Tumor vaccine, and preparation method therefor and use thereof

Abstract

The present application belongs to the technical field of medicine. Disclosed are a tumor vaccine, and a preparation method therefor and the use thereof. The raw materials of the tumor vaccine comprise a bacterial cell membrane and a liposome complex, wherein the raw materials of the liposome complex comprise a liposome and an aromatic lipid compound. The tumor vaccine provided includes the bacterial cell membrane and the liposome compound, wherein the liposome compound can fuse cells, the bacterial cell membrane can activate immunity, and the two used in combination can generate an excellent synergistic effect. Therefore, the immune escape state of the tumor cells is changed, DC cells infiltrating the tumor tissue can recognize and phagocytize tumor cells, and tumor antigens are presented, so that an in situ tumor vaccine is formed to achieve the anti-tumor effect. Moreover, the tumor vaccine provided has conventional raw materials, a simple preparation method, and good uniformity, which are beneficial to practical production and application.

Description

A tumor vaccine and its preparation method and application

[0001] Related cross-references

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on July 3, 2023, with application number "202310801545.1" and public name "A tumor vaccine, its preparation method and application", the entire content of which is incorporated by reference into this application. Technical Field

[0003] The present application relates to the field of medical technology, and in particular to a tumor vaccine and a preparation method and application thereof. Background Art

[0004] Cancer vaccines, due to their ability to induce effective anti-tumor responses with minimal systemic toxicity, are a promising alternative or supplement to traditional cancer therapies and have been a research hotspot in the oncology field in China and abroad in recent years. However, since the world's first cancer vaccine, Sipuleucel-T, was launched for prostate cancer treatment in 2010, no new therapeutic cancer vaccine products have been approved for marketing. Consequently, the market for vaccines applicable to various cancers is virtually blank, suggesting a relatively late start in global cancer vaccine research and development. Cancer vaccines with proven clinical efficacy represent not only an unmet need for the health and well-being of Chinese residents, but also represent significant investment value. However, due to the inherent immune escape mechanisms of tumor cells and their complex interactions with the immunosuppressive tumor microenvironment, patient response rates to cancer vaccines, and more generally to immunotherapy, are generally low. Adjuvants are key vaccine components that enhance the intensity, breadth, and durability of immune responses. Effective delivery of tumor antigens and adjuvants is a crucial prerequisite for generating robust and durable anti-tumor immune responses and remains a major challenge in the field of cancer vaccines.

[0005] Existing tumor vaccines achieve their anti-tumor therapeutic goals by directly presenting tumor antigens to dendritic cells (DCs). However, tumors are inherently heterogeneous, and some tumor cells lack the ability to directly activate immune cells. Therefore, even if tumor antigens can be presented to DCs to activate anti-tumor immunity, the weak tumor antigenicity may result in immune neglect, significantly reducing the efficacy of tumor vaccines.

[0006] Summary of the Invention

[0007] The present application discloses a tumor vaccine and its preparation method and application, which can overcome the shortcomings of the above-mentioned prior art.

[0008] To achieve the above objectives, the present application discloses a tumor vaccine in a first aspect, wherein the raw materials of the tumor vaccine include bacterial cell membrane and liposome complex; the raw materials of the liposome complex include liposome and aromatic lipid compounds.

[0009] How to transform a "cold" tumor with a poor response to immunotherapy into a "hot" tumor that can induce an immune response and is infiltrated with a large number of immune cells is the key to improving the effect of immunotherapy; therefore, how to construct an in situ microenvironment that allows the tumor to turn from "cold" to "hot" is the main point of regulating the immune response. The inventors' previous studies have found that exogenous bacterial components can effectively regulate tumor immune responses, can be effectively taken up by antigen presenting cells (APCs), and promote the maturation of APCs. Based on the previous research foundation, the present application provides a tumor vaccine, the raw materials of which include bacterial cell membranes and liposome complexes, wherein the raw materials of the liposome complexes include liposomes and aromatic lipid compounds; that is, the tumor vaccine provided by the present application is a nano-hybrid vesicle system with membrane fusion liposomes as the main body and hybridized bacterial inner membrane components. The tumor vaccine provided in the present application comprises a bacterial cell membrane and a liposome complex, wherein the liposome complex can fuse cells and the bacterial cell membrane can activate immunity. The combination of the two can achieve an excellent synergistic effect, that is, the characteristics of membrane fusion liposomes can be utilized to fuse bacterial components to the surface of the tumor cell membrane, thereby changing the state of tumor cells from immune escape, allowing DC cells infiltrating the tumor tissue to recognize and phagocytize tumor cells, and then present tumor antigens to form an in situ tumor vaccine to achieve an anti-tumor effect.

[0010] As an optional embodiment, the particle size distribution of the tumor vaccine is 142nm-435nm, and the particle size distribution coefficient is 0.166-0.657.

[0011] Preferably, the Zeta potential of the tumor vaccine is (-33) mV-(+23) mV.

[0012] As an optional embodiment, the mass ratio of the bacterial cell membrane and the liposome complex is bacterial cell membrane:liposome complex=1:(0.5-20).

[0013] Exemplarily, the mass ratio of the bacterial cell membrane to the liposome complex can be 1:0.5, 1:1, 1:2, 1:4, 1:5, 1:6, 1:8, 1:10, 1:12, 1:16, 1:18, or 1:20.

[0014] The inventors have discovered that when the mass ratio of the bacterial cell membrane and liposome complex is within the range given above, the advantages of the bacterial cell membrane and liposome complex can be significantly amplified, that is, the two have a significant synergistic effect, thereby achieving the maximum fusion rate of tumor cells to tumor vaccines and the highest antigen presentation rate of DCs to tumors. In other words, the resulting product has a better inhibitory effect on tumors, which is reflected in slower tumor growth and longer survival of the research subjects.

[0015] As an optional embodiment, in the liposome complex, the mass ratio of liposome to aromatic lipid compound is liposome: aromatic lipid compound = (1.5-2.5):0.1.

[0016] For example, the mass ratio of liposomes to aromatic lipid compounds can be 1.5:0.1, 2:0.1, or 2.5:0.1.

[0017] The inventors have found that when the mass ratio of liposomes to aromatic lipid compounds in the liposome complex is within the above range, the overall effect of the obtained product is better.

[0018] As an optional embodiment, the liposome is at least one of DOPE and DOTAP.

[0019] Preferably, the liposome is a mixture of DOPE and DOTAP.

[0020] More preferably, the liposome is a mixture of DOPE and DOTAP in a mass ratio of 1:1.

[0021] The inventors have found that when the liposomes are preferably at least one of DOPE and DOTAP, especially a mixture of the two, the resulting product has a better inhibitory effect on tumors. Among them, when the liposomes are a mixture of DOPE and DOTAP in a mass ratio of 1:1, the resulting product exhibits the best anti-tumor effect.

[0022] As an optional embodiment, the aromatic lipid compound is at least one of DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide), DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and DiO (3-octadecyl-2-[3-(3-octadecyl-2(3H)-benzoxazol-2-ylidene)-1-propen-1-yl]benzoxazole perchlorate).

[0023] The inventors have found that when the aromatic lipid compound is further selected as at least one of DiR, DiI, and DiO, the resulting product can more significantly inhibit the growth rate of tumors and prolong the survival of the research subjects.

[0024] As an optional embodiment, the method for preparing the bacterial cell membrane is as follows: the frozen bacterial liquid is inoculated into LB medium for culturing for 8h-12h, and then the culture is taken and inoculated into LB medium again for constant temperature shaking culture. When the OD600 is 0.7-1, the culture is terminated and the bacteria are collected and washed; then lysozyme is added to the washed product for treatment. After the treatment, the protoplasts are collected, washed, and resuspended with PBS, and then an extraction solution equal in volume to the bacterial liquid is added for incubation. After the incubation is completed, the mixture is centrifuged, the supernatant is taken, resuspended with PBS, and frozen for storage.

[0025] Because the bacterial inner membrane itself is an extracellular substance with extremely strong immunogenicity, it can be used as a natural adjuvant to activate the body's anti-tumor immune response. Therefore, the present application does not limit the bacterial cell membrane. For example, the bacterial liquid can be any bacterial liquid such as Escherichia coli liquid and Bifidobacterium liquid.

[0026] Preferably, the volume ratio of the frozen bacterial solution to the LB culture medium is (4-6) μL:10 mL.

[0027] Illustratively, the volume ratio of the frozen bacterial solution to the LB culture medium may be 4 μL:10 mL, 4.5 μL:10 mL, 5 μL:10 mL, 5.5 μL:10 mL, or 6 μL:10 mL.

[0028] Preferably, the culture conditions for 8-12 hours are: culture at 37°C ± 2°C and 180-220 rpm.

[0029] Illustratively, the culture rotation speed may be 180 rpm, 190 rpm, 200 rpm, 210 rpm, or 220 rpm.

[0030] Preferably, when the culture is taken out and inoculated into LB medium again, the mass ratio of the culture to the LB medium is (0.8-1.2):100.

[0031] Illustratively, the mass ratio of the culture medium to the LB medium may be 0.8:100, 0.9:100, 1.0:100, 1.1:100, or 1.2:100.

[0032] Preferably, the constant temperature shaking culture conditions are: culture at 37°C ± 2°C, 180 rpm-220 rpm for 2 h-4 h.

[0033] Illustratively, the culture rotation speed may be 180 rpm, 190 rpm, 200 rpm, 210 rpm, or 220 rpm, and the culture time may be 2 h, 2.5 h, 3 h, 3.5 h, or 4 h.

[0034] Preferably, the centrifugation conditions for the bacteria collection centrifugation are: centrifugation at 4°C±1°C, 2800g-3200g for 15min-25min.

[0035] Illustratively, the centrifugal force in the centrifugation conditions may be 2800g, 2850g, 2900g, 2950g, 3000g, 3050g, 3100g, 3150g, or 3200g, and the centrifugation time may be 15 min, 18 min, 20 min, 22 min, or 25 min.

[0036] Preferably, in the lysozyme treatment, the mass concentration of lysozyme in the mixed system is 1.8 mg / mL-2.2 mg / mL, and the treatment conditions are shaking digestion at 37°C±2°C for 55min-65min, followed by centrifugation at 4°C±1°C and 2800g-3200g for 15min-25min.

[0037] Exemplarily, the mass concentration of the lysozyme in the mixed system can be 1.8 mg / mL, 1.9 mg / mL, 2.0 mg / mL, 2.1 mg / mL, 2.2 mg / mL, the shaking digestion time can be 55 min, 57 min, 59 min, 61 min, 63 min, 65 min, the centrifugal force in the centrifugation conditions can be 2800 g, 2850 g, 2900 g, 2950 g, 3000 g, 3050 g, 3100 g, 3150 g, 3200 g, and the centrifugation time can be 15 min, 18 min, 20 min, 22 min, 25 min.

[0038] Preferably, an appropriate amount of PBS is added to resuspend the protoplasts obtained above to obtain a bacterial solution, and an extraction solution of equal volume to the bacterial solution is added, wherein the extraction solution is a 2% Triton X-100 solvent, MgCl2, DNAse, and Tris-HCl with a pH value of 8.0 are added thereto, wherein the molar concentration of Tris-HCl in the extraction solution is 50 mM, the molar concentration of MgCl2 is 10 mM, and the mass concentration of DNAse is 10 mg / mL.

[0039] Preferably, the incubation is carried out on ice for 25 min to 35 min.

[0040] Preferably, the centrifugation after incubation is: first centrifugation at 4°C±1°C, 3800g-4200g for 4min-6min, then taking the supernatant after centrifugation and centrifuging again at 4°C±1°C, 17000g-19000g for 8min-12min.

[0041] Illustratively, the centrifugal force under centrifugation conditions at 4°C±1°C may be 3800g, 3850g, 3900g, 3950g, 4000g, 4050g, 4100g, 4150g, or 4200g, and the centrifugal force for re-centrifuging the supernatant after centrifugation may be 17000g, 17500g, 18000g, 18500g, or 19000g.

[0042] Preferably, the freezing storage temperature is -80°C ± 2°C.

[0043] As an optional embodiment, the liposome complex is prepared by adding liposomes and aromatic lipid compounds to chloroform, then evaporating the chloroform, then dispersing the evaporated product in 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid (HEPES) buffer, vortexing, and finally ultrasonically homogenizing to obtain the liposome complex.

[0044] Preferably, the ratio of the total mass of the liposomes and the aromatic lipid compound to the mass volume of chloroform is (5-15) mg:1 mL.

[0045] Illustratively, the ratio of the total mass of the liposomes and the aromatic lipid compound to the mass of chloroform is 5 mg:1 mL, 10 mg:1 mL, or 15 mg:1 mL.

[0046] Preferably, the evaporation is carried out using a rotary evaporator in a water bath at 55° C.-65° C., and the chloroform evaporation time is 0.5 h-1 h.

[0047] Preferably, the concentration of the HEPES buffer is 18 mM-22 mM; the sum of the mass concentrations of the liposomes and the aromatic lipid compound in the buffer is 2 mg / mL-10 mg / mL.

[0048] Illustratively, the sum of the mass concentrations of the liposomes and the aromatic lipid compound in the buffer solution may be 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, or 10 mg / mL.

[0049] Preferably, the vortex time is 1 min-2 min.

[0050] Preferably, the ultrasonic homogenization time is 10 min-20 min, and the ultrasonic homogenization power is 25W-35W.

[0051] Illustratively, the time for ultrasonic homogenization can be 10 min, 12 min, 14 min, 16 min, 18 min, or 20 min, and the power can be 25 W, 27 W, 29 W, 30 W, 32 W, or 35 W.

[0052] The second aspect of the present application discloses a method for preparing a tumor vaccine, which comprises: mixing bacterial cell membranes and liposome complexes, incubating the mixture, and extruding the mixture after the incubation to obtain the tumor vaccine.

[0053] As an optional embodiment, the extrusion is performed through a liposome extruder, the pore size of the filter membrane of the extruder is 200nm-400nm; and the number of extrusions is 8-12 times.

[0054] Illustratively, the pore size of the filter membrane may be 200 nm, 300 nm, or 400 nm, and the number of extrusions may be 8 times, 9 times, 10 times, 11 times, or 12 times.

[0055] The third aspect of the present application discloses the use of tumor vaccines in the preparation of drugs for in situ enhancement of tumor cell autoimmunogenicity.

[0056] Compared with the prior art, this application has the following beneficial effects:

[0057] This application provides a tumor vaccine, preparation method, and application thereof. The tumor vaccine comprises a bacterial cell membrane and liposome complex. The liposome complex is capable of cell fusion, while the bacterial cell membrane is capable of immune activation. The combination of the two creates a synergistic effect, shifting tumor cells from a state of immune escape. This allows DCs infiltrating tumor tissue to recognize and phagocytose the tumor cells, subsequently presenting tumor antigens, thereby forming an in situ tumor vaccine and achieving an anti-tumor effect. Furthermore, the tumor vaccine provided herein utilizes conventional raw materials, a simple preparation method, and a uniform and excellent quality, making it suitable for practical production and application. DETAILED DESCRIPTION

[0058] In order to better illustrate the purpose, technical solutions and advantages of this application, this application will be further described below in conjunction with specific embodiments.

[0059] Example 1

[0060] The present invention provides a tumor vaccine, and the preparation method of the tumor vaccine is as follows:

[0061] (1) Preparation of bacterial cell membrane: Frozen Escherichia coli liquid (5 μL) was inoculated into 10 mL of LB medium and cultured overnight at 37°C and 200 rpm for 10 hours; then the overnight culture was inoculated into LB medium at a mass ratio of 1:100. After inoculation, the culture was cultured at 37°C and 200 rpm for 3 hours. When the OD600 value was monitored to be between 0.7 and 1, the bacteria were harvested (centrifuged at 4°C and 3000 g for 20 minutes) and washed three times with PBS. After washing, lysozyme (final concentration of 2 mg / mL) was added to digest the bacterial cell wall and the culture was digested at 37°C and shaken for 1 hour. After digestion, the culture was centrifuged at 4°C and 3000 g for 20 minutes. The protoplasts were collected and washed three times with PBS. After washing, the cells were resuspended in an appropriate amount of PBS. Next, add an equal volume of extraction solution (2% Triton X-100, MgCl2, DNAse, and Tris-HCl (pH 8.0) added to the solution; the molar concentration of Tris-HCl is 50 mM, the molar concentration of MgCl2 is 10 mM, and the mass concentration of DNAse is 10 mg / mL) and incubate on ice for 30 minutes. After incubation, centrifuge the lysate at 4000 rpm for 5 minutes at 4°C. The supernatant is then centrifuged at 18000 rpm for 10 minutes at 4°C. Resuspend the supernatant in PBS and assay the protein concentration using a BCA protein quantification kit. Store at -80°C until further use.

[0062] (2) Preparation of liposome complexes: DOPE, DOTAP, and DiR were mixed in a mass ratio of 1:1:0.1 and dissolved in chloroform, wherein the mass volume ratio of the total mass of liposomes and aromatic lipid compounds to the mass volume of chloroform was 10 mg:1 mL. Chloroform was then rotary evaporated at 60°C for 30 min. The evaporation product was then dispersed in a 20 mM HEPES buffer and vortexed for 1.5 min. The product was then homogenized under ultrasound at 30 W for 15 min to obtain a liposome complex, wherein the total mass concentration of DOPE, DOTAP, and DiR in the buffer was 2 mg / mL.

[0063] (3) Preparation of tumor vaccine: The bacterial cell membrane prepared in step (1) and the liposome complex prepared in step (2) were mixed at a mass ratio of 1:10, incubated at 37°C for 10 min, and then extruded through a 200 nm filter membrane using a liposome extruder for 10 times to obtain a tumor vaccine.

[0064] Example 2

[0065] This embodiment of the present application provides a tumor vaccine. The only difference between the tumor vaccine and Example 1 is that the aromatic lipid compound used in step (2) is DiI.

[0066] Example 3

[0067] This embodiment of the present application provides a tumor vaccine. The only difference between the tumor vaccine and Example 1 is that the aromatic lipid compound used in step (2) is DiO.

[0068] Example 4

[0069] This embodiment of the present application provides a tumor vaccine. The only difference between the tumor vaccine and Example 1 is that the mass ratio of the bacterial cell membrane and the liposome complex in step (3) is 2:1.

[0070] Example 5

[0071] This embodiment of the present application provides a tumor vaccine. The only difference between the tumor vaccine and Example 1 is that the mass ratio of the bacterial cell membrane and the liposome complex in step (3) is 1:1.

[0072] Example 6

[0073] This embodiment of the present application provides a tumor vaccine. The only difference between the tumor vaccine and Example 1 is that the mass ratio of the bacterial cell membrane and the liposome complex in step (3) is 1:2.

[0074] Example 7

[0075] This embodiment of the present application provides a tumor vaccine. The only difference between the tumor vaccine and Example 1 is that the mass ratio of the bacterial cell membrane and the liposome complex in step (3) is 1:5.

[0076] Example 8

[0077] This embodiment of the present application provides a tumor vaccine. The only difference between the tumor vaccine and Example 1 is that the mass ratio of the bacterial cell membrane and the liposome complex in step (3) is 1:20.

[0078] Example 9

[0079] This embodiment of the present application provides a tumor vaccine. The only difference between the tumor vaccine and Example 1 is that the Escherichia coli bacterial solution is replaced by Bifidobacterium bacterial solution in step (1).

[0080] Example 10

[0081] The present embodiment provides a tumor vaccine. The only difference between the tumor vaccine and Example 1 is that in step (2), DOPE, DOTAP, and DiR are mixed in a mass ratio of 2:0.5:0.1.

[0082] Comparative Example 1

[0083] The comparative example of the present application provides a tumor vaccine, the only difference between the tumor vaccine and Example 1 is that DiR is not included in step (2).

[0084] Effect Example 1

[0085] The particle size, particle size distribution coefficient (PDI) and Zeta potential value of the tumor vaccine prepared in Examples 1-10 and Comparative Example 1 of the present application were tested. The particle size and particle size distribution coefficient were measured by dynamic light scattering, and the Zeta potential was measured by a surface potential meter. The test results are shown in Table 1.

[0086] Table 1

[0087] As can be seen from Table 1, the tumor vaccine prepared by the preparation method of the present application has uniform particle size and excellent dispersibility.

[0088] Effect Example 2

[0089] The present application test examples 1-10 and comparative example 1 prepared a tumor vaccine to activate dendritic cells in vitro, specifically comprising the following steps:

[0090] The B16-OVA tumor cells were divided into 13 groups, specifically the tumor vaccines prepared in Examples 1-10 and Comparative Example 1, the bacterial cell membrane group (prepared by the preparation method in Example 1), and the blank group; the tumor cells were mixed and incubated with the tumor vaccines and bacterial cell membranes prepared in Examples 1-10 and Comparative Example 1, respectively, wherein the blank group was not added with any substance, and the expression changes of MHCI-SIINFEKL on the surface of DC cells at different incubation times were recorded, and the secretion of cytokines IL-6, IL-1β, and TNF-α of DC cells was measured at the same time; specifically, the prepared products of each group were incubated with tumor cells for 4 hours, and the number of dendritic cells and tumor cells was co-cultured for 24 hours at a ratio of 5:1. After that, the cell culture supernatant was collected for cytokine (IL-6, IL-1β, TNF-α) determination. The cells were then collected for flow cytometry determination, and after screening the dendritic cells by CD11c staining, the expression of MHCI-SIINFEKL therein was determined, and the results were recorded as shown in Table 2.

[0091] Table 2

[0092] As can be seen from Table 2, compared with the blank group, the technical solution of the present application can significantly improve the antigen presentation of dendritic cells, while increasing the release of cytokines such as IL-6, IL-1β, and TNF-α by dendritic cells. It can be seen from the examples, the bacterial cell membrane group, and Comparative Example 1 that the bacterial cell membrane and liposome complex of the present application are indispensable, and good results can only be achieved when the two are prepared at the same time.

[0093] Effect Example 3

[0094] The efficacy example of this application uses 4T1 breast cancer-bearing mice to evaluate the tumor vaccines prepared in Examples 1-10 and Comparative Example 1, specifically comprising the following steps:

[0095] 130 mice bearing 4T1 breast cancer were divided into 13 groups, with 10 mice in each group; specifically, the tumor vaccine and bacterial cell membrane groups prepared in Examples 1-10 and Comparative Example 1 (prepared by the preparation method in Example 1), and a blank group; 50 μL of tumor vaccine or bacterial cell membrane was injected intratumorally on days 4, 6, and 8 after inoculation, and the tumor volume of the mice was measured on days 0, 6, 8, 10, 12, 14, 18, and 20 after tumor inoculation, and the average volume was recorded. The tumor volume was determined by multiplying the length by the square of the width divided by 2. The tumor volume of the mouse was greater than 2000 mm 3 Death was determined after the mice were killed. The weight and survival period of the mice were observed. The survival rate was the number of days when the survival rate of the mice reached 50% and 0%. The tumor volume (mm 3 ) test results are shown in Table 3, and the survival rate (d) test results are shown in Table 4.

[0096] Table 3

[0097] It can be seen from Table 3 that, compared with the blank group, the product obtained by adopting the technical solution of the present application can significantly slow down the growth rate of tumors; it can be seen from the examples, bacterial cell membrane group and comparative example 1 that when the technical solution of the present application is not adopted, the growth of tumors cannot be effectively slowed down.

[0098] Table 4

[0099] It can be seen from Table 4 that, compared with the blank group, the product obtained by adopting the technical solution of the present application can significantly prolong the survival of mice; it can be seen from the examples, bacterial cell membrane group and comparative example 1 that, when the technical solution of the present application is not adopted, there is no significant effect of prolonging the survival of tumor mice compared with the blank group.

[0100] Effect Example 4

[0101] The efficacy example of this application uses 4T1 breast cancer-bearing mice to conduct in vivo evaluation of the tumor vaccines prepared in Examples 1-10 and Comparative Example 1, specifically comprising the following steps:

[0102] 130 mice bearing 4T1 breast cancer were taken and divided into 13 groups, with 10 mice in each group; specifically, the tumor vaccine prepared in Examples 1-10 and Comparative Example 1, the bacterial cell membrane group (prepared by the preparation method in Example 1), and the blank group; 50 μL of tumor vaccine or bacterial cell membrane was injected into the tumor on the 4th, 6th, and 8th days after tumor inoculation, respectively. The growth of the tumor was observed on days 6, 8, 10, 12, 14, 16, and 18 after tumor inoculation. On the 18th day, the mice were sacrificed and the mice, tumors, spleens and other organs were taken for subsequent analysis. CD3, CD4, CD8, etc. were labeled and statistically analyzed for tumor-infiltrating T cells. After taking equal mass of tumor tissue, the tumor cells were ground and extracted. After treating the red blood cells in the tumor with red blood cell lysis solution, the cells were stained with antibodies and then measured by flow cytometry. At the same time, for mouse spleen cells, 100,000 mouse spleen cells were inoculated into a 96-well plate for ELISPOT test at each well. After stimulation with 10 μg / mL 4T1 tumor lysate for 24 hours, antigen-specific T cells were analyzed using ELISPOT. The results are shown in Table 5.

[0103] Table 5

[0104] As can be seen from Table 5, compared with the blank group, the product obtained by adopting the technical solution of the present application can significantly increase the infiltration of CD8+ T cells in the tumor, and at the same time increase the number of tumor-specific T cells in the spleen; it can be seen from the examples, bacterial cell membrane group and comparative example 1 that when the technical solution of the present application is not adopted, there is no obvious improvement in the infiltration of CD8+ T cells in the tumor and the number of spleen-specific T cells.

[0105] Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A tumor vaccine, characterized in that: The raw materials of the tumor vaccine include bacterial cell membrane and liposome complex; The raw materials of the liposome complex include liposome and aromatic lipid compound.

2. The tumor vaccine according to claim 1, characterized in that The particle size of the tumor vaccine is 142nm-435nm, and the particle size distribution coefficient is 0.166-0.

657.

3. The tumor vaccine according to claim 1, characterized in that The mass ratio of the bacterial cell membrane to the liposome complex is bacterial cell membrane:liposome complex=1:(0.5-20).

4. The tumor vaccine according to claim 1, characterized in that In the liposome complex, the mass ratio of liposome to aromatic lipid compound is liposome:aromatic lipid compound=(1.5-2.5):0.

1.

5. The tumor vaccine according to claim 4, characterized in that The liposome is at least one of DOPE and DOTAP.

6. The tumor vaccine according to claim 1, characterized in that The aromatic lipid compound is at least one of DiR, DiI and DiO.

7. The tumor vaccine according to claim 1, characterized in that The preparation method of the bacterial cell membrane is: The frozen bacterial liquid was inoculated into LB medium for culturing for 8h-12h, and then the culture was taken and inoculated into LB medium again for constant temperature shaking culture. When OD600 was 0.7-1, the culture was terminated and the bacteria were collected and washed. Then lysozyme was added to the washed product for treatment. After the treatment, the protoplasts were collected, washed, and resuspended with PBS. Then, an extraction solution with an equal volume to the bacterial liquid was added for incubation. After the incubation, the mixture was centrifuged, and the supernatant was taken, resuspended with PBS, and frozen for storage.

8. The tumor vaccine according to claim 1, characterized in that The preparation method of the liposome complex is: The liposomes and aromatic lipid compounds are added to chloroform, and then the chloroform is evaporated. The evaporated product is then dispersed in 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid buffer and vortexed, and finally ultrasonically homogenized to obtain a liposome complex.

9. The method for preparing a tumor vaccine according to any one of claims 1 to 8, characterized in that: The preparation method is: The bacterial cell membrane and liposome complex are mixed and incubated, and then extruded after the incubation to obtain the tumor vaccine.

10. Use of the tumor vaccine according to any one of claims 1 to 8 in the preparation of a drug for in situ enhancing the autoimmunogenicity of tumor cells.

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