Preparation method for autologous tumor vaccine based on cancerous vesicle modification and use

Abstract

The present invention provides a preparation method for an autologous tumor vaccine based on cancerous vesicle modification. The preparation method comprises: extracting a cancerous vesicle, and modifying the surface thereof with a metal oxide. The metal oxide is one or more of FeOOH, Fe3O4, Fe2O3, Mn3O4, MnO2, MnOOH, AlOOH, and Al3O4. The modification method uses a vesicle surface adsorption compounding method, or in situ growth of a metal oxide on the surface of a vesicle. The present invention also provides an autologous tumor vaccine and use of the vaccine in the preparation of a combined drug for preventing or treating a tumor. The vaccine is prepared by using the described preparation method. In the present invention, by means of extracting a cancerous vesicle and subjecting it to surface modification, the vesicle not only maintains a tumor-specific antigen, but also shields an anti-phagocytic signal, which significantly enhances the uptake and activation effects of DC cells, thereby improving the immune activation effect. Also, the preparation is simple, and the cost is low.

Description

A method for preparing and applying an autologous tumor vaccine based on cancerous vesicle modification Technical Field

[0001] This invention relates to the field of tumor immunotherapy, and in particular to a method for preparing and applying an autologous tumor vaccine based on cancerous vesicle modification. Background Technology

[0002] Autologous tumor vaccines are personalized immunotherapies developed based on a patient's own tumor cells or their derivatives. Their primary goal is to control or eliminate tumors by stimulating the patient's own immune system to recognize and attack these cells. Because they utilize the patient's own antigens, autologous tumor vaccines are highly personalized, enabling precise treatment targeting the unique tumor characteristics of each individual patient.

[0003] In recent years, autologous tumor vaccine technology has made significant progress in the field of tumor immunotherapy, and it is mainly divided into tumor cell lysate vaccine technology, dendritic cell vaccine technology, and cancerous vesicle vaccine technology.

[0004] Tumor cell lysate vaccine technology primarily involves extracting the patient's own tumor cells, lysing them, and then using the lysate as a vaccine. The advantage of this technology is the utilization of tumor-specific antigens, but its disadvantages include a complex preparation process and heterogeneous antigen composition: a) Complex preparation: A large number of tumor cells need to be extracted and processed, making the preparation process complex and time-consuming; b) Heterogeneous antigen composition: The antigenic composition produced after tumor cell lysis is complex and heterogeneous, affecting the vaccine's effectiveness; c) Significant side effects: Due to the presence of a large amount of non-specific antigens, it may cause unnecessary immune responses and side effects.

[0005] Dendritic cell vaccine technology primarily utilizes the patient's own dendritic cells, which are cultured in vitro, loaded with antigens, and then injected back into the patient. This vaccine can effectively activate T-cell responses, but its preparation is costly and complex: a) High cost: The process of culturing dendritic cells in vitro and loading them with antigens is complex and expensive; b) Complex preparation: Dendritic cells need to be extracted from the patient, undergo complex in vitro operations, and then injected back into the body; c) Unstable efficacy: Due to differences in cellular responses among individual patients, the vaccine's effectiveness is inconsistent.

[0006] Cancerous vesicles are nanoparticles derived from tumor cells that carry a large number of bioactive molecules. Cancerous vesicle vaccine technology extracts tumor-derived cancerous vesicles and effectively utilizes the tumor-specific antigens they carry for immunotherapy. Current cancerous vesicle vaccines mainly activate the immune response through direct injection of cancerous vesicles, but there are problems with insufficient immunogenicity and immune escape: a) Insufficient immunogenicity: Simple injection of cancerous vesicles may not be able to fully activate the immune system, resulting in an insufficient immune response; b) Immune escape: Cancerous vesicles may carry immunosuppressive molecules, leading to immune escape and reducing the effectiveness of the vaccine.

[0007] In summary, existing autologous tumor vaccine technologies have limitations in antigen preparation, personalized treatment, immunogenicity, and immune escape. Specifically, existing vaccines typically rely on tumor-associated antigens or synthetic peptides, but these methods fail to fully reflect the specificity and heterogeneity of tumors. Furthermore, existing vaccines have limited effectiveness in activating dendritic cells (DCs) and other immune cells, and struggle to overcome immunosuppressive signals in the tumor microenvironment, resulting in poor efficacy in clinical applications.

[0008] Therefore, there is an urgent need to develop a new type of autologous tumor vaccine technology.

[0009] Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a method for preparing an autologous tumor vaccine based on cancerous vesicle modification and its application. By extracting cancerous vesicles and modifying their surface with metal oxides such as iron oxide, manganese oxide and aluminum oxide, the modified cancerous vesicles not only retain tumor-specific antigens, but also shield anti-phagocytic signals, significantly enhance the uptake and activation effect of DC cells, thereby improving the effect of immune activation. At the same time, the preparation process is simple and low cost.

[0011] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0012] A method for preparing an autologous tumor vaccine based on cancerous vesicle modification involves extracting cancerous vesicles and modifying their surface with metal oxides.

[0013] As one of the preferred embodiments of the present invention, the cancerous vesicles are derived from malignant pleural effusion or ascites.

[0014] As one of the preferred embodiments of the present invention, the metal oxide is one or more of FeOOH, Fe3O4, Fe2O3, Mn3O4, MnO2, MnOOH, AlOOH, and Al3O4.

[0015] As one of the preferred embodiments of the present invention, the modification method employs a vesicle surface adsorption composite method, or uses cancerous vesicles as templates to grow metal oxides in situ on the vesicle surface.

[0016] As one of the preferred embodiments of the present invention, the specific steps of the vesicle surface adsorption composite method are as follows: cancerous vesicles are taken and dispersed in deionized water or PBS solution, with a concentration not exceeding 10 mg / mL; then, under ice bath conditions, an aqueous dispersion of metal oxide is added dropwise, with a concentration not exceeding 10 mg / mL; after stirring for 30 min to 12 h, the product is collected by centrifugation.

[0017] As one of the preferred embodiments of the present invention, the specific steps of the method for in-situ growth of metal oxides on the vesicle surface are as follows: cancerous vesicles are taken and dispersed in deionized water or PBS solution with a concentration not exceeding 10 mg / mL; then, under ice bath conditions, metal oxide precursors are added dropwise; a nonionic surfactant is added, and after stirring for 30 min to 12 h, the product is collected by centrifugation.

[0018] As one of the preferred embodiments of the present invention, the metal oxide precursor is selected from one or more of ferric chloride, ferrous sulfate, ferric sulfate, ferric nitrate, potassium ferrate, potassium permanganate, manganese chloride, aluminum chloride, aluminum sulfate, and aluminum nitrate; wherein ferric chloride, ferrous sulfate, ferric sulfate, ferric nitrate, and potassium ferrate are used as precursors for the preparation of FeOOH, Fe3O4, and Fe2O3; potassium permanganate and manganese chloride are used as precursors for the preparation of Mn3O4, MnO2, and MnOOH; and aluminum chloride, aluminum sulfate, and aluminum nitrate are used as precursors for the preparation of AlOOH and Al3O4.

[0019] As one of the preferred embodiments of the present invention, the nonionic surfactant is selected from one or more of PVP and PEG.

[0020] An autologous tumor vaccine, wherein the vaccine is prepared using the above-described method for preparing an autologous tumor vaccine based on cancerous vesicle modification.

[0021] The application of the above-mentioned autologous tumor vaccine in the preparation of combination drugs for the prevention or treatment of tumors.

[0022] The advantages of this invention compared to the prior art are:

[0023] (1) Personalized treatment: This invention utilizes the patient's own tumor cancerous vesicles to ensure the individualization and specificity of tumor-specific antigens, thereby enhancing the targeted nature of treatment. Compared with traditional uniform antigen or synthetic peptide vaccines, this invention can more accurately reflect the specificity of individual tumors and improve the specificity and effectiveness of vaccines.

[0024] (2) Enhanced immune activation: The surface-modified metal oxide of this invention can shield anti-phagocytic signals, reduce non-specific immune responses and side effects; in addition, through surface modification, it can also enhance the immunogenicity of cancerous vesicles, enhance the uptake and activation effect of dendritic cells (DCs) (reduce immune escape), thereby increasing the intensity of the immune response.

[0025] (3) Simplified preparation process: The extraction and modification process of cancerous vesicles in this invention is relatively simple, reducing preparation costs and time;

[0026] In summary, based on existing autologous tumor vaccine technology, this invention solves the problems of complex antigen preparation, inability to meet individualized treatment, insufficient immunogenicity, and immune escape in existing technologies through innovative surface modification methods, and has significant potential for clinical application. Attached Figure Description

[0027] Figure 1 is a characterization diagram of the composite structure of AlOOH adsorbed on the surface of cancerous vesicles in Example 1;

[0028] Figure 2 is a characterization diagram of the composite structure of FeOOH grown in situ on the surface of cancerous vesicles in Example 2;

[0029] Figure 3 is a characterization diagram of the composite structure of MnO2 grown in situ on the surface of cancerous vesicles in Example 3;

[0030] Figure 4 is a characterization diagram of the composite structure of FeOOH adsorbed on the surface of cancerous vesicles in Example 4;

[0031] Figure 5 is a characterization diagram of the composite structure of Fe3O4 adsorbed on the surface of cancerous vesicles in Example 5;

[0032] Figure 6 is a schematic diagram of the extraction of cancerous vesicles, surface metal oxide functionalization modification, and autologous vaccine reinfusion treatment process in Experiment Example 1.

[0033] Figure 7 is a schematic diagram of the cancerous vesicle structure before and after modification in Experimental Example 1 (in the figure, TEV: cancerous vesicle; mTEV: cancerous vesicle modified with surface metal oxide);

[0034] Figure 8 shows the results of metal oxide modification and characterization of cancerous vesicles in Experiment 1 (Figure a is a schematic diagram of TEV of cancerous vesicles and mTEV of cancerous vesicles with surface modified metal oxide; Figure b is the energy dispersive spectroscopy characterization of cancerous vesicles with surface modified iron oxide; Figure c is the energy dispersive spectroscopy quantitative characterization of cancerous vesicles with surface modified iron oxide; Figure d is the exosome concentration detection characterization of particle size change of cancerous vesicles before and after surface modification with iron oxide; Figure e is the dynamic light scattering detection characterization of particle size change of cancerous vesicles before and after surface modification with iron oxide; Figure f is the surface potential change of cancerous vesicles before and after surface modification with iron oxide).

[0035] Figure 9 shows the results of DC uptake promoted by autologous cancer vesicle vaccines modified with surface metal oxides in Experiment Example 2 (Figure a shows the schematic diagram and actual results of co-incubation and endocytosis of cancer vesicle TEV and surface-modified cancer vesicle mTEV with DC cells, respectively; Figure b shows the flow cytometry results of DC cells uptake of cancer vesicles modified with surface metal oxides from different sources).

[0036] Figure 10 shows the intracellular degradation results of the autologous vaccine modified with surface metal oxide in Experiment Example 2 (Figure a shows the intracellular degradation of cancerous vesicles mTEV modified with surface metal oxide, exposing cancerous vesicles; Figure b shows the color of cancerous vesicles under different pH conditions; Figure c shows the degree of disintegration of the autologous vaccine modified with surface metal oxide under different pH conditions; Figure d shows the morphological observation of degradation under acidic conditions by TEM; Figure e shows the ROS generated during the degradation of metal oxide in a simulated intracellular environment).

[0037] Figure 11 shows the results of promoting DC cell maturation with autologous cancer vesicle vaccines modified with surface metal oxides in Experiment Example 2 (Figure a shows that surface metallization modification significantly promoted the expression of MHC on the surface of DC cells compared with TEVs without surface metallization modification; Figure b shows the flow cytometry results; Figure c shows the quantitative analysis of the flow cytometry results; Figure d shows that mTEVs modified with metal oxides in cancer vesicles more easily promoted the secretion of maturation-related cytokines by DCs).

[0038] Figure 12 shows how the autologous cancerous vesicle vaccine modified with surface metal oxides promoted macrophage uptake and macrophage inflammatory polarization in Experiment Example 2 (Figure a shows the results of fluorescence microscopy; Figure b shows the results of cytokines; Figure c shows the expression of inflammation-related genes in macrophages after treatment with different materials).

[0039] Figure 13 shows the immune activation and efficacy of autologous tumor vaccines in the 4T1 breast cancer model of Experiment Example 3 (Figure a shows the animal experimental design of the tumor-bearing mouse model; Figure b shows the tumor volume growth curve of mice; Figure c shows the tumor growth curve of mice in different treatment groups; Figure d shows the lung tumor metastasis of mice in different treatment groups; Figure e shows the lung nodule statistics: cancerous vesicles with surface metal oxide modification significantly inhibited lung metastasis; Figure f shows the mouse survival curve; Figure g shows the lymph nodes of mice in different treatment groups; Figures h-j show the activation effect of different cancerous vesicle treatments on macrophages in mice; Figure k shows the activation of intratumoral DCs under different cancerous vesicle vaccine treatments by flow cytometry; Figure l shows the quantitative analysis of surface markers of DC activation; Figure m shows the level of IFN-γ secreted by T cells promoted by cancerous vesicle vaccines with different surface states). Detailed Implementation

[0040] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and experimental methods used in the following embodiments are all conventional reagents or methods in the art and will not be described again.

[0041] Example 1: AlOOH-modified autologous tumor vaccine (AlOOH adsorbed on the surface of cancerous vesicles):

[0042] (1) Extract cancerous vesicles from malignant pleural effusion and disperse them in deionized water at a concentration of 6 mg / mL.

[0043] (2) Under ice bath conditions, add AlOOH aqueous dispersion (concentration of 6 mg / mL) dropwise.

[0044] (3) Stir for 6 hours.

[0045] (4) Collect the product by centrifugation at 9000 rpm for 40 min.

[0046] (5) Wash the collected product three times with PBS ice water solution and store at -80℃ for later use.

[0047] Figure 1 shows the characterization of the composite structure of AlOOH adsorbed on the surface of cancerous vesicles in this embodiment.

[0048] Example 2: FeOOH-modified autologous tumor vaccine (FeOOH grown in situ on the surface of cancerous vesicles):

[0049] (1) Extract cancerous vesicles from malignant pleural effusion and disperse them in deionized water at a concentration of 10 mg / mL.

[0050] (2) Add potassium ferrate solution (concentration of 10 mg / mL) dropwise under ice bath conditions.

[0051] (3) Add PVP to achieve a final concentration of 0.1%.

[0052] (4) Stir for 3 hours.

[0053] (5) Collect the product by centrifugation at 10,000 rpm for 35 min.

[0054] (6) Wash the collected product three times with PBS ice water solution and store at -80℃ for later use.

[0055] Figure 2 shows the characterization of the composite structure of FeOOH grown in situ on the surface of cancerous vesicles in this embodiment.

[0056] Example 3: MnO2-modified autologous tumor vaccine (MnO2 grown in situ on the surface of cancerous vesicles):

[0057] (1) Extract cancerous vesicles from malignant pleural effusion and disperse them in PBS solution at a concentration of 10 mg / mL.

[0058] (2) Add potassium permanganate (KMnO4) solution (concentration 10 mg / mL) dropwise under ice bath conditions.

[0059] (3) Add PEG to achieve a final concentration of 0.1%.

[0060] (4) Stir for 5 hours.

[0061] (5) Collect the product by centrifugation at 9500 rpm for 40 min.

[0062] (6) Wash the collected product three times with PBS ice water solution and store at -80℃ for later use.

[0063] Figure 3 shows the characterization of the composite structure of MnO2 grown in situ on the surface of cancerous vesicles in this embodiment.

[0064] Example 4: FeOOH-modified autologous tumor vaccine (FeOOH adsorbed on the surface of cancerous vesicles):

[0065] (1) Extract cancerous vesicles from malignant pleural effusion and disperse them in deionized water at a concentration of 5 mg / mL.

[0066] (2) Add FeOOH nanoparticle aqueous dispersion (concentration of 5 mg / mL) dropwise under ice bath conditions.

[0067] (3) Stir for 4 hours.

[0068] (4) Collect the product by centrifugation at 10,000 rpm for 30 min.

[0069] (5) Wash the collected product three times with deionized water and freeze-dry for later use.

[0070] Figure 4 shows the characterization of the composite structure of FeOOH adsorbed on the surface of cancerous vesicles in this embodiment.

[0071] Example 5: Fe3O4 modified autologous tumor vaccine (Fe3O4 adsorbed on the surface of cancerous vesicles)

[0072] (1) Extract cancerous vesicles from malignant pleural effusion and disperse them in PBS solution at a concentration of 8 mg / mL.

[0073] (2) Add Fe3O4 nanoparticle aqueous dispersion (concentration of 8 mg / mL) dropwise under ice bath conditions.

[0074] (3) Stir for 12 hours.

[0075] (4) Collect the product by centrifugation at 8000 rpm for 45 min.

[0076] (5) Wash the collected product three times with PBS solution and store it at -80℃ for later use.

[0077] Figure 5 shows the characterization of the composite structure of Fe3O4 adsorbed on the surface of cancerous vesicles in this embodiment.

[0078] Example 6: Fe2O3 modified autologous tumor vaccine (Fe2O3 adsorbed on the surface of cancerous vesicles):

[0079] (1) Extract cancerous vesicles from malignant peritoneal effusion and disperse them in PBS solution at a concentration of 10 mg / mL.

[0080] (2) Add Fe2O3 aqueous dispersion (concentration of 10 mg / mL) dropwise under ice bath conditions.

[0081] (3) Stir for 12 hours.

[0082] (4) Collect the product by centrifugation at 10,000 rpm for 35 min.

[0083] (5) Wash the collected product three times with PBS ice water solution and store at -80℃ for later use.

[0084] Example 7: Mn3O4-modified autologous tumor vaccine (Mn3O4 adsorbed on the surface of cancerous vesicles):

[0085] (1) Extract cancerous vesicles from malignant pleural effusion and disperse them in deionized water at a concentration of 5 mg / mL.

[0086] (2) Add Mn3O4 aqueous dispersion (concentration of 5 mg / mL) dropwise under ice bath conditions.

[0087] (3) Stir for 30 minutes.

[0088] (4) Collect the product by centrifugation at 9000 rpm for 40 min.

[0089] (5) Wash the collected product three times with PBS ice water solution and store at -80℃ for later use.

[0090] Example 8: Al3O4-modified autologous tumor vaccine (Al3O4 grown in situ on the surface of cancerous vesicles):

[0091] (1) Extract cancerous vesicles from malignant peritoneal effusion and disperse them in deionized water at a concentration of 6 mg / mL.

[0092] (2) Add aluminum chloride solution (concentration of 6 mg / mL) dropwise under ice bath conditions.

[0093] (3) Add PVP to achieve a final concentration of 0.1%.

[0094] (4) Stir for 12 hours.

[0095] (5) Collect the product by centrifugation at 9500 rpm for 40 min.

[0096] (6) Wash the collected product three times with PBS ice water solution and store at -80℃ for later use.

[0097] Example 9: MnOOH-modified autologous tumor vaccine (MnOOH grown in situ on the surface of cancerous vesicles):

[0098] (1) Extract cancerous vesicles from malignant peritoneal effusion and disperse them in PBS solution at a concentration of 5 mg / mL.

[0099] (2) Add manganese chloride solution (concentration of 5 mg / mL) dropwise under ice bath conditions.

[0100] (3) Add PVP to achieve a final concentration of 0.1%.

[0101] (4) Stir for 30 minutes.

[0102] (5) Collect the product by centrifugation at 9000 rpm for 40 min.

[0103] (6) Wash the collected product three times with PBS ice water solution and store at -80℃ for later use.

[0104] The following experimental examples are used to verify the effectiveness of the vaccine technology of the present invention.

[0105] Experimental Example 1: Extraction and Surface Modification of Cancerous Vesicles:

[0106] Cancerous vesicles were successfully extracted from pleural effusions of multiple patients with malignant tumors and then surface-modified with iron oxide (see Examples 2, 4, and 5), manganese oxide (see Example 3), and aluminum oxide (see Example 1).

[0107] The extraction of cancerous vesicles, surface metal oxide functionalization modification, and autologous vaccine reinfusion treatment process of this invention are shown in Figure 6 (vaccine reinfusion: the surface-modified cancerous vesicle vaccine is reinfused into the patient's body to stimulate an immune response against the tumor using the patient's own immune system). The schematic diagram of the cancerous vesicle structure before and after modification is shown in Figure 7 (in the figure, TEV: cancerous vesicle; mTEV: cancerous vesicle modified with surface metal oxide).

[0108] Figure 8 shows the results of surface metal oxide modification and characterization of cancerous vesicles in this experiment (taking surface-modified iron oxide FeOOH from Example 2 as an example). In the figure, a) is a schematic diagram of cancerous vesicles (TEVs) and surface-modified cancerous vesicles (mTEVs) with metal oxides; b) is the energy dispersive spectroscopy (EDS) characterization of surface-modified iron oxide cancerous vesicles, showing obvious surface element colocalization; c) is the EDS characterization of surface-modified iron oxide cancerous vesicles; d) is the exosome concentration detection characterization of particle size changes before and after surface modification with iron oxide in cancerous vesicles; e) is the dynamic light scattering detection characterization of particle size changes before and after surface modification with iron oxide in cancerous vesicles; f) is the surface potential change before and after surface modification with iron oxide in cancerous vesicles.

[0109] The results above show that the modified cancerous vesicles have stable surfaces and uniform metal oxide modification.

[0110] Experiment Example 2: Antigen-presenting cells (DCs) and macrophages uptake experiment:

[0111] The experimental results are shown in Figures 9-12 (taking surface-modified iron oxide FeOOH from Example 2 as an example).

[0112] Figure 9 shows how autologous vaccines modified with surface metal oxides promote DC uptake. In the figure, Figure a shows the co-incubation and endocytosis of cancerous vesicles (TEV) and surface-modified cancerous vesicles (mTEV) with DC cells, indicating that surface metal oxide modification significantly promotes DC uptake of cancerous vesicles. Figure b is a flow cytometry plot of DC uptake of cancerous vesicles modified with surface metal oxides from different sources, showing that surface modification of cancerous vesicles from different tumor cell sources has a similar trend, that is, DCs have a stronger phagocytic capacity for surface-modified cancerous vesicles.

[0113] Figure 10 shows the intracellular degradation results of the autologous vaccine modified with surface metal oxides. In the figure, a) is a schematic diagram showing that the cancerous vesicles modified with surface metal oxides (mTEVs) can be degraded in cells, exposing the cancerous vesicles; b) shows the color of cancerous vesicles under different pH conditions, showing that they are significantly degraded to colorless under acidic conditions; c) shows the degree of disintegration of the autologous vaccine modified with surface metal oxides under different pH conditions; d) shows the morphological observation of degradation under acidic conditions by TEM; e) shows the ROS generated during the degradation of metal oxides in a simulated intracellular environment.

[0114] Figure 11 shows how surface metal oxide-modified cancerous vesicles (mTEVs) promote DC cell maturation. In the figure, Figure a shows the results of surface metallization modification significantly promoting the expression of MHC on the surface of DC cells compared with unmodified cancerous vesicles (TEVs); Figure b shows the flow cytometry results, demonstrating that metal oxide-modified cancerous vesicles (mTEVs) more readily promote the expression of DC maturation markers CD80 and CD86; Figure c shows the quantitative analysis of the flow cytometry results; Figure d shows that metal oxide-modified cancerous vesicles (mTEVs) more readily promote the secretion of maturation-related cytokines by DCs.

[0115] Figure 12 shows how autologous cancer vesicles (mTEVs) modified with surface metal oxides promote macrophage uptake and macrophage inflammatory polarization. In the figure, Figure a shows the results of fluorescence microscopy, demonstrating that mTEVs modified with metal oxides are more easily taken up by macrophages; Figure b shows the cytokine results, demonstrating that mTEVs modified with metal oxides easily promote macrophage inflammatory polarization; Figure c shows the expression of inflammation-related genes in macrophages after treatment with different materials.

[0116] The results above show that: (1) Compared with unmodified cancerous vesicles, the uptake rate of modified cancerous vesicles by DC cells is significantly increased; (2) The metal oxide materials used for modification also promote the acceleration of the self-antigen processing ability of DC and macrophages.

[0117] Experiment Example 3: Animal Model Experiment

[0118] The experimental results are shown in Figure 13 (taking surface-modified iron oxide FeOOH from Example 2 as an example).

[0119] Figure 13 shows the immune activation and efficacy of autologous tumor vaccines in the 4T1 breast cancer model. In the figure, a) shows the animal experimental design of the tumor-bearing mouse model: mice were injected with different cancer vesicle-related vaccines after breast cancer was constructed; b) shows the tumor volume growth curve in mice: compared with simple cancer vesicles (TEV), surface metal oxide-modified cancer vesicles (mTEV) significantly inhibited tumor growth; c) shows the tumor growth curves in mice under different treatment groups; d) shows the lung tumor metastasis in mice under different treatment groups; e) shows the lung nodule statistics: surface metal oxide-modified cancer vesicles significantly inhibited lung metastasis; f) shows the mouse survival curve; g) shows the lymph nodes in mice under different treatment groups; h-j show the activation effect of different cancer vesicle treatments on macrophages in mice; k) shows the activation of intratumoral DCs under different cancer vesicle vaccine treatments by flow cytometry; l) shows the quantitative analysis of surface markers of DC activation; m) shows the level of IFN-γ secreted by T cells promoted by cancer vesicle vaccines with different surface states.

[0120] The results above indicate that in the mouse tumor model, the tumor growth of the modified cancerous vesicle autovaccine group was significantly inhibited and the survival time was significantly prolonged compared with the control group and the unmodified cancerous vesicle group. Immunohistochemical analysis showed that the activation levels of DC cells and T cells were significantly increased.

[0121] In summary, this invention develops a novel autologous tumor vaccine by extracting cancerous vesicles from pleural or peritoneal effusions of malignant tumors and modifying their surfaces with metal oxides. This vaccine not only retains tumor-specific antigen information but also significantly enhances the uptake and immune activation capabilities of dendritic cells (DCs), demonstrating significant advantages in personalized treatment and promising clinical application prospects.

[0122] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an autologous tumor vaccine based on cancerous vesicle modification, characterized in that, Cancerous vesicles were extracted and their surfaces were modified with metal oxides.

2. The method for preparing an autologous tumor vaccine based on cancerous vesicle modification according to claim 1, characterized in that, The cancerous vesicles originate from malignant pleural effusion or ascites.

3. The method for preparing an autologous tumor vaccine based on cancerous vesicle modification according to claim 1, characterized in that, The metal oxide is one or more of FeOOH, Fe3O4, Fe2O3, Mn3O4, MnO2, MnOOH, AlOOH, and Al3O4.

4. The method for preparing an autologous tumor vaccine based on cancerous vesicle modification according to any one of claims 1 to 3, characterized in that, The modification method employs a vesicle surface adsorption composite method, or uses cancerous vesicles as templates to grow metal oxides in situ on the vesicle surface.

5. The method for preparing an autologous tumor vaccine based on cancerous vesicle modification according to claim 4, characterized in that, The specific steps of the vesicle surface adsorption complexation method are as follows: cancerous vesicles are taken and dispersed in deionized water or PBS solution with a concentration not exceeding 10 mg / mL; then, under ice bath conditions, an aqueous dispersion of metal oxide is added dropwise with a concentration not exceeding 10 mg / mL; after stirring for 30 min to 12 h, the product is collected by centrifugation.

6. The method for preparing an autologous tumor vaccine based on cancerous vesicle modification according to claim 4, characterized in that, The specific steps of the method for in-situ growth of metal oxides on the surface of vesicles are as follows: cancerous vesicles are taken and dispersed in deionized water or PBS solution with a concentration not exceeding 10 mg / mL; then, under ice bath conditions, metal oxide precursors are added dropwise; a nonionic surfactant is added, and after stirring for 30 min to 12 h, the product is collected by centrifugation.

7. The method for preparing an autologous tumor vaccine based on cancerous vesicle modification according to claim 6, characterized in that, The metal oxide precursor is selected from one or more of ferric chloride, ferrous sulfate, ferric sulfate, ferric nitrate, potassium ferrate, potassium permanganate, manganese chloride, aluminum chloride, aluminum sulfate, and aluminum nitrate; wherein ferric chloride, ferrous sulfate, ferric sulfate, ferric nitrate, and potassium ferrate are used as precursors for the preparation of FeOOH, Fe3O4, and Fe2O3; potassium permanganate and manganese chloride are used as precursors for the preparation of Mn3O4, MnO2, and MnOOH; and aluminum chloride, aluminum sulfate, and aluminum nitrate are used as precursors for the preparation of AlOOH and Al3O4.

8. The method for preparing an autologous tumor vaccine based on cancerous vesicle modification according to claim 6, characterized in that, The nonionic surfactant is selected from one or more of PVP and PEG.

9. An autologous tumor vaccine, characterized in that, The vaccine is prepared using the autologous tumor vaccine preparation method based on cancerous vesicle modification as described in any one of claims 1 to 8.

10. The use of an autologous tumor vaccine as described in claim 9 in the preparation of a combination drug for the prevention or treatment of tumors.

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