Tumor vaccine as well as preparation method and application thereof

Abstract

The invention discloses a tumor vaccine as well as a preparation method and application thereof, and belongs to the technical field of medicines. The tumor vaccine is prepared from the following raw materials: a bacterial cell membrane and a lipidosome compound, the lipidosome compound is prepared from the following raw materials: lipidosome and an aromatic lipid compound. According to the tumor vaccine provided by the invention, the bacterial cell membrane and the liposome compound are included, the liposome compound can fuse cells, the bacterial cell membrane can activate immunity, and the combination of the liposome compound and the bacterial cell membrane can achieve an excellent synergistic effect, so that tumor cells are changed from an immune escape state; the DC cells infiltrated in the tumor tissue can recognize and phagocytize the tumor cells, and then the tumor antigen is presented, so that the in-situ tumor vaccine is formed, and the anti-tumor effect is achieved. The tumor vaccine provided by the invention has the advantages of conventional raw materials, simple preparation method and good uniformity, and is beneficial to actual production and application.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a tumor vaccine and a preparation method and application thereof. Background Art

[0002] Because of its ability to induce effective anti-tumor responses with low systemic toxicity, tumor vaccines are a very promising strategy to replace or supplement traditional cancer therapies and have been one of the research hotspots in the field of tumors at home and abroad in recent years. However, since the world's first tumor vaccine Sipuleucel-T was used to treat prostate cancer in 2010, no new therapeutic tumor vaccine products have been approved for marketing. Therefore, the market for vaccines that can be used to treat various tumors is almost blank, suggesting that the global tumor vaccine research and industry started late. Tumor vaccines with good clinical therapeutic effects not only represent the unmet needs of the life and health of Chinese residents, but also breed this heavy investment value. However, due to the immune escape mechanism of tumor cells themselves and the complex interaction with the immunosuppressive tumor microenvironment, the clinical response rate of patients to tumor vaccines and more general immunotherapy is generally low. Adjuvants are key vaccine components that enhance the intensity, breadth and durability of immune responses. The effective delivery of tumor antigens and adjuvants is an important prerequisite for generating strong and lasting anti-tumor immune responses, and it is also a major challenge in the field of tumor vaccines.

[0003] Existing tumor vaccines achieve the purpose of anti-tumor treatment by directly presenting tumor antigens to dendritic cells (DC cells). However, tumors themselves are highly heterogeneous, and some tumor cells lack direct activation of immune cells. Therefore, even if tumor antigens can be presented to DC cells to activate anti-tumor immunity, it is possible that the weak tumor antigenicity will result in immune neglect, greatly reducing the efficacy of tumor vaccines. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and to provide a tumor vaccine and a preparation method and application thereof.

[0005] To achieve the above objectives, in a first aspect of the present invention, the present invention provides a tumor vaccine, the raw materials of which include bacterial cell membrane and liposome complex; the raw materials of the liposome complex include liposome and aromatic lipid compounds.

[0006] 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 effectiveness of immunotherapy; therefore, how to construct a microenvironment in situ that allows the tumor to turn from "cold" to "hot" is the main point in 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 invention 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 invention is a nano-hybrid vesicle system with membrane fusion liposomes as the main body and hybridized bacterial inner membrane components. The tumor vaccine provided by the present invention includes bacterial cell membrane and liposome complex, wherein the liposome complex can fuse cells and the bacterial cell membrane can activate immunity, and the combination of the two can achieve excellent synergistic effect, that is, the characteristics of membrane fusion liposomes can be used to fuse bacterial components to the surface of tumor cell membranes, thereby changing the state of tumor cells from immune escape, allowing DC cells infiltrating in tumor tissues to recognize and phagocytize tumor cells, and then present tumor antigens to form an in situ tumor vaccine to achieve an anti-tumor effect.

[0007] As a preferred embodiment of the tumor vaccine of the present invention, the particle size distribution of the tumor vaccine is 142-435 nm, and the particle size distribution coefficient is 0.166-0.657.

[0008] Preferably, the Zeta potential of the tumor vaccine is (-33)-(+23) mV.

[0009] As a preferred embodiment of the tumor vaccine of the present invention, the mass ratio of the bacterial cell membrane to the liposome complex is bacterial cell membrane:liposome complex=1:(0.5-20).

[0010] Exemplarily, the mass ratio of the bacterial cell membrane to the liposome complex may 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.

[0011] The inventors have found that when the mass ratio of the bacterial cell membrane and the liposome complex is within the range given above, the advantages of the bacterial cell membrane and the liposome complex can be significantly amplified, that is, the two have an obvious synergistic effect, thereby achieving the maximum fusion rate of tumor cells to tumor vaccines and the highest antigen presentation rate of DC to tumors, that is, the obtained product has a better inhibitory effect on tumors, which is reflected in the slower growth rate of tumors and longer survival of the research subjects.

[0012] As a preferred embodiment of the tumor vaccine of the present invention, in the liposome complex, the mass ratio of liposome to aromatic lipid compound is liposome: aromatic lipid compound = (1.5-2.5): 0.1.

[0013] Exemplarily, the mass ratio of liposome to aromatic lipid compound may be 1.5:0.1, 2:0.1, or 2.5:0.1.

[0014] 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.

[0015] As a preferred embodiment of the tumor vaccine of the present invention, the liposome is at least one of DOPE and DOTAP.

[0016] Preferably, the liposome is a mixture of DOPE and DOTAP.

[0017] More preferably, the liposome is a mixture of DOPE and DOTAP in a mass ratio of 1:1.

[0018] 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.

[0019] As a preferred embodiment of the tumor vaccine of the present invention, 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).

[0020] 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 the tumor and prolong the survival period of the research subject.

[0021] As a preferred embodiment of the tumor vaccine described in the present invention, the preparation method of the bacterial cell membrane is as follows: the frozen bacterial liquid is inoculated into the LB medium for culturing for 8-12 hours, and then the culture is taken and inoculated into the 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 extraction solution is centrifuged, and the supernatant is taken, resuspended with PBS, and then frozen for storage.

[0022] Because the bacterial inner membrane itself is an extracellular substance with strong immunogenicity, it can be used as a natural adjuvant to activate the body's anti-tumor immune response. Therefore, the present invention is not limited to the bacterial cell membrane. Exemplarily, the bacterial liquid can be any bacterial liquid such as Escherichia coli liquid and Bifidobacterium liquid.

[0023] Preferably, the volume ratio of the frozen bacterial solution to the LB culture medium is (4-6) μL:10 mL.

[0024] 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.

[0025] Preferably, the culture conditions for 8-12 hours are: culture at 37±2°C and 180-220 rpm.

[0026] Illustratively, the culture rotation speed may be 180 rpm, 190 rpm, 200 rpm, 210 rpm, or 220 rpm.

[0027] 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.

[0028] Exemplarily, 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.

[0029] Preferably, the constant temperature shaking culture conditions are: culture at 37±2°C and 180-220 rpm for 2-4 hours.

[0030] 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.

[0031] Preferably, the centrifugation conditions in the bacteria collection centrifugation are: centrifugation at 4±1°C and 2800-3200g for 15-25 min.

[0032] Exemplarily, the centrifugal force in the centrifugal conditions may be 2800g, 2850g, 2900g, 2950g, 3000g, 3050g, 3100g, 3150g, 3200g, and the centrifugation time may be 15min, 18min, 20min, 22min, 25min.

[0033] Preferably, in the lysozyme treatment, the mass concentration of lysozyme in the mixed system is 1.8-2.2 mg / mL, and the treatment conditions are shaking digestion at 37±2°C for 55-65 min, followed by centrifugation at 4±1°C and 2800-3200g for 15-25 min.

[0034] 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, and 2.2 mg / mL, the shaking digestion time can be 55 min, 57 min, 59 min, 61 min, 63 min, and 65 min, the centrifugal force in the centrifugal conditions can be 2800 g, 2850 g, 2900 g, 2950 g, 3000 g, 3050 g, 3100 g, 3150 g, and 3200 g, and the centrifugation time can be 15 min, 18 min, 20 min, 22 min, and 25 min.

[0035] 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 uses 2% Triton X-100 as a solvent, and MgCl, DNAse and Tris-HCl with a pH value of 8.0 are added thereto, wherein in the extraction solution, the molar concentration of Tris-HCl is 50 mM, the molar concentration of MgCl is 10 mM, and the mass concentration of DNAse is 10 mg / mL.

[0036] Preferably, the incubation is carried out on ice for 25-35 min.

[0037] Preferably, the centrifugation after incubation is: first centrifugation at 4±1°C and 3800-4200g for 4-6 min, then taking the supernatant after centrifugation and centrifuging it again at 4±1°C and 17000-19000g for 8-12 min.

[0038] Exemplarily, the centrifugal force under centrifugation conditions at 4±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.

[0039] Preferably, the cryopreservation temperature is -80±2°C.

[0040] As a preferred embodiment of the tumor vaccine of the present invention, the preparation method of the liposome complex is: 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 and vortexing, and finally ultrasonically homogenizing to obtain the liposome complex.

[0041] Preferably, the ratio of the total mass of the liposomes and aromatic lipid compounds to the mass volume of chloroform is (5-15) mg:1 mL.

[0042] Exemplarily, the ratio of the total mass of the liposome and the aromatic lipid compound to the mass of chloroform is 5 mg:1 mL, 10 mg:1 mL, 15 mg:1 mL.

[0043] Preferably, the evaporation is carried out using a rotary evaporator in a water bath at 55-65° C., and the evaporation time of chloroform is 0.5-1 h.

[0044] Preferably, the concentration of the HEPES buffer is 18-22 mM; the sum of the mass concentrations of the liposomes and the aromatic lipid compounds in the buffer is 2-10 mg / mL.

[0045] Illustratively, the sum of the mass concentrations of the liposome 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.

[0046] Preferably, the vortex time is 1-2 min.

[0047] Preferably, the ultrasonic homogenization time is 10-20 min, and the ultrasonic homogenization power is 25-35W.

[0048] Illustratively, the time of ultrasonic homogenization can be 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, and the power can be 25 W, 27 W, 29 W, 30 W, 32 W, 35 W.

[0049] In the second aspect of the present invention, the present invention provides a method for preparing the tumor vaccine, wherein the preparation method comprises: mixing the bacterial cell membrane and the liposome complex, incubating the mixture, and extruding the mixture after the incubation to obtain the tumor vaccine.

[0050] As a preferred embodiment of the preparation method of the present invention, the incubation temperature is 37±1° C. and the time is 8-12 min.

[0051] As a preferred embodiment of the preparation method of the present invention, the extrusion is performed through a liposome extruder, the pore size of the filter membrane of the extruder is 200-400 nm; and the number of extrusions is 8-12 times.

[0052] Exemplarily, 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.

[0053] In the third aspect of the present invention, the present invention provides the use of the tumor vaccine in preparing a drug for in situ enhancing the autoimmunogenicity of tumor cells.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] The present invention discloses a tumor vaccine and its preparation method and application. The tumor vaccine provided by the present invention includes bacterial cell membrane and liposome complex, wherein the liposome complex can fuse cells, and the bacterial cell membrane can activate immunity. The combination of the two can achieve excellent synergistic effect, allowing tumor cells to change from the state of immune escape, allowing DC cells infiltrating in tumor tissue to recognize and phagocytize tumor cells, and then present tumor antigens to form an in situ tumor vaccine to achieve anti-tumor effect. In addition, the tumor vaccine provided by the present invention has conventional raw materials, simple preparation method, uniform and excellent, which is conducive to actual production and application. DETAILED DESCRIPTION

[0056] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0057] Example 1

[0058] An embodiment of the present invention provides a tumor vaccine, and the preparation method of the tumor vaccine is:

[0059] (1) Preparation of bacterial cell membrane: The frozen Escherichia coli liquid (5 μL) was inoculated into 10 mL 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, and after inoculation, the OD600 value was monitored to be between 0.7 and 1 at 37°C and 200 rpm for 3 hours. The bacteria were collected (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 digestion was shaken at 37°C for 1 hour. After digestion, the protoplasts were collected and washed three times with PBS, and then resuspended with an appropriate amount of PBS. Then add an extraction solution of equal volume to the bacterial solution (the extraction solution is a 2% Triton X-100 solvent, MgCl, DNAse and Tris-HCl with a pH value of 8.0 are added, wherein in the extraction solution, the molar concentration of Tris-HCl is 50mM, the molar concentration of MgCl is 10mM, and the mass concentration of DNAse is 10mg / mL) and incubate on ice for 30 minutes. After the incubation, centrifuge the lysate at 4°C, 4000rpm for 5min. Take the supernatant after centrifugation and then centrifuge at 4°C, 18000rpm for 10 minutes, take the supernatant after centrifugation and resuspend it with PBS; use the BCA protein quantitative detection kit to detect the protein concentration, and store it at -80°C for future use;

[0060] (2) Preparation of liposome complex: DOPE, DOTAP and DiR were mixed in a mass ratio of 1:1:0.1 and then added to chloroform to dissolve, 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. The chloroform was then rotary evaporated at 60°C for 30 min. The product after evaporation was then dispersed in a HEPES buffer with a mass concentration of 20 mM and vortexed for 1.5 min. The product was then homogenized at 30 W for 15 min under ultrasound to obtain a liposome complex, wherein the total mass concentration of DOPE, DOTAP and DiR in the buffer was 2 mg / mL.

[0061] (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.

[0062] Example 2

[0063] The embodiment of the present invention provides a tumor vaccine. The only difference between the tumor vaccine and the embodiment 1 is that the aromatic lipid compound used in step (2) is DiI.

[0064] Example 3

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

[0066] Example 4

[0067] The embodiment of the present invention provides a tumor vaccine. The only difference between the tumor vaccine and the embodiment 1 is that the mass ratio of the bacterial cell membrane and the liposome complex in step (3) is 2:1.

[0068] Example 5

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

[0070] Example 6

[0071] The embodiment of the present invention provides a tumor vaccine. The only difference between the tumor vaccine and the embodiment 1 is that the mass ratio of the bacterial cell membrane and the liposome complex in step (3) is 1:2.

[0072] Example 7

[0073] The embodiment of the present invention provides a tumor vaccine. The only difference between the tumor vaccine and the embodiment 1 is that the mass ratio of the bacterial cell membrane and the liposome complex in step (3) is 1:5.

[0074] Example 8

[0075] The embodiment of the present invention provides a tumor vaccine. The only difference between the tumor vaccine and the embodiment 1 is that the mass ratio of the bacterial cell membrane to the liposome complex in step (3) is 1:20.

[0076] Example 9

[0077] The embodiment of the present invention provides a tumor vaccine. The only difference between the tumor vaccine and the embodiment 1 is that the Escherichia coli bacterial solution is replaced by the Bifidobacterium bacterial solution in step (1).

[0078] Example 10

[0079] The embodiment of the present invention provides a tumor vaccine. The only difference between the tumor vaccine and the embodiment 1 is that in step (2), DOPE, DOTAP and DiR are mixed in a mass ratio of 2:0.5:0.1.

[0080] Comparative Example 1

[0081] The comparative example of the present invention provides a tumor vaccine, the only difference between the tumor vaccine and Example 1 is that DiR is not contained in step (2).

[0082] Effect Example 1

[0083] The particle size, particle size distribution coefficient (PDI) and Zeta potential value of the tumor vaccine prepared by the test examples 1-10 and the comparative example 1 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.

[0084] Table 1

[0085] Particle size / nm PDI Zeta potential / mV Example 1 158±15 0.185±0.005 -16±3 Example 2 155±12 0.168±0.013 -18±3 Example 3 165±15 0.176±0.016 -16±3 Example 4 350±70 0.506±0.100 -30±3 Example 5 370±65 0.466±0.100 -24±2 Example 6 340±86 0.557±0.100 -18±3 Example 7 220±35 0.304±0.054 -10±2 Example 8 160±18 0.188±0.022 +20±3 Example 9 200±15 0.240±0.034 -18±3 Example 10 160±13 0.160±0.010 -16±3 Comparative Example 1 150±10 0.150±0.020 -15±4

[0086] It can be seen from Table 1 that the tumor vaccine prepared by the preparation method of the present invention has uniform particle size and excellent dispersibility.

[0087] Effect Example 2

[0088] The activation of dendritic cells by the tumor vaccine prepared in vitro by testing examples 1-10 and comparative example 1 of the present invention specifically includes the following steps:

[0089] 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 did not add 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 determined at the same time; specifically, the prepared products of each group were incubated with tumor cells for 4 hours, and the number ratio of dendritic cells to tumor cells was 5:1. After co-culture for 24 hours, the supernatant of cell culture was collected for cytokine (IL-6, IL-1β, TNF-α) determination. Then the cells were collected for flow cytometry determination, and after the dendritic cells were screened out by CD11c staining, the expression of MHCI-SIINFEKL therein was determined, and the results were recorded as shown in Table 2;

[0090] Table 2

[0091]

[0092]

[0093] As can be seen from Table 2, compared with the blank group, the technical solution of the present invention can significantly improve the antigen presentation of dendritic cells, and at the same time improve the release of cytokines such as IL-6, IL-1β, TNF-α, etc. of dendritic cells. It can be seen from the examples, the bacterial cell membrane group and the comparative example 1 that the bacterial cell membrane and the liposome complex of the present invention are indispensable, and only when the two are prepared at the same time can a good effect be achieved.

[0094] Effect Example 3

[0095] The effect example of the present invention uses 4T1 breast cancer-bearing mice to evaluate the tumor vaccines prepared in Examples 1-10 and Comparative Example 1, which specifically includes the following steps:

[0096] 130 mice bearing 4T1 breast cancer were 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 intratumorally on the 4th, 6th, and 8th days after inoculation, and the tumor volume of the mice was measured on the 0th, 6th, 8th, 10th, 12th, 14th, 18th, and 20th days after tumor inoculation, and the average volume was recorded. The tumor volume was measured by length multiplied by width squared divided by 2. The tumor volume of the mouse was greater than 2000 mm 3 Death was determined after the mice were treated. 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 was 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;

[0097] Table 3

[0098] 0d 6d 8d 10d 12d 14d 18d 20d Blank Group 0 343±105 535±122 910±230 1910±350 NA NA NA Bacterial cell membrane group 0 182±74 234±88 338±101 553±142 1053±324 1574±678 NA Example 1 0 165±63 217±46 287±73 365±77 436±51 478±54 578±62 Example 2 0 178±36 241±63 313±84 388±106 466±78 505±43 566±57 Example 3 0 143±42 210±36 276±46 343±62 411±53 447±56 522±53 Example 4 0 164±62 228±61 308±55 377±93 454±66 557±72 608±69 Example 5 0 148±33 261±52 318±42 368±81 423±34 511±35 572±58 Example 6 0 155±43 239±43 331±57 397±86 467±59 588±93 669±78 Example 7 0 172±54 242±58 326±43 412±81 503±54 611±65 699±81 Example 8 0 181±47 266±41 345±51 425±77 515±67 624±79 713±87 Example 9 0 177±42 252±44 337±43 402±45 510±51 574±53 653±51 Example 10 0 188±41 266±57 368±52 422±51 502±47 626±56 721±77 Comparative Example 1 0 198±43 262±86 358±117 589±134 1162±423 1668±576 NA

[0099] It can be seen from Table 3 that, compared with the blank group, the product obtained by adopting the technical solution of the present invention 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 invention is not adopted, the growth of tumors cannot be effectively slowed down.

[0100] Table 4

[0101]

[0102] It can be seen from Table 4 that, compared with the blank group, the product obtained by adopting the technical scheme of the present invention can significantly prolong the survival period of mice; it can be seen from the embodiments, the bacterial cell membrane group and the comparative example 1 that, when the technical scheme of the present invention is not adopted, there is no obvious effect of prolonging the survival period of tumor mice compared with the blank group.

[0103] Effect Example 4

[0104] The effect example of the present invention uses 4T1 breast cancer-bearing mice to conduct in vivo evaluation of the tumor vaccines prepared in Examples 1-10 and Comparative Example 1, which specifically includes the following steps:

[0105] 130 mice bearing 4T1 breast cancer were 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 intratumorally on the 4th, 6th, and 8th days after tumor inoculation. 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 to obtain organs such as mice, tumors, and spleens for subsequent analysis. CD3, CD4, CD8, etc. were labeled and statistically analyzed for tumor-infiltrating T cells. After taking equal masses of tumor tissue, the tumor cells were ground and extracted. After the red blood cells in the tumor were treated with red blood cell lysis solution, the cells were stained with antibodies and then measured by flow cytometry. Meanwhile, for mouse spleen cells, 100,000 spleen cells were inoculated into a 96-well plate for ELISPOT test at each well, and after being stimulated 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;

[0106] Table 5

[0107]

[0108] It can be seen from Table 6 that, compared with the blank group, the product obtained by adopting the technical solution of the present invention can significantly increase the infiltration of CD8+T cells in the tumor, and at the same time can increase the number of tumor-specific T cells in the spleen; it can be seen from the embodiments, the bacterial cell membrane group and the comparative example 1 that when the technical solution of the present invention 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.

[0109] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

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 142-435 nm, 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 8-12 hours, 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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