Engineering bacterial outer membrane vesicle with function of inducing memory-like NK cells, preparation and application

Abstract

The invention discloses an engineered bacterial outer membrane vesicle with a function of inducing memory-like NK cells, and preparation and application thereof, and belongs to the technical field of pharmaceutical preparations and nano materials. The preparation method comprises the following steps: firstly, extracting bacterial outer membrane vesicles, then entrapping an immunologic adjuvant Poly (I: C) in the bacterial outer membrane vesicles by adopting an electroporation method, and finally modifying phagocytic peptide Tuftsin on the membrane surface by adopting a membrane insertion method so as to realize precise targeting of tumor-related macrophages in liver, spleen and tumor tissues. The preparation induces polarization of tumor-related macrophages through the synergistic effect of inherent immunocompetence of bacterial outer membrane vesicles and Poly (I: C), promotes secretion of proinflammatory cytokines at the same time, effectively reverses a tumor immunosuppression microenvironment, and enhances tumor infiltration and targeted killing efficiency of memory-like NK cells and cytotoxic T lymphocytes. The whole scheme has the advantages of simple process, accurate targeting, high biological safety and the like, provides an innovative drug delivery system for tumor immunotherapy, and has wide application prospect and commercial value.

Description

Technical Field

[0001] The present invention belongs to the technical fields of pharmaceutical preparations and nanomaterials, and particularly relates to an engineered bacterial outer membrane vesicle with the function of inducing memory-like NK cells, and simultaneously discloses a preparation method and related applications thereof. Background Art

[0002] The tumor immunosuppressive microenvironment is one of the important factors affecting the efficacy of clinical immunotherapy. The tumor immune microenvironment is very complex and consists of a variety of immune cells and cytokines secreted by them. Among them, immunosuppressive cells account for more than 50% of the total number of immune cells, and they promote the growth, proliferation and migration of tumor cells by secreting immunosuppressive cytokines. At the same time, high levels of immunosuppressive cytokines assist tumor tissues to resist the infiltration of immune effector cells such as cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells and inhibit their killing functions to a certain extent, so that tumor tissues can escape the attack of the body's immune system, ultimately leading to a reduction or even failure in the efficiency of clinical immunotherapy based on CTLs and NK cells.

[0003] In addition to individual cytokines that can directly participate in the activation and proliferation of CTLs and NK cells, studies have shown that the combination of these three cytokines, IL-12, IL-15 and IL-18, can preactivate NK cells in the absence of antigens, endowing NK cells with characteristics similar to memory cells, called cytokine-induced memory-like (CIML) NK cells. Compared with traditional NK cells, CIML NK cells have the advantages of strong proliferation ability, high target cell killing efficiency and long-lasting in vivo activity. First, CIML NK cells are preactivated in vitro with IL-12, IL-15 and IL-18 and then transferred into the body, with stronger proliferation ability and IFN-γ secretion ability, and this effect is inherent in CIML NK cells and still exists in the daughter cells after cell division. Subsequently, CIML NK cells will enter the resting stage, and IFN-γ secretion will return to normal levels. When stimulated again by cytokines, activating receptors or tumor antigens, CIML NK cells will initiate a rapid and strong immune response.

[0004] However, the CIML NK cell therapy that preactivates NK cells in vitro with cytokines and transfuses them back to patients is significantly limited in its clinical application due to inherent defects such as complex operations and low cell survival rates. If a drug delivery system that can directly activate immune cells in vivo and induce the secretion of key cytokines such as IL-12, IL-15 and IL-18 can be developed, it will be expected to simultaneously achieve tumor growth inhibition and immune memory formation, providing a new strategy for tumor immunotherapy.

[0005] Bacterial outer membrane vesicles (OMVs) have unique multi-component characteristics: their bilayer membrane structure not only contains bacterial components such as lipids, proteins, and nucleic acids, but also carries pathogen-associated molecular patterns (PAMPs). This enables OMVs to serve as vaccine carriers to trigger specific immune responses and also become potential anti-tumor tools through intercellular information transmission mechanisms.

[0006] In the field of tumor treatment, OMVs have become ideal drug carriers due to their passive targeting ability and easy modification characteristics brought about by their natural nano-size. For example, Chinese Patent CN 118078772 A discloses a drug-loaded bacterial outer membrane vesicle and its preparation method and application. It uses magnetotactic bacterial outer membrane vesicles as carriers, with chemotherapy drugs and iron ions encapsulated in the carrier and tumor-targeting peptides modified on the surface, having both therapeutic and diagnostic functions. However, this document does not record the ability of this drug-loaded bacterial outer membrane vesicle to induce the secretion of multiple cytokines including IL-12, IL-15, and IL-18 in vivo, unable to activate CIML NK cells and lacking a continuous production mechanism of IFN-γ.

[0007] Therefore, to construct engineered bacterial outer membrane vesicles with the function of highly inducing memory-like NK cells, three core elements need to be systematically optimized: the selection of the parental strain, the compatibility of the loaded drugs, and the design of the targeting peptide segments, so as to synergistically achieve cytokine induction and immune microenvironment remodeling. Summary of the Invention

[0008] Aiming at the above existing problems, the present invention aims to provide engineered bacterial outer membrane vesicles (TOMP) with the function of inducing memory-like NK cells and discloses a specific preparation method. The TOMP uses OMVs with a special particle size derived from Gram-negative bacteria as the core carrier, targets macrophages by surface modification with the phagocytosis-promoting peptide Tuftsin, and simultaneously encapsulates immune adjuvants inside to synergistically activate the cytokine release pathways such as IL-15, IL-12, and IL-6. Based on the in-situ cytokine release mechanism mediated by macrophage polarization in the tumor microenvironment (TME), this application reverses the immune suppression state of the TME, promotes the activation and expansion of CIML NK cells and tumor infiltration, and finally forms a new immune therapy system that combines highly efficient solid tumor / hematological tumor killing, postoperative recurrence prevention, and dynamic monitoring based on imaging / biomarkers.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows: An engineered bacterial outer membrane vesicle with the function of inducing memory-like NK cells, wherein the bacterial outer membrane vesicle encapsulates an immune adjuvant and is surface-modified with a phagocytosis-promoting peptide; the bacterial outer membrane vesicle is selected from any one of Escherichia coli outer membrane vesicles, Shigella flexneri outer membrane vesicles, attenuated Salmonella outer membrane vesicles, and Vibrio cholerae outer membrane vesicles, preferably attenuated Salmonella outer membrane vesicles (VNP20009); the amino acid sequence of the phagocytosis-promoting peptide (Tuftsin peptide) is Thr-Lys-Pro-Arg (SEQ ID NO: 1).

[0010] Furthermore, the immune adjuvant is polyinosinic acid-polycytidylic acid Poly(I:C), and the encapsulation efficiency of the immune adjuvant exceeds 50%. Poly(I:C) is an artificially synthesized immune agonist that can bind to Toll-like receptor 3 of antigen-presenting cells, activate the interferon regulatory factor 3 signaling pathway, and effectively induce the release of cytokines such as IL-15, IL-12, and IL-6.

[0011] The preparation method of the above-mentioned engineered bacterial outer membrane vesicle with the function of inducing memory-like NK cells is as follows:

[0012] S1. Inoculate bacteria in a liquid medium, collect the culture solution after two-stage amplification culture, centrifuge (7000 - 9000g, 10 - 20 min) to obtain a sterile supernatant, filter, concentrate, and perform ultra-high-speed centrifugation on the concentrate (150000 - 200000g, 2 - 4 h), and collect the precipitate to obtain bacterial outer membrane vesicles OMV;

[0013] S2. Use the electroporation method to encapsulate Poly(I:C) into OMV to obtain OMV with an internal encapsulated immune adjuvant, denoted as OMV-Poly(I:C);

[0014] S3. Synthesize the phagocytosis-promoting peptide Tuftsin by solid-phase polypeptide synthesis method and connect it to distearoyl phosphatidylethanolamine to obtain DSPE-Tuftsin; the lipid structure of DSPE can help the complex penetrate the cell membrane and be delivered to the diseased tissue directionally. Tuftsin enhances the local immune response, and DSPE improves the drug stability. After binding to the drug delivery carrier, it is more suitable for cancer immunotherapy.

[0015] S4. Use the membrane insertion method to insert DSPE-Tuftsin into the vesicle membrane of OMV-Poly(I:C) to obtain the final product, denoted as TOMP.

[0016] Furthermore, the synthesis process of DSPE-Tuftsin is as follows:

[0017] S31. Add 2-Cl-Trt resin to NMP for swelling and perform suction filtration;

[0018] S32. Put the swollen 2-Cl-Trt resin, FMOC-AA-OH, HATU, HOAt, and DIPEA into DMF together. After mixing evenly, carry out the reaction in a solid-phase synthesis tube, wash it, take a small amount of the resin for ninhydrin detection, and it is colorless; add a piperidine solution with a mass concentration of 20% and react for 30 min, then wash it. Take a small amount of the resin for ninhydrin detection, and it is blue or reddish-brown, thus obtaining the phagocytosis-promoting peptide.

[0019] S33. Weigh Tuftsin, succinic anhydride, DMAP, and DIPEA, add them into DMF and mix well, then add them into a solid-phase synthesis tube, and react overnight at room temperature. Wash it, take a small amount of the resin for ninhydrin detection. If it is colorless or light yellow, the reaction is complete, and compound 8 is obtained.

[0020] S34. Mix compound 8 with HATU and DIPEA, react at room temperature for 1 - 3 h, then add an excess of DSPE, and continue to react for 18 - 30 h. Wash it and carry out ninhydrin detection, and it is colorless, thus obtaining compound 9.

[0021] S35. Treat compound 9 with a cleavage solution at room temperature for 2 - 5 h, concentrate it, add low-temperature ether, centrifuge it, collect the light yellow precipitate at the bottom of the centrifuge tube, repeat 5 times, and purify it by HPLC to obtain the target product DSPE-Tuftsin.

[0022] Further, in step S4, the mass ratio of DSPE-Tuftsin to OMV-Poly(I:C) is 1:0.2 - 1.5, and the particle size of the obtained TOMP is 130 - 170 nm.

[0023] Further, in step S32, the equivalent ratio of 2-Cl-Trt resin, FMOC-AA-OH, HATU, HOAt, and DIPEA is 1:3 - 5:2 - 3:2 - 3:5 - 8; the addition sequence of FMOC-AA-OH is Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH in turn.

[0024] Further, in step S33, the equivalent ratio of Tuftsin, succinic anhydride, DMAP, and DIPEA is 1:2 - 4:2 - 4:4 - 6.

[0025] Further, in step S34, the equivalent ratio of compound 8, HATU, and DIPEA is 1:2 - 3:2 - 3.

[0026] Based on its active targeting function, the engineered bacterial outer membrane vesicles with the function of inducing memory-like NK cells can precisely deliver the co-loading system of OMV and Poly(I:C) to tumor-associated macrophages, stimulate the secretion of inflammatory cytokines such as IL-12, IL-15 and IL-18, and then specifically activate the tumor-killing functions of cytokine-induced memory-like NK cells (CIMLNK) and cytotoxic T lymphocytes (CTL), and induce the programmed apoptosis of regulatory T cells and myeloid-derived suppressor cells, thereby realizing the reprogramming of the tumor immunosuppressive microenvironment and ultimately achieving the synergistic therapeutic effect of enhancing the anti-tumor immune response while reducing systemic toxicity.

[0027] Based on the above principle, the engineered bacterial outer membrane vesicles with the function of inducing memory-like NK cells can be effectively applied in the preparation of tumor immunotherapy agents.

[0028] The tumor immunotherapy agents include drugs for the treatment of hematological malignancies and drugs for the treatment of solid tumors.

[0029] The beneficial effects of the present invention are as follows:

[0030] 1. The engineered bacterial outer membrane vesicles (TOMP) disclosed in this application use bacterial outer membrane vesicles with special particle sizes and structures derived from Gram-negative bacteria as the core body. While modifying the phagocytosis-promoting peptide Tuftsin on the surface of the bacterial outer membrane vesicles, the immunoadjuvant Poly(I:C) is encapsulated inside. The phagocytosis-promoting peptide can actively target macrophages and mediate their phagocytosis, and finally precisely deliver the immunostimulants OMV and Poly(I:C) to macrophages in the NK cell-enriched region, stimulate the release of key cytokines such as IL-12, IL-15 and IL-18, and then activate the memory-like phenotype (CIMLNK) of NK cells, significantly enhancing the cell proliferation ability and IFN-γ secretion ability, helping to construct a multiple immune network, and significantly enhancing the anti-tumor immune response of the body;

[0031] 2. This application uses bacterial outer membrane vesicles as a carrier to construct TOMP. Its preparation method is simple, does not involve overly complex steps and equipment, and it has high stability, excellent encapsulation efficiency, and good transformation value;

[0032] 3. This application realizes the in-situ release of cytokines such as IL-12 / IL-15 / IL-18 by regulating the interaction between macrophages and NK cells in the tumor microenvironment and using Tuftsin-targeted induction of tumor-associated macrophage polarization, and finally realizes safe, long-lasting and efficient cytokine immunotherapy, which is expected to provide new ideas and references for the treatment of patients with hematological tumors and solid tumors in the future. Description of the Drawings

[0033] Figure 1Schematic diagram of the synthesis of engineered bacterial outer membrane vesicles TOMP with induced memory-like NK cell function;

[0034] Figure 2 Chemical flow chart for the synthesis of DSPE-Tuftsin in Example 1;

[0035] Figure 3 LC-MS spectrum of DSPE-Tuftsin prepared in Example 1;

[0036] Figure 4 Poly(I:C) concentration-fluorescence intensity calibration curve;

[0037] Figure 5 Expression of FITC in A20 cells after different treatments;

[0038] Figure 6 Transmission electron micrograph (subfigure a) and particle size distribution map (subfigure b) of TOMP prepared in Example 1;

[0039] Figure 7 Secretion of different cytokines in the cell supernatant after co-incubation of each group of materials with mouse bone marrow-derived macrophages (BMDM). Among them, subfigure a shows the secretion of IL-12, subfigure b shows the secretion of IL-15, and subfigure c shows the secretion of IL-18;

[0040] Figure 8 Secretion of IFN-γ after co-incubating NK cells with the supernatant of BMDM stimulated in vitro and then stimulating the supernatant of tumor cells;

[0041] Figure 9 Body weight curve of mice in different treatment groups;

[0042] Figure 10 Tumor volume curve of mice in different treatment groups. Detailed implementation manners

[0043] To enable those of ordinary skill in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples.

[0044] Example 1

[0045] This example discloses a preparation method of engineered bacterial outer membrane vesicles with induced memory-like NK cell function. The overall preparation process refers to Figure 1, specifically, first extract bacterial outer membrane vesicles (OMVs), then encapsulate the immunoadjuvant polyinosinic acid cytidylic acid Poly(I:C) in the OMVs by electroporation to obtain OMV-Poly(I:C), and finally connect the phagocytosis-promoting peptide Tuftsin to the surface of the vesicles by the membrane insertion method, that is, the engineered bacterial outer membrane vesicles (TMOP) with the function of inducing memory-like NK cells are obtained.

[0046] The specific preparation process is as follows:

[0047] S1. Synthesis of phagocytosis-promoting peptide Tuftsin and DSPE-Tuftsin (the synthesis process refers to Figure 2 ):

[0048] S11. Take 1 g of 2-chlorotrityl chloride resin (2-Cl-Trt) resin (substitution degree 0.337 g / mol), add it to 10 mL of N-methylpyrrolidone (NMF) and swell for 1 h, then filter by suction;

[0049] S12. Weigh 2-Cl-Trt resin, amino acid FMOC-AA-OH with N-terminal protection, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-hydroxy-7-azabenzotriazole (HOAt) and N,N-diisopropylethylamine (DIPEA) according to the equivalent ratio of 1:3:2.5:3:6, mix them in DMF, add them to a solid-phase synthesis tube and react at room temperature for 2 h. During the polypeptide synthesis process, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Thr(tBu)-OH are successively coupled to the resin to obtain intermediate products compound 1, 3, 5, and 7 respectively. After each coupling is completed, the resin is washed alternately with dichloromethane (DCM) and DMF 6 times, and the coupling efficiency is verified by ninhydrin detection (the detection result is colorless, indicating that the amino group is completely protected). After each intermediate product is obtained, the resin is treated with a 20% piperidine solution for 30 minutes to stepwise remove the Fmoc protecting groups of each intermediate product (compound 1, 3, 5, 7) to obtain the deprotected compounds 2, 4, 6, and Tuftsin. Take a small amount of resin for ninhydrin detection. If it is blue or reddish-brown (indicating that the amino group is exposed), it is confirmed that the target product - phagocytosis-promoting peptide Tuftsin is successfully obtained, and its amino acid sequence is Thr-Lys-Pro-Arg (TKPR, SEQ ID NO.1);

[0050] S13. Weigh Tuftsin, succinic anhydride, 4-dimethylaminopyridine (DMAP), and DIPEA according to an equivalent ratio of 1:3:3.3:6, add them to DMF and mix well, then add them to a solid-phase synthesis tube. React overnight at room temperature, wash, and take a small amount of resin for ninhydrin detection. If it is colorless or light yellow, the reaction is complete, and compound 8 is obtained.

[0051] S14. Mix compound 8 with HATU and DIPEA according to an equivalent ratio of 1:2:3, react at room temperature for 2 h, then add an excess of distearoyl phosphatidylethanolamine (DSPE), continue to react for 24 h, wash, and perform ninhydrin detection. If it is colorless, compound 9 is obtained.

[0052] S15. Treat compound 9 with 10 mL of cleavage solution (prepared by mixing trifluoroacetic acid (TFA), trimethylsilyl (TIS), H2O, and 1,2-ethanedithiol (EDT) according to a mass ratio of 94:1:2.5:2.5) at room temperature for 3 h, concentrate, transfer it to a 50 mL centrifuge tube, add cold ether, centrifuge for 10 min, collect the light yellow precipitate at the bottom of the centrifuge tube, repeat the ether washing - centrifugation operation 5 times, and finally purify it by high-performance liquid chromatography (HPLC) to obtain the target product DSPE-Tuftsin.

[0053] S2. Synthesis of OMV-Poly(I:C):

[0054] S21. Take 50 μL of attenuated Salmonella (VNP20009, stored at -80 °C), add it to 10 mL of LB liquid medium, and grow it overnight in a shaker at 37 °C to obtain the original bacterial solution.

[0055] S22. Inoculate the original bacterial solution into fresh LB medium at a volume ratio of 1:50, and further incubate it in a shaker at 37 °C for 12 h to obtain the diluted bacterial solution.

[0056] S23. Centrifuge the diluted bacterial solution at 4 °C (8000 g) for 15 min, collect the supernatant, filter it through a 0.45 μm polyethersulfone filter, concentrate it to 100 mL using a 100 kDa ultrafiltration membrane, and then filter and concentrate it through a 0.22 μm filter to obtain the concentrated solution.

[0057] S24. Centrifuge the concentrated solution at 4 °C at ultra-high speed (150000 g) for 3 h to obtain outer membrane vesicles (OMV), and use a BCA protein quantification kit to quantify the total protein in OMV.

[0058] S25. Dissolve OMV in phosphate buffer (PBS), mix it with polyinosinic acid cytidylic acid Poly(I:C) at a mass ratio of 1:1.25, and use electroporation (1500 V, 20 ms pulse width, and 3 pulse numbers) to introduce Poly(I:C) into OMV. Then, ultracentrifuge at 4 °C (150000 g) for 1 h to remove the unencapsulated Poly(I:C), obtaining OMV loaded with an immune adjuvant, denoted as OMV-Poly(I:C).

[0059] S3. Preparation of TOMP:

[0060] Dissolve the DSPE-Tuftsin prepared in S15 and the OMV-Poly(I:C) prepared in S25 in PBS respectively, mix the two, and the mass ratio of DSPE-Tuftsin to OMV-Poly(I:C) is 1:0.6. Place it on a shaker at 37 °C for 2 h, then centrifuge at 13000 rpm for 30 min to finally obtain TOMP.

[0061] Corresponding performance tests

[0062] 1. Figure 3 This is the LC-MS spectrum of DSPE-Tuftsin. The peak with a molecular mass of 1327.62 shown in the figure is consistent with the actual molecular weight, proving that DSPE-Tuftsin was successfully synthesized.

[0063] 2. The steps for measuring the drug loading and encapsulation of OMV with Poly(I:C) are as follows:

[0064] (1) Dissolve OMV in PBS, mix it with Poly(I:C) at a mass ratio of 1:1.25, and use electroporation (1500 V, 20 ms pulse width, and 3 pulse numbers) to introduce Poly(I:C) into OMV. Ultracentrifuge at 4 °C (150000 g) for 1 h, and collect the supernatant, which contains the unencapsulated Poly(I:C);

[0065] (2) Prepare a Poly(I:C) solution with a concentration of 2 μg / mL using PBS, and use the working solution in the RNA quantitative detection kit to serially dilute the Poly(I:C) solution to 0, 2, 10, 20, 50, 100 ng / mL, measure its fluorescence intensity, and make a standard curve ( Figure 4 );

[0066] (3) Measure the fluorescence intensity of the supernatant obtained in step (1) to obtain the concentration of the unencapsulated Poly(I:C). Conduct three parallel repeated experiments, and further calculate the drug loading rate and encapsulation rate.

[0067] The fluorescence intensities of the supernatants obtained from three parallel experiments and the concentration of unencapsulated Poly(I:C) are shown in Table 1.

[0068] Table 1. Fluorescence intensities of the supernatants and the concentration of unencapsulated Poly(I:C)

[0069]

[0070]

[0071] It was calculated that the drug loading rate reached 95.54% and the encapsulation efficiency was 54.44%.

[0072] 3. To verify the insertion of DSPE-Tuftsin into the membrane of OMV-Poly(I:C) by detecting the expression of FITC in A20 cells after different treatments, the specific detection steps are as follows:

[0073] (1) Dissolve the DSPE-Tuftsin prepared in Example 1 and OMV in PBS respectively, mix DSPE-Tuftsin and OMV according to a mass ratio of 1:0.6, shake at 37 °C for 2 h, then centrifuge at 13000 rpm for 30 min to collect OMV-Tuftsin;

[0074] (2) Seed A20 cells in a 6-well plate and grow them in a constant temperature incubator for 12 h;

[0075] (3) Add OMV conjugated with fluorescein isothiocyanate (FITC) and OMV-Tuftsin, and incubate for 1 h, 2 h, and 4 h respectively;

[0076] (4) Detect the expression of FITC on the cells by flow cytometry.

[0077] For the expression situation, see Figure 5 , as the incubation time extended, the fluorescence intensities of both the OMV group and the OMV-Tuftsin group increased, indicating that regardless of whether the phagocytosis-promoting peptide was modified, the endocytosis of OMV by A20 cells was time-dependent. The longer the incubation time, the more opportunities for OMV to contact the cells, and the endocytosis process gradually accumulated, resulting in an increase in intracellular fluorescence signal. At the same time period, compared with the unmodified OMV group, the fluorescence intensity of the OMV-Tuftsin group was significantly higher, indicating the successful insertion of DSPE-Tuftsin, and the modification of Tuftsin significantly enhanced the uptake efficiency of A20 cells for OMV.

[0078] As can be seen from the above results, Tuftsin, as an immunologically active tetrapeptide (Thr-Lys-Pro-Arg), can bind to specific receptors on the surface of A20 cells, triggering active endocytosis. The modification of Tuftsin can significantly improve the targeted delivery efficiency of OMV, indicating that Tuftsin-modified OMV has the potential for drug delivery and has good application prospects in tumor treatment or immunomodulation.

[0079] 4. As can be seen from Figure 6 subfigure a, the TOMP prepared in this example has a uniform size, and the particle size is about 150 nm; the dynamic light scattering analysis of TOMP shows its particle size distribution ( Figure 6 subfigure b).

[0080] 5. After incubating each group of materials with mouse bone marrow-derived macrophages (BMDM), the secretion of IL-12 (a), IL-15 (b), and IL-18 (c) in the cell supernatant was measured, including the following steps:

[0081] 1) Extract BMDM cells from mouse bone marrow and culture the BMDM cells in a 6-well plate at a density of 1×10 6 for 7 days;

[0082] 2) Aspirate the old medium and add 1 mL of medium (15 μg / mL) containing PBS, OMV, Poly(I:C), OMV-Poly(I:C), OMV-Tuftsin, and TOMP, respectively, and co-incubate with mouse BMDM cells in a 37°C incubator for 24 h;

[0083] 3) Digest the cells from the 6-well plate, collect them into different centrifuge tubes, centrifuge to remove the cells, and collect the supernatant of each group;

[0084] 4) Detect the secretion of IL-12, IL-15, and IL-18 in the cell supernatant after incubation by an enzyme-linked immunosorbent assay (ELISA) kit.

[0085] The results are shown in Figure 7 . As can be seen from the figure, compared with other materials, the secretion levels of IL-12, IL-15, and IL-18 in mouse bone marrow-derived macrophages can be significantly upregulated after co-incubation with TOMP, thus proving that TOMP can help form a pro-inflammatory microenvironment by autostimulating cytokines such as IL-12, IL-15, and IL-18.

[0086] 6. After co-incubating NK cells with the supernatant of BMDM stimulated in vitro, the secretion of IFN-γ in the cell supernatant was measured, including the following steps:

[0087] 1) Extract BMDM cells from mouse bone marrow and culture the BMDM cells in a 6-well plate at a density of 1×10 per well for 7 days; 6 ;

[0088] 2) Aspirate the old medium and add 1 mL of medium (15 μg / mL) containing PBS, OMV, Poly(I:C), OMV-Poly(I:C), OMV-Tuftsin, and TOMP to each well, and co-incubate with mouse BMDM cells in a 37 °C incubator for 24 h;

[0089] 3) Digest the cells from the 6-well plate, collect them into different centrifuge tubes, centrifuge to remove the cells, and collect the supernatant of each group;

[0090] 4) Extract primary NK cells from mouse spleen and evenly inoculate the NK cells into a 6-well plate for culture;

[0091] 5) Add the supernatant collected in step 3) to the well plate containing NK cells, and co-incubate the supernatant with NK cells for 24 h;

[0092] 6) Aspirate the medium, add fresh medium and A20 tumor cells with a density of 5×10 to the well plate containing NK cells in step 5), and co-incubate again for 24 h; 5 ;

[0093] 7) Centrifuge and collect the supernatant, and detect the secretion of IFN-γ in the supernatant of NK cells after co-incubation by ELISA kit.

[0094] The results are shown in Figure 8 , from Figure 8 it can be seen that compared with other groups, after co-incubating NK cells with the supernatant of BMDM stimulated by TOMP and then stimulating A20 tumor cells, the strongest IFN-γ secretion ability is shown, verifying that TOMP has a good memory function. It may be that the cytokines secreted by BMDM stimulated by TOMP directly activate the receptors on the surface of NK cells through receptor-ligand interaction, making NK cells have stronger proliferation ability and IFN-γ secretion ability. When memory-like NK cells are stimulated by cytokines, activating receptors or tumor antigens again, they will initiate a rapid and strong immune response. And high concentrations of IFN-γ can not only inhibit pathogen replication, but also enhance antigen presentation efficiency by up-regulating the expression of MHC class I molecules, promoting the cooperative killing of T cells and NK cells, which will provide a new theoretical basis for the development of NK cell-based immunotherapy.

[0095] 7. Test the in vivo anti-tumor effect of TOMP, including the following steps:

[0096] (1) Subcutaneously inoculate the right thigh of BALB / c mice with 1×105 One A20-luc cell was used to establish a subcutaneous tumor model;

[0097] (2) On the 12th day after inoculation, the tumor-bearing mice for treatment were randomly divided into 5 groups (5 mice in each group). Mice in different groups were intravenously injected with 100 μL of different materials (PBS, OMV, Poly(I:C), OMV-Poly(I:C), TOMP) at the same concentration. Mice in the same group were injected with the same material, and the injection was performed once every two days for a total of 3 times;

[0098] (3) The body weight and tumor size of the mice were measured every other day. The formula for calculating the tumor volume (V) was V = W 2 ×L / 2 (W and L are the shortest and longest diameters of the tumor, respectively).

[0099] Figure 9 and Figure 10 are the body weight curve and tumor volume curve of mice in different treatment groups, respectively. The body weight change curves of mice in all treatment groups showed no significant difference compared with the PBS control group, and no toxic reaction with a body weight decrease exceeding 10% was observed, demonstrating that each preparation had good systemic safety ( Figure 9 ); in terms of anti-tumor effect, the Poly(I:C) group showed limited tumor growth inhibition, the OMV and OMV-Poly(I:C) groups showed moderate tumor growth inhibition, while the TOMP group could significantly inhibit the growth of subcutaneous tumors in mice, indicating that TOMP had significant in vivo anti-tumor activity ( Figure 10 ).

[0100] The above shows and describes the basic principles, main features and advantages of the present invention. However, the above are only specific embodiments of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments obtained by those skilled in the art without departing from the technical solution of the present invention should be covered within the scope of the present invention.

Claims

1. An engineered bacterial outer membrane vesicle with the function of inducing memory-like NK cells, characterized in that, The bacterial outer membrane vesicles encapsulate an immune adjuvant and are surface-modified with a phagocytosis-promoting peptide; The bacterial outer membrane vesicles are selected from any one of Escherichia coli outer membrane vesicles, Shigella flexneri outer membrane vesicles, attenuated Salmonella outer membrane vesicles, and Vibrio cholerae outer membrane vesicles; The amino acid sequence of the phagocytosis-promoting peptide is as shown in SEQ ID NO:

1.

2. The engineered bacterial outer membrane vesicles with the function of inducing memory-like NK cell function according to claim 1, characterized in that, The bacterial outer membrane vesicles are attenuated Salmonella outer membrane vesicles, and the immune adjuvant is polyinosinic acid-polycytidylic acid Poly(I:C), and the encapsulation rate of the immune adjuvant exceeds 50%.

3. The preparation method of the engineered bacterial outer membrane vesicles with the function of inducing memory-like NK cell function as described in any one of claims 1-2, characterized in that, It includes the following steps: S1. Inoculate bacteria into a liquid medium, collect the culture solution after two-stage amplification culture, centrifuge to obtain a sterile supernatant, filter, concentrate, perform ultra-high-speed centrifugation on the concentrate, and collect the precipitate to obtain bacterial outer membrane vesicles OMV; S2. Use the electroporation method to encapsulate Poly(I:C) into OMV to obtain OMV internally encapsulating the immune adjuvant, denoted as OMV-Poly(I:C); S3. Synthesize the phagocytosis-promoting peptide by solid-phase peptide synthesis method and connect it to distearoyl phosphatidylethanolamine to obtain DSPE-Tuftsin; S4. Use the membrane insertion method to insert DSPE-Tuftsin into the vesicle membrane of OMV-Poly(I:C) to obtain the final product, denoted as TOMP.

4. The preparation method of the engineered bacterial outer membrane vesicles with the function of inducing memory-like NK cells as claimed in claim 3, wherein, The synthesis process of DSPE-Tuftsin is as follows: S31. Add 2-Cl-Trt resin to NMP for swelling, and perform suction filtration; S32. Put the swollen 2-Cl-Trt resin, FMOC-AA-OH, HATU, HOAt, and DIPEA together into DMF, mix well, react in a solid-phase synthesis tube, wash, take a small amount of resin for ninhydrin detection, which is colorless; add a 20% piperidine solution by mass concentration and react for 30 min, then wash, take a small amount of resin for ninhydrin detection, which is blue or reddish-brown, to obtain Tuftsin peptide; S33. Weigh Tuftsin peptide, succinic anhydride, DMAP, and DIPEA, add them to DMF and mix well, then add them into the solid-phase synthesis tube, react overnight at room temperature, wash, take a small amount of resin for ninhydrin detection, and if it is colorless or light yellow, the reaction is complete to obtain compound 8; S34. Mix compound 8 with HATU and DIPEA, react at room temperature for 1 - 3 h, then add an excessive amount of DSPE, continue to react for 18 - 30 h, wash and perform ninhydrin detection, which is colorless, to obtain compound 9; S35. Treat compound 9 with a cleavage solution at room temperature for 2 - 5 h, concentrate, add low-temperature ether, centrifuge, collect the precipitate, repeat 5 times, and purify by HPLC to obtain the target product DSPE-Tuftsin.

5. The preparation method of the engineered bacterial outer membrane vesicles with the function of inducing memory-like NK cells according to claim 3, characterized in that In step S4, the mass ratio of DSPE-Tuftsin to OMV-Poly(I:C) is 1:0.2 - 1.5, and the particle size of the obtained TOMP is 130 - 170 nm.

6. The preparation method of the engineered bacterial outer membrane vesicles with the function of inducing memory-like NK cells as described in claim 4, characterized in that, In step S32, the equivalent ratio of 2-Cl-Trt resin, FMOC-AA-OH, HATU, HOAt and DIPEA is 1:3-5:2-3:2-3:5-8; the addition sequence of FMOC-AA-OH is Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH in turn.

7. The preparation method of the engineered bacterial outer membrane vesicles with induced memory-like NK cell function according to claim 4, characterized in that, In step S33, the equivalent ratio of Tuftsin peptide, succinic anhydride, DMAP and DIPEA is 1:2-4:2-4:4-6.

8. The preparation method of the engineered bacterial outer membrane vesicles with the function of inducing memory-like NK cell function according to claim 4, characterized in that, In step S34, the equivalent ratio of compound 8, HATU and DIPEA is 1:2-3:2-3.

9. Use of the engineered bacterial outer membrane vesicles with induced memory-like NK cell function according to any one of claims 1-2 in the preparation of a tumor immunotherapy preparation.

10. The application according to claim 9, characterized in that, The tumor immunotherapy preparation includes drugs for treating hematological malignancies and drugs for treating solid tumors.

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