A tumor vaccine based on bacterial outer membrane vesicles and tumor-associated sugar antigens, and a preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, and more particularly to a tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens, its preparation method, and its application. The tumor vaccine provided by this invention induces increased levels of IL-6 and TNF-α secretion in RAW 264.7 and DC 2.4 cells in vitro, promoting phagocytosis and maturation; in vivo, it induces the maturation of T cells and DC cells in the spleen and lymph nodes, significantly inhibiting tumor growth. The tumor vaccine achieves stable antigen loading, induces a strong immune response, significantly inhibits tumor growth, and demonstrates good efficacy and safety in mouse models.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Malignant tumors pose a severe public health challenge due to their high incidence and mortality rates, as well as the limitations of traditional therapies in advanced-stage tumors caused by drug resistance and recurrence. Against this backdrop, tumor vaccines, with their immense potential to elicit specific and durable anti-tumor immune responses, have become a key direction for overcoming treatment challenges. Tumor-associated glycoantigens (TACAs), due to their high specific expression on the surface of cancer cells, have become ideal targets for developing tumor vaccines. Although naturally occurring TACAs have relatively weak immunogenicity, their immunogenicity can be significantly enhanced through chemical structural modification, combination with novel adjuvants, and nanodelivery techniques, providing promising new therapies for conquering malignant tumors. However, existing technologies have the following problems and limitations: TACAs, due to their small molecular weight and simple structure, cannot be recognized by the immune system on their own and therefore cannot elicit a specific immune response. They must be cross-linked with carrier proteins to become complete antigens. Traditional carriers such as KLH (keyhole limpethemocyanin), while capable of conjugating tumor-associated glycoantigens, still exhibit weak immunogenicity and primarily induce humoral immune responses, resulting in limited antitumor efficacy. For instance, in mouse models, the sTn-KLH vaccine, obtained by conjugating KLH with sialylated Tn antigen (sTn), showed low tumor inhibition rates, and clinical trials demonstrated no significant survival benefit. Physical mixing of sTn with carriers fails to achieve efficient conjugation, resulting in low antigen-specific immune responses and an inability to effectively inhibit tumor growth.

[0004] These technological shortcomings result in current tumor-associated glycoantigen vaccines being costly, complex in manufacturing, and of poor performance, failing to strongly activate the immune response. Summary of the Invention

[0005] In view of this, the present invention provides a tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens, its preparation method, and its application. The present invention utilizes a rapid and stable ligation technology to achieve efficient covalent coupling of OMV and TACA, thereby improving the immunogenicity and antitumor efficacy of this type of tumor vaccine.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens, comprising: nanobacterial outer membrane vesicles (OMV) and tumor-associated glycoantigens; The nano-OMV and tumor-associated glycoantigens are coupled via click chemical conjugation.

[0007] Furthermore, nano-OMVs with azide were coupled with alkyne-based tumor-associated glycoantigens via click chemical coupling.

[0008] Furthermore, tumor-associated glycoantigens include sTn, Tn, TF (Thomsen-Friendreich), LewisX, LewisY, sLeX (sialyl Lewisx), sLewisY, sialyl LewisA, KH-1, Globo H, SSEA-3, GD2, GD3, GM2, and GM3.

[0009] Furthermore, tumor-associated glycoantigens include sTn.

[0010] The OMV-sTn nanovaccine provided by this invention overcomes the challenge of weak immunogenicity of tumor-associated glycoantigens through a synergistic, multi-level immune activation mechanism. This vaccine utilizes OMV as a natural immune adjuvant and a highly efficient delivery carrier. Its nanoscale size enables passive targeting to lymphoid organs, and the high-density sTn antigen on its surface significantly enhances the capture efficiency of antigen-presenting cells (APCs). Pathogen-associated molecular patterns (PAMPs) carried by OMV, such as lipopolysaccharides, lipoproteins, and bacterial nucleic acids, comprehensively activate Toll-like receptors (TLR2 / 4 / 9) and the intracellular cGAS-STING pathway, driving APC maturation (upregulating CD80 / CD86) and the secretion of pro-inflammatory factors such as IL-6 and TNF-α, while simultaneously polarizing macrophages to an anti-tumor M1 phenotype. Against this backdrop of potent innate immune signaling, the sTn antigen activates CD8+ via a cross-presentation pathway. + T cells are induced to produce sTn-specific cytotoxic T lymphocytes (CTLs) under Th1 polarization. Simultaneously, the densely displayed sTn antigens on the surface of OMV-sTn can be directly recognized by B cell receptors, inducing humoral immunity and aiding in the clearance of tumor cells. This integrated design ultimately forms specific immune surveillance and memory against sTn-positive tumors, providing an innovative solution for the development of tumor vaccines based on weakly immunogenic glycoantigens.

[0011] Furthermore, the OMV is an outer membrane vesicle of Escherichia coli.

[0012] In a second aspect, the present invention provides a method for preparing a tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens as described in the first aspect, comprising the following steps: (1) Preparation of azide-modified bacterial outer membrane vesicles OMVs-N3; (2) Preparation of alkyne-based tumor-associated glycoantigens; (3) OMVs-N3 is obtained by clicking chemical coupling with alkynylated tumor-associated glycoantigen and then purification.

[0013] Further, the specific steps of step (1) are as follows: Escherichia coli K5ASSH is cultured in LB liquid medium containing chloramphenicol, kanamycin and GlcNAc, and the bacterial cells are collected; then transferred to LB liquid medium containing chloramphenicol and kanamycin for culture, and after 1-2 h of culture, IPTG inducer and GlcNAz are added to induce bacterial surface glycosylation and introduce azide; culture is continued until OD 600 If the concentration is above 1.0, collect the bacterial solution, centrifuge, filter, and concentrate to obtain OMVs-N3.

[0014] Furthermore, no specific limitations are made on the preparation method of alkynylated tumor-associated glycoantigens, as long as those skilled in the art can prepare them.

[0015] Furthermore, the preparation method of alkynylated sTn can be as follows: react alkynylated Tn with CTP (cellular triphosphate), Neu5Ac, MgCl2·6H2O and enzyme in Tris-HCl buffer. After the reaction is completed, alkynylated sTn is obtained through post-processing.

[0016] Furthermore, the pH of the Tris-HCl buffer is 8.0–9.0.

[0017] Furthermore, the mass ratio of alkynylated Tn, CTP, Neu5Ac, and MgCl2·6H2O is 1:1.3~1.5:0.8~0.9:7~8.

[0018] Furthermore, the enzymes are NMCSS and Psp2,6ST; the volume ratio of NMCSS to Psp2,6ST is 1:1~3.

[0019] Further, the preparation method of alkynylated Tn is as follows: (2-1) N-acetylgalactosamine (GalNAc) is reacted in acetic anhydride and pyridine solution to protect the hydroxyl group of GalNAc by acetylation. After purification, acetylated Tn antigen is obtained. (2-2) The acetylated Tn antigen and alkynyl PEG were dissolved in dichloroethane, and CaSO4 and FeCl3 were added to react. The reaction product was treated with alkali and then purified to obtain alkynylated Tn.

[0020] Further, in step (2-1), the volume ratio of acetic anhydride to pyridine is 1:1.5~2.5.

[0021] Further, in step (2-1), the purification operation is as follows: after evaporation to dryness, it is dissolved in dichloromethane, then washed with saturated NaHCO3 and water in sequence, then dried, filtered, and evaporated to dryness to obtain acetylated Tn antigen.

[0022] Further, in step (2-2), the mass ratio of acetylated Tn antigen to alkynyl PEG is 1:0.3~0.4.

[0023] Furthermore, in step (2-2), the mass ratio of acetylated Tn antigen to CaSO4 is 1:0.6~0.8.

[0024] Furthermore, in step (2-2), the mass ratio of acetylated Tn antigen to FeCl3 is 1:0.3~0.4.

[0025] Further, in step (2-2), the specific operation of the alkali treatment is as follows: the reaction product is dissolved in methanol, and the pH value is adjusted to 8-10 with sodium methoxide.

[0026] Furthermore, in step (3), the mass ratio of OMVs-N3 to alkyd tumor-associated glycoantigen is 0.2-0.3:1.

[0027] Further, in step (3), the click chemical coupling catalyst is ascorbic acid and copper sulfate; the mass ratio of ascorbic acid to alkynylated tumor-associated glycoantigen is 0.7~0.8:1; the mass ratio of copper sulfate to alkynylated tumor-associated glycoantigen is 0.7~0.8:1.

[0028] Furthermore, in step (3), the chemical coupling temperature is 20~30 ℃ and the time is 10~15 h.

[0029] Thirdly, the present invention provides the application of the tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens as described in the first aspect or the tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens prepared by the preparation method described in the second aspect in the preparation of drugs for the prevention or treatment of tumors.

[0030] Furthermore, the tumors are colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, glioblastoma, neuroblastoma, cholangiocarcinoma, and osteosarcoma.

[0031] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention provides a method for preparing OMV-sTn conjugates based on click chemistry, which enables stable antigen loading, induces a strong immune response, and significantly inhibits tumor growth. It demonstrates good efficacy and safety in mouse models. The OMV-sTn conjugates prepared by this invention exhibit enhanced immunomodulatory activity. In vitro, they induce increased levels of IL-6 and TNF-α secreted by RAW 264.7 and DC 2.4 cells, promoting phagocytosis and maturation. In vivo, they induce the maturation of T cells and DC cells in the spleen and lymph nodes, significantly inhibiting tumor growth. No significant changes in mouse body weight were observed, and H&E staining of major organs showed no toxicity, indicating that this vaccine has high safety. Attached Figure Description

[0032] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0033] Figure 1 This is a route design diagram for the present invention; Figure 2 Characterization diagrams of OMV and OMV-sTn are shown below; (A) is the particle size characterization diagram of OMV by DLS; (B) is the particle size characterization diagram of OMV-sTn by DLS; (C) is the potential characterization diagram of OMV and OMV-sTn by DLS; (D) is the transmission electron microscope image of OMV; (E) is the transmission electron microscope image of OMV-sTn; and (F) is the characterization diagram of sTn on the surface of OMV-sTn by flow cytometry. Figure 3 The following are flow cytometry representations of the phagocytic activity of OMV-sTn: (A) shows the uptake of OMV-sTn by RAW264.7 macrophages; (B) shows the fluorescence intensity of OMV-sTn after uptake by RAW264.7 macrophages; (C) shows the uptake of OMV-sTn by DC2.4 dendritic cells; and (D) shows the fluorescence intensity of OMV-sTn after uptake by DC2.4 dendritic cells. Figure 4 The images show laser confocal microscopy characterizations of OMV-sTn phagocytosis. (A) shows the laser confocal microscopy characterization of OMV-sTn uptake by RAW264.7 macrophages; (B) shows the laser confocal microscopy characterization of OMV-sTn uptake by DC2.4 dendritic cells. Figure 5 The figure shows the effect of OMV-sTn on the activation of RAW 264.7 macrophages; where (A) represents CD86; (B) represents CD80; (C) represents CD163; (D) represents IL-6; and (E) represents TNF-α. Figure 6The image shows the in vivo immunological activity evaluation results of OMV-sTn; where (A) represents the effect on CD3+ of mouse spleen T cells. + CD4 + Effect diagram; (B) shows the effect on CD3+ of mouse spleen T cells. + CD8 + Effect diagram; (C) shows the effect on CD11c in mouse spleen DC cells. + CD86 + Effect diagram; (D) shows the effect on CD11c in mouse spleen DC cells. + MHCⅡ + Effect diagram; (E) shows the effect on CD3+ of mouse lymphocytes. + CD4 + Effect diagram; (F) shows the effect on mouse lymphocytes CD3 + CD8 + Effect diagram; (G) shows the effect on mouse lymphocyte DC cells CD11c + CD86 + Effect diagram; (H) shows the effect on mouse lymphocyte DC cells CD11c + MHCⅡ + Impact diagram; Figure 7 Image showing the results of HE staining; Figure 8 This is a graph showing the changes in body weight of mice during drug administration. Figure 9 Image of a tumor in a mouse model for colon cancer prevention; Figure 10 The following are tumor growth curves for a colon cancer prevention model mouse: (A) Tumor growth curves for each group of mice; (B) Tumor growth curves for the Control group; (C) Tumor growth curves for the sTn group; (D) Tumor growth curves for the OMV group; (E) Tumor growth curves for the OMV+sTn group; and (F) Tumor growth curves for the OMV-sTn group. Detailed Implementation

[0034] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0035] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0036] Example 1 Preparation of OMV-sTn (1) Preparation of OMV azide: Based on Escherichia coli K5ASSH, it was cultured in LB liquid medium containing chloramphenicol (34 μg / mL), kanamycin (100 μg / mL) and GlcNAc (100 μg / mL) until the OD of the strain was obtained. 600 After reaching a concentration of 0.6–0.8, the bacterial cells were collected by centrifugation at 6000 r / min for 5 min. The supernatant was discarded, and the cells were washed three times with 1×PBS buffer. The cells were then transferred to LB liquid medium containing chloramphenicol (34 μg / mL) and kanamycin (100 μg / mL) for further culture to consume the GlcNAc naturally present in the cells. After 1 h of culture, IPTG inducer (0.2 mmol / L) and the non-natural substrate GlcNAz (100 μg / mL) were added to induce glycosylation on the bacterial surface, introducing azide (N3). The cells were then cultured until OD200 reached. 600 If the concentration is above 1.0, collect the bacterial culture, centrifuge at 8000 g for 15 min to remove bacteria, collect the supernatant, filter it through 0.45 μm and 0.22 μm filter membranes in sequence, concentrate it with an ultrafiltration tube, and collect OMV by ultracentrifugation (150,000 g, 3 h) to obtain nano-OMV with azide.

[0037] (2) Synthesis of alkynylated sTn: First, the Tn antigen containing an alkynyl group was synthesized. N-acetylgalactosamine (GalNAc) was reacted in a solution of acetic anhydride and pyridine (volume ratio 1:2) to acetylate and protect the hydroxyl group of GalNAc. After evaporation to dryness, it was dissolved in dichloromethane, washed three times with saturated NaHCO3, washed three times with water, dried with anhydrous Na2SO4, filtered, and evaporated to dryness to obtain the acetylated Tn antigen. The acetylated Tn antigen (620 mg) and alkynyl PEG (250 mg) were dissolved in dichloroethane, and CaSO4 (400 mg) and FeCl3 (200 mg) were added. The mixture was heated under reflux in an oil bath at 110 °C for 12 h, and the single-component product was collected by silica gel column chromatography. The product was then dissolved in methanol, and the pH was adjusted to 8-10 with sodium methoxide. The product was purified by silica gel column chromatography and P2 column chromatography to obtain the alkynylated Tn antigen.

[0038] Subsequently, sialylated alkyne Tn antigen, i.e., alkyne-sTn, was synthesized. Using a one-pot two-enzyme system, alkyne-sTn antigen (100 mg), CTP (cellular triphosphate, 147.86 mg), Neu5Ac (86.79 mg), MgCl2·6H2O (760.34 mg), and enzymes (NMCSS, Psp2,6ST; NMCSS to Psp2,6ST volume ratio 1:2) were added to Tris-HCl buffer (pH 8.5). The mixture was incubated at 37 °C until complete, and the reaction was terminated with an equal volume of ethanol. The mixture was centrifuged, evaporated to dryness, and purified using a P2 column to obtain alkyne-sTn.

[0039] (3) Click chemical coupling: OMV (2.53 mg protein) with an azide group and sTn (10 mg) with an alkyne group were reacted overnight at room temperature in PBS buffer (pH 7.4) under the catalysis of ascorbic acid (7.25 mg) and copper sulfate (7.32 mg) to achieve bioorthogonal click chemical coupling. After the reaction was completed, the reaction solution was centrifuged (10000 g, 30 min), the precipitate was collected, and the purified conjugate was obtained by washing three times with PBS. The reaction route is shown below:

[0040] Example 2 Characterization, Experiment and Results Analysis (1) Characterization: sTn was labeled with sialic acid antibody SNA, incubated at 4 °C for 1 h, centrifuged at 10000 r / min for 30 min, washed once with PBS, resuspended in PBS, and then incubated with DyLight488 streptavidin at 4 °C in the dark for 40 min. OMV was collected by centrifugation at 10000 r / min for 30 min, washed three times with PBS, and the fluorescence intensity was detected by flow cytometry. The coupling rate of sTn was detected by a total sugar content assay kit. The particle size, potential, and PDI of OMV and OMV-sTn were detected by dynamic light scattering (DLS); the morphology was detected by transmission electron microscopy (TEM).

[0041] (2) Phagocytosis of OMV-sTn by antigen-presenting cells: Fluorescent labeling of OMV and OMV-sTn: OMV and OMV-sTn were labeled according to the instructions of Beyotime DiO dye. After incubation at room temperature in the dark for 20 min, OMV and OMV-sTn were collected by centrifugation at 10000 g for 30 min. The samples were washed three times with PBS to obtain DiO-labeled OMV and OMV-sTn.

[0042] Phagocytosis assay: RAW 264.7 or DC 2.4 cells were phagocytosed at a concentration of 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / well in confocal microscopy dishes and cultured for 24 h. Then, fluorescently labeled OMV-sTn (10 μg / mL) or other control substances were added, and the cells were incubated for another 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 min, washed three times with PBS, and stained with DAPI at room temperature for 10 min to stain the nuclei. After washing three times with PBS, 500 μL of PBS was added, and cell uptake was observed using a laser confocal microscope.

[0043] RAW 264.7 or DC 2.4 cells were cultured at 2 × 10⁻⁶. 5Cells were seeded at a density of cells / well in 12-well plates and cultured for 24 h. Then, fluorescently labeled OMV-sTn (10 μg / mL) or other control substances were added, and the cells were incubated for another 24 h. Cells were collected by trypsin digestion, washed three times with PBS, and the fluorescence intensity of the cells was detected by flow cytometry. The mean fluorescence intensity (MFI) was used as the quantitative indicator.

[0044] (3) Activation effect of OMV-sTn on macrophages: RAW264.7 cells were injected with 2×10 5 Cells were seeded at a density of [number] cells / well in 12-well plates and cultured for 24 h. OMV-sTn (10 μg / mL) or other control substances were added, and the plates were incubated for 48 h. Subsequently, cells and cell supernatants were collected separately. The expression of cell surface markers, including CD86, CD80, and CD163, was detected by flow cytometry. The levels of IL-6 and TNF-α in the cell supernatant were detected using an ELISA kit.

[0045] (4) Immunization protocol for animal experiments: Mice were randomly divided into 5 groups (n=5): PBS, sTn (186 μg / mL), OMV (100 μg / mL), OMV+sTn (100 μg+186 μg / mL) and OMV-sTn (100 μg+186 μg / mL) groups, with 100 μL per mouse. Immunization was performed subcutaneously 5 times (on days 0, 5, 10, 15 and 20). On day 25, the mice were euthanized and the spleen, lymph nodes and major organs were collected.

[0046] (5) Detection of cellular immunomodulatory activity: Single-cell suspensions were isolated from the spleen and lymph nodes, blocked, and labeled with antibodies bearing different fluorescent tags. T cells (CD3+) were detected by flow cytometry. + CD4 + and CD3 + CD8 + ) and DC cells (CD11c + CD86 + and CD11c + MHCII + The activation level of ).

[0047] (6) Safety evaluation: After the initial immunization, the weight of the mice was monitored every other day until the end of the experiment. After the mice were euthanized, the heart, liver, spleen, lung and kidney tissues were collected, H&E staining was performed, and histopathological changes were observed under an optical microscope.

[0048] (7) Preventive effect of OMV-sTn on colorectal cancer: BALB / c mice were randomly divided into 5 groups, with 5 mice in each group. These groups were designated as Control group, sTn group, OMV group, OMV+sTn group, and OMV-sTn group. On days 0, 5, 10, and 15, each group was subcutaneously injected with 100 μL of the corresponding vaccine per mouse. On day 20, 100 μL of CT26 cell suspension (10×10⁻⁶ cells) was injected into each mouse. 5 The tumor (volume / mL) was subcutaneously injected into the lower left abdomen of mice. Subsequently, the mouse's weight and the length and width of the tumor were measured every other day, and the tumor volume was calculated using the formula: Tumor Volume = Length × Width. 2 ×0.5, calculate tumor volume. When the tumor volume in the Control group exceeds 1500 mm... 3 At that time, the mice were euthanized, and the tumor tissue was removed for photographing.

[0049] The route design diagram of this invention is as follows: Figure 1 As shown.

[0050] Figure 2 Characterization diagrams of OMV and OMV-sTn are shown below; (A) shows the particle size characterization of OMV by DLS; (B) shows the particle size characterization of OMV-sTn by DLS; (C) shows the potential characterization of OMV and OMV-sTn by DLS; (D) shows the transmission electron microscope image of OMV; (E) shows the transmission electron microscope image of OMV-sTn; and (F) shows the characterization of sTn on the surface of OMV-sTn by flow cytometry. As shown in the figure, the particle size characterization results of OMV and OMV-sTn by DLS indicate that the particle size of OMV remains stable after reacting with sTn. Figure 2 A, B); In the DLS potential characterization results of OMV and OMV-sTn, the encapsulation of sTn slightly reduced the membrane potential value of OMV, but still remained negative. Figure 2 C); OMV and OMV-sTn were characterized by transmission electron microscopy. The results showed that the reaction did not change the morphology of OMV, and OMV-sTn still maintained the bilayer film structure of OMV. Figure 2 D, E); Flow cytometry characterization of sTn on the OMV-sTn surface demonstrated the successful introduction of sTn onto the OMV surface ( Figure 2 F).

[0051] Figure 3 The following are flow cytometry representations of the phagocytic activity of OMV-sTn: (A) shows the uptake of OMV-sTn by RAW264.7 macrophages; (B) shows the fluorescence intensity of OMV-sTn by RAW264.7 macrophages after uptake; (C) shows the uptake of OMV-sTn by DC2.4 dendritic cells; and (D) shows the fluorescence intensity of OMV-sTn by DC2.4 dendritic cells after uptake. Figure 3 In the middle, compared to the Control group, P <0.01, P <0.0001; compared with the OMV group, P && <0.01, P &&&& <0.0001; compared with the OMV+sTn group, P ！ <0.05, P ！！！！ <0.0001. Figure 4 These are laser confocal microscopy images characterizing the phagocytic activity of OMV-sTn; where (A) is a laser confocal microscopy image characterizing the uptake of OMV-sTn by RAW264.7 macrophages; and (B) is a laser confocal microscopy image characterizing the uptake of OMV-sTn by DC2.4 dendritic cells. Figure 3-4 As shown: The results indicate that, compared to the OMV and OMV+sTn groups, OMV-sTn significantly enhanced the uptake of RAW264.7 macrophages ( Figure 3 A, B, Figure 4 A); Compared to OMV and OMV+sTn, OMV-sTn significantly enhances the uptake of DC2.4 by dendritic cells ( Figure 3 C, D, Figure 4 B).

[0052] Figure 5 The figure shows the effect of OMV-sTn on the activation of RAW 264.7 macrophages; where (A) is CD86; (B) is CD80; (C) is CD163; (D) is IL-6; and (E) is TNF-α. Figure 5 In the middle, compared to the Control group, P <0.0001; compared with the sTn group, P #### <0.0001; compared with the OMV group, P && <0.01, P &&& <0.001, P &&&& <0.0001; compared with the OMV+sTn group, P ！！！ <0.01, P ！！！！<0.0001; compared with the OMV-sTn group, P @ <0.05. Compared with the OMV and OMV+sTn groups, OMV-sTn significantly enhanced the expression of macrophage surface molecules CD80 and CD86, and decreased the expression of cell surface CD163, indicating that OMV-sTn can promote macrophage polarization towards M1 type and inhibit its polarization towards M2 type. Figure 5 AC); Compared to OMV and OMV+sTn, OMV-sTn can significantly enhance the secretion of pro-inflammatory cytokines IL-6 and TNF-α by dendritic cells. Figure 5 DE).

[0053] Figure 6 The image shows the in vivo immunological activity evaluation results of OMV-sTn; where (A) represents the effect on CD3+ of mouse spleen T cells. + CD4 + Effect diagram; (B) shows the effect on CD3+ of mouse spleen T cells. + CD8 + Effect diagram; (C) shows the effect on CD11c in mouse spleen DC cells. + CD86 + Effect diagram; (D) shows the effect on CD11c in mouse spleen DC cells. + MHCⅡ + Effect diagram; (E) shows the effect on CD3+ of mouse lymphocytes. + CD4 + Effect diagram; (F) shows the effect on mouse lymphocytes CD3 + CD8 + Effect diagram; (G) shows the effect on mouse lymphocyte DC cells CD11c + CD86 + Effect diagram; (H) shows the effect on mouse lymphocyte DC cells CD11c + MHCⅡ + The impact diagram. Figure 6 In the middle, compared to the Control group, P <0.05, P <0.01, P <0.0001; compared with the sTn group, P ### <0.001, P #### <0.0001; compared with the OMV group, P & <0.05,P && <0.01, P &&& <0.001; compared with the OMV+sTn group, P ！！ <0.01, P ！！！ <0.001, P ！！！！ <0.0001. As shown in the figure, OMV-sTn affects the activation of T cells and DC cells in the mouse spleen, and OMV-sTn can significantly increase CD3+ in the mouse spleen. + CD4 + CD3 + CD8 + The proportion of T cells and CD11c + CD86 + CD11c + MHCⅡ + The proportion of DC cells indicates that OMV-sTn can promote the maturation and activation of T cells and DC cells in the mouse spleen. Figure 6 AD); OMV-sTn affects the activation of T cells and DC cells in mouse lymph nodes; OMV-sTn can significantly increase CD3+ in lymph nodes. + CD4 + CD3 + CD8 + The proportion of T cells and CD11c + CD86 + CD11c + MHCⅡ + The proportion of dendritic cells (DCs) indicates that OMV-sTn can promote the maturation and activation of T cells and DCs in lymph nodes. Figure 6 EH).

[0054] Figure 7 This is an image showing the results of HE staining. (See figure.) Figure 7 The images show the HE staining results of the heart, liver, spleen, lungs, and kidneys of five groups of mice. The images show that there was no obvious damage or inflammatory response in the organs of each group of mice, which proves the safety of the preparation.

[0055] Figure 8 The graph shows the changes in mouse body weight during drug administration. As shown, the body weight of mice in each group showed a relatively stable upward trend, further indicating that the formulation has good safety for mice.

[0056] Figure 9 This is an image of a tumor in a mouse model for colon cancer prevention. Figure 10The following are tumor growth curves for a colon cancer prevention model mouse: (A) Tumor growth curves for each group of mice; (B) Tumor growth curves for the Control group; (C) Tumor growth curves for the sTn group; (D) Tumor growth curves for the OMV group; (E) Tumor growth curves for the OMV+sTn group; and (F) Tumor growth curves for the OMV-sTn group. Figure 10 In the middle, compared to the Control group, P <0.001; compared with the OMV+sTn group, P ！ <0.05. For example... Figure 9-10 As shown, compared with other groups, OMV-sTn can significantly inhibit tumor growth, proving that it has a good anti-tumor effect.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens, characterized in that, This includes: nano-OMV and tumor-associated glycoantigens; The nano-OMV and tumor-associated glycoantigens are coupled via click chemical conjugation.

2. The tumor vaccine as described in claim 1, characterized in that, Azide-containing nano-OMVs are coupled with alkyne-based tumor-associated glycoantigens via click chemistry; or, the tumor-associated glycoantigens include sTn, Tn, TF, LewisX, LewisY, sLeX, sLewisY, sialyl LewisA, KH-1, Globo H, SSEA-3, GD2, GD3, GM2, and GM3.

3. The tumor vaccine as described in claim 2, characterized in that, The tumor-associated glycoantigen is sTn; the OMV mentioned is the outer membrane vesicle of Escherichia coli.

4. The method for preparing a tumor vaccine as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of azide-modified bacterial outer membrane vesicles OMVs-N3; (2) Preparation of alkyne-based tumor-associated glycoantigens; (3) OMVs-N3 is obtained by clicking chemical coupling with alkynylated tumor-associated glycoantigen and then purification.

5. The preparation method according to claim 4, characterized in that, The specific steps of step (1) are as follows: Escherichia coli K5ASSH is cultured in LB liquid medium containing chloramphenicol, kanamycin and GlcNAc, and the bacterial cells are collected; then transferred to LB liquid medium containing chloramphenicol and kanamycin for culture. After 1-2 h of culture, IPTG inducer and GlcNAz are added to induce bacterial surface glycosylation and introduce azide; culture is continued until OD 600 If the concentration is above 1.0, collect the bacterial solution, centrifuge, filter, and concentrate to obtain OMVs-N3.

6. The preparation method according to claim 4, characterized in that, The preparation method of alkynylated sTn is as follows: alkynylated Tn is reacted with CTP, Neu5Ac, MgCl2·6H2O and enzyme in Tris-HCl buffer. After the reaction is completed, alkynylated sTn is obtained through post-processing. Alternatively, the preparation method of acetylated Tn is as follows: (1-1) N-acetylgalactosamine is reacted in acetic anhydride and pyridine solution to acetylate and protect the GalNAc hydroxyl group. After purification, the acetylated Tn antigen is obtained. (1-2) The acetylated Tn antigen and alkynyl PEG were dissolved in dichloroethane, and CaSO4 and FeCl3 were added to react. The reaction product was treated with alkali and then purified to obtain alkynylated Tn.

7. The preparation method according to claim 6, characterized in that, The pH of the Tris-HCl buffer solution is 8.0~9.0; Alternatively, the mass ratio of alkynylated Tn, CTP, Neu5Ac, and MgCl2·6H2O is 1:1.3~1.5:0.8~0.9:7~8; Alternatively, the enzymes are NMCSS and Psp2,6ST; the volume ratio of NMCSS to Psp2,6ST is 1:1~3; Alternatively, in step (2-1), the volume ratio of acetic anhydride to pyridine is 1:1.5~2.5; Alternatively, in step (2-1), the purification operation is as follows: after evaporation, dissolve in dichloromethane, then wash with saturated NaHCO3 and water in sequence, then dry, filter, and evaporate to obtain acetylated Tn antigen; Alternatively, in step (2-2), the mass ratio of acetylated Tn antigen to alkynyl PEG is 1:0.3~0.4; Alternatively, in step (2-2), the mass ratio of acetylated Tn antigen to CaSO4 is 1:0.6~0.8; Alternatively, in step (2-2), the mass ratio of acetylated Tn antigen to FeCl3 is 1:0.3~0.4; Alternatively, in step (2-2), the specific operation of the alkali treatment is as follows: the reaction product is dissolved in methanol, and the pH value is adjusted to 8-10 with sodium methoxide.

8. The preparation method according to claim 4, characterized in that, In step (3), the mass ratio of OMVs-N3 to alkynylated tumor-associated glycoantigen is 0.2-0.3:1; or, in step (3), the click chemical coupling catalyst is ascorbic acid and copper sulfate; or, the mass ratio of ascorbic acid to alkynylated tumor-associated glycoantigen is 0.7-0.8:1; or, the mass ratio of copper sulfate to alkynylated tumor-associated glycoantigen is 0.7-0.8:1; or, in step (3), the click chemical coupling temperature is 20-30 ℃ and the time is 10-15 h.

9. The use of the tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens as described in any one of claims 1 to 3, or the tumor vaccine based on bacterial outer membrane vesicles and tumor-associated glycoantigens prepared by the preparation method described in any one of claims 4 to 8, in the preparation of drugs for the prevention or treatment of tumors.

10. The application as described in claim 9, characterized in that, The tumors mentioned are colon cancer, rectal cancer, gastric cancer, breast cancer, ovarian cancer, pancreatic cancer, lung cancer, prostate cancer, bladder cancer, glioblastoma, neuroblastoma, cholangiocarcinoma, and osteosarcoma.

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