse hepatocellular carcinoma after treatment with the genetically engineered bacterial outer membrane vesicles of the present invention. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Bacterial outer membrane vesicles overexpressing the CLEC9A-scFv / CD3-scFv bispecific antibody were obtained by transforming the CLEC9A-scFv / CD3-scFv plasmid into E. coli W3110 (ΔmsbB) and inducing and ultracentrifuging.

[0031] Bacterial outer membrane vesicles (BMVs) are biomembrane vesicles naturally secreted during bacterial growth. They are approximately 20-250 nm in size and naturally carry pathogen-associated molecular patterns (PAMs) from the parent bacteria. Studies have shown that these PAMs can act as immune adjuvants, recruiting large numbers of immune cells to infiltrate tumor sites and activating the immune microenvironment. Furthermore, BMVs have high biocompatibility, good stability, and a long half-life, making them suitable as drug carriers and easily crossing biological barriers. These nanoparticles can also carry antibodies, receptors, or ligands while maintaining their original structure and function, enabling them to perform their biological functions.

[0032] Therefore, in order to achieve better immunotherapy effects against hepatocellular carcinoma, we overexpressed the CLEC9A-scFv / CD3-scFv bispecific antibody on the surface of bacterial outer membrane vesicles, which is of great significance for the immunotherapy of hepatocellular carcinoma.

[0033] Preferably, the method for extracting bacterial outer membrane vesicles overexpressing the CLEC9A-scFv / CD3-scFv bispecific antibody is:

[0034] (1) The plasmid of the CLEC9A-scFv / CD3-scFv bispecific antibody was transformed into competent E. coli W3110 (ΔmsbB), and monoclonal colonies were picked using LB plates with corresponding resistance;

[0035] (2) Take the monoclonal colony from (1) and transfer it to the corresponding resistance LB liquid culture medium, shake and culture at 180 rpm and 37°C until the OD value reaches 0.6;

[0036] (3) Take the culture medium in (2), add 1 mM IPTG inducer, and culture with shaking at 180 rpm and 16°C for 16 hours;

[0037] (4) The culture medium in (3) was centrifuged at 4000 rpm for 15 min at 4°C, the supernatant was collected, and the supernatant was filtered through a 0.22 μm filter membrane;

[0038] (5) The supernatant after filtration in step (4) was ultracentrifuged at 150,000 g for 2 h, the supernatant was discarded, the precipitate was resuspended with PBS, and the resuspended solution was collected to obtain bacterial outer membrane vesicles overexpressing the CLEC9A-scFv / CD3-scFv bispecific antibody.

[0039] Bacterial outer membrane vesicles overexpressing the CLEC9A-scFv / CD3-scFv bispecific antibody can accumulate at the tumor site of hepatocellular carcinoma. The CLEC9A-scFv / CD3-scFv bispecific antibody carried on the surface of the bacterial outer membrane vesicles can enhance the interaction between cDC1 and T cells and effectively activate CD8+ T cells; the pathogen-associated molecular patterns carried by the bacterial outer membrane vesicles can increase the infiltration of immune cells at the tumor site, reverse the transformation of tumor-associated macrophages from M2 to M1, reduce the immunosuppression of the tumor microenvironment, and ultimately play a synergistic killing role against hepatocellular carcinoma.

[0040] Therefore, the present invention also provides use of the bacterial outer membrane vesicles overexpressing the CLEC9A-scFv / CD3-scFv bispecific antibody in the preparation of tumor drugs for the treatment of hepatocellular carcinoma.

[0041] Example 1 Synthesis of genetically engineered bacterial outer membrane vesicles

[0042] The ClyA gene was amplified from the genome of Escherichia coli (strain W3110) and cloned into the pET28a vector. Next, the CLEC9A-scFv-CD3-scFv bispecific antibody and 3HA tag were placed downstream of ClyA, respectively, where the CLEC9A-scFv-CD3-scFv bispecific antibody was fused to the C-terminus of ClyA, and the HA tag was fused to the C-terminus of the CLEC9A / CD3 bispecific antibody, ultimately obtaining the recombinant protein ClyA-CLEC9A-scFv-CD3-scFv-3HA sequence. In addition, a flexible linker GGGGSGGGGSGGGGS was inserted between the VH and VL of CLEC9A-scFv and between the VH and VL of CD3-scFv. Figure 1The pET28a-ClyA-CLEC9A-scFv-CD3-scFv-3HA plasmid was transformed into E. coli W3110 (ΔmsbB). The CLEC9A-scFv-CD3-scFv bispecific antibody was fixed to the membrane of bacterial outer membrane vesicles in series with the ClyA protein, thereby achieving the engineered bacterial outer membrane vesicles to simultaneously target CLEC9A on the surface of cDC1 and CD3 on the surface of CD8+ T cells in the tumor, enhancing the interaction between cDC1 and CD8+ T cells.

[0043] The Anti-CLEC9A scFv and Anti-CD3 scFv gene sequences were obtained from existing technologies. After the recombinant protein ClyA-CLEC9A-scFv-CD3-scFv-3HA sequence was designed, it was synthesized by BGI. The Anti-CLEC9A scFv gene sequence is shown in the sequence listing as SEQ ID NO. 1. The Anti-CD3 scFv gene sequence is shown in the sequence listing as SEQ ID NO. 2.

[0044] The method for preparing genetically engineered bacterial outer membrane vesicles comprises the following steps:

[0045] Step 1: Transform the plasmid of the CLEC9A-scFv / CD3-scFv bispecific antibody into competent E. coli W3110 (ΔmsbB), and pick monoclonal colonies using LB plates with corresponding resistance;

[0046] Step 2: Take the monoclonal colony obtained in step 1 and transfer it to LB liquid medium of corresponding resistance, shake and culture at 180 rpm and 37°C until its OD value reaches 0.6;

[0047] Step 3: Take the culture medium obtained in step 2, add 1 mM IPTG inducer, and culture with shaking at 180 rpm and 16°C for 16 hours;

[0048] Step 4: Centrifuge the culture medium obtained in step 3 at 4000 rpm for 15 min at 4°C, collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to obtain a filtered supernatant;

[0049] Step 5: The filtered supernatant obtained in step 4 was subjected to ultracentrifugation at 150,000 g for 2 h, the supernatant was discarded, the precipitate was resuspended with PBS, and the resuspension was collected to obtain bacterial outer membrane vesicles overexpressing the CLEC9A-scFv / CD3-scFv bispecific antibody, which was stored at -80°C. Example 2 Western-blot characterization of the surface of genetically engineered bacterial outer membrane vesicles containing the CLEC9A-scFv / CD3-scFv bispecific antibody

[0050] (1) Collect a certain amount of bacterial outer membrane vesicles, wash them twice with pre-cooled PBS, discard the supernatant, add the prepared protein lysis buffer, shake and mix, and place on ice for 30 minutes;

[0051] (2) Centrifuge at 12,000 g for 15 min at 4°C and transfer the supernatant to a new EP tube;

[0052] (3) Protein concentration was determined using a BCA protein quantification kit;

[0053] (4) Add denaturation buffer to adjust the sample protein concentration so that the concentration of each sample is consistent, and place in a metal bath at 100°C for 10 minutes to fully denature the protein;

[0054] (5) SDS-PAGE electrophoresis: Prepare the corresponding ratio of separation gel and stacking gel according to the molecular weight of the target protein. Load 20 μL of sample into each well, perform electrophoresis on the stacking gel at a constant voltage of 80 V, and on the separation gel at a constant voltage of 120 V until the target protein is separated and then perform electroblotting;

[0055] (6) Transfer: Transfer the proteins from the SDS-PAGE gel to a PVDF membrane using wet electrophoresis transfer in pre-cooled electrophoresis transfer buffer at a constant current of 250 mA for 90 min.

[0056] (7) Blocking: Prepare 5% BSA·PBST and block the transferred PVDF membrane at room temperature for 1-2 hours;

[0057] (8) Antibody incubation: Cut the membrane according to the molecular weight of the target protein, place it in the prepared primary antibody dilution solution (diluted according to the recommended ratio in the instructions), and incubate it on a shaker at 4°C overnight (12 hours). The next day, remove the PVDF membrane and wash it three times with PBST, each time for 10 minutes. Place it in the secondary antibody dilution solution and incubate it at room temperature for 1.5 hours, and then wash it three times with PBST, each time for 10 minutes.

[0058] (9) Development: ECL chemical development method was used, and imaging analysis was performed using a gel imaging system;

[0059] The results are as follows Figure 2 As shown, the recombinant protein band with the tag protein HA is clearly displayed, indicating that the genetically engineered bacterial outer membrane vesicles have successfully expressed the CLEC9A-scFv / CD3-scFv bispecific antibody.

[0060] Example 3 Electron microscopy and NTA characterization of genetically engineered bacterial outer membrane vesicles

[0061] Transmission electron microscopy (TEM) was used for morphological characterization. The bacterial outer membrane vesicles were adsorbed on a copper grid and negatively stained with 2% uranyl acetate solution for 10 seconds, and the excess dye was removed with absorbent paper. Figure 3 As shown, the bacterial outer membrane vesicles are round structures, which are consistent with the morphology of traditional bacterial outer membrane vesicles.

[0062] NanoSight NS300 system was then used to perform nanoparticle tracking analysis to measure particle size distribution and concentration. Figure 4 As shown, the vesicle size is about 35 nm, which is consistent with the size of bacterial outer membrane vesicles.

[0063] Example 4 ELISA test of specific binding between the CLEC9A-scFv / CD3-scFv bispecific antibody carried on the surface of genetically engineered bacterial outer membrane vesicles and the corresponding antigen

[0064] (1) ELISA plates were coated with the extracellular domain of recombinant mouse CLEC9A or mouse CD3 (0.1 μg / well) overnight at 4°C. All subsequent steps were performed at room temperature.

[0065] (2) After washing, the cells were blocked with 2% BSA for 1 hour and then incubated with serially diluted OMV-CLEC9A, OMV-CD3, and OMV-CLEC9A / CD3 for 1 hour.

[0066] (3) After washing, the cells were incubated with horseradish peroxidase-conjugated anti-3x HA tag antibody for 1 hour.

[0067] (4) Add 100 μL of single-component substrate solution (TMB) to each well, react for 30 min, and then add 100 μL of 2 M sulfuric acid to terminate the reaction.

[0068] (5) Immediately record the absorbance at 450 nm using a Thermo Fisher spectrophotometer.

[0069] The ELISA results of the specific binding of the CLEC9A-scFv / CD3-scFv bispecific antibody carried on the surface of genetically engineered bacterial outer membrane vesicles to the corresponding antigen are shown in Figure 5. Example 5 Genetically Engineered Bacterial Outer Membrane Vesicles Promote the Interaction between cDC1 and CD8+ T Cells via the CLEC9A-scFv / CD3-scFv Bispecific Antibody Carried on Their Surface

[0070] (1) Induction of mouse bone marrow-derived dendritic cells: Resuspend the isolated bone marrow cells in RPMI 1640 medium containing 20 ng / mL GM-CSF to induce BMDCs differentiation; observe under a microscope and culture in a 37°C incubator.

[0071] (2) After 3 days, observe the adhesion and perform a half-medium change. Perform a half-medium change every other day. The cells can be used for subsequent experiments around the 8th day.

[0072] (3) CD3+ T cells were isolated by negative selection using a mouse CD3+ T cell isolation kit, and then 5×10 5 Cells were seeded in 24-well plates and cultured in RPMI 1640 complete medium containing IL-15 (5 ng / ml) and IL-7 (5 ng / ml).

[0073] (4) CD3+ T cells and BMDCs were co-cultured in a U-shaped 96-well plate at a ratio of 1:10. After treatment with PBS, OMV-CLEC9A, OMV-CD3, or OMV-CLEC9A / CD3 (20 μg vesicle protein / mL) for 24 hours, the coupling ratio of T lymphocytes and BMDCs in the co-culture systems with different treatments was detected by flow cytometry.

[0074] Representative flow cytometry images of bacterial outer membrane vesicles overexpressing CLEC9A-scFv / CD3-scFv bispecific antibodies enhancing the interaction between cDC1 and T cells are shown in Figure 2. Figure 6 shown.

[0075] Example 6 Efficacy of Genetically Engineered Bacterial Outer Membrane Vesicles in Treating Hepatocellular Carcinoma

[0076] 1. Establishment of mouse hepatocellular carcinoma subcutaneous tumor model and experimental grouping: 6-8 week old healthy male C57BL / 6 mice were selected, Hepa 1-6 cells cultured under standard conditions were digested and centrifuged, and the obtained 1×10 6 The cells were resuspended in 50ul PBS and inoculated subcutaneously in the right inguinal region of the mice. The tumor formation in the mice was dynamically observed. The long diameter (a) and short diameter (b) of the subcutaneous tumor were measured with a vernier caliper, and the tumor volume (mm) was calculated according to the following formula. 3 )=a×b 2 / 2, tumor volume is about 100mm 3 The mice were randomly divided into 4 groups, 5 in each group, including PBS group, OMV-CLEC9A group, OMV-CD3 group and OMV-CLEC9A / CD3 group.

[0077] 2. Treatment of subcutaneous hepatocellular carcinoma mouse models: Place the mouse in a tail vein injection device, secure the mouse, and locate the tail vein. Before injection, confirm that there are no bubbles in the needle. During injection, insert the needle in the forward direction. The presence of blood in the aspirated needle indicates successful entry into the tail vein. The treatment dose is 200 μg of vesicle protein per mouse, administered once every three days for a total of four doses. Tumor volume is measured every three days.

[0078] Depend on Figure 7 It can be seen that the therapeutic effect of bacterial outer membrane vesicles overexpressing CLEC9A-scFv / CD3-scFv bispecific antibodies is the most obvious.

[0079] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent replacements for some of the technical features therein. 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 genetically engineered bacterial outer membrane vesicle, characterized in that: The genetically engineered bacterial outer membrane vesicles are bacterial outer membrane vesicles that overexpress CLEC9A-scFv / CD3-scFv bispecific antibodies through genetic engineering.

2. The method for preparing genetically engineered bacterial outer membrane vesicles according to claim 1, characterized in that: The following steps are involved: Step 1: Transform the plasmid of the CLEC9A-scFv / CD3-scFv bispecific antibody into competent E. coli W3110 (ΔmsbB), and pick monoclonal colonies using LB plates with corresponding resistance; Step 2: Take the monoclonal colony obtained in step 1 and transfer it to LB liquid medium of corresponding resistance, shake and culture at 180 rpm and 37°C until its OD value reaches 0.6; Step 3: Take the culture medium obtained in step 2, add 1 mM IPTG inducer, and culture with shaking at 180 rpm and 16°C for 16 hours; Step 4: Centrifuge the culture medium obtained in step 3 at 4000 rpm for 15 min at 4°C, collect the supernatant, and filter the supernatant through a 0.22 μm filter membrane to obtain a filtered supernatant; Step 5: The filtered supernatant obtained in step 4 was ultracentrifuged at 150,000 g for 2 h, the supernatant was discarded, the precipitate was resuspended with PBS, and the resuspended solution was collected to obtain bacterial outer membrane vesicles overexpressing the CLEC9A-scFv / CD3-scFv bispecific antibody.

3. Use of the genetically engineered bacterial outer membrane vesicles according to claim 1 in promoting the interaction between cDC1 and CD8+ T lymphocytes.

4. Use of the genetically engineered bacterial outer membrane vesicles according to claim 1 in the preparation of tumor drugs for the treatment of hepatocellular carcinoma.

Images