Broad-spectrum multivalent tumor cell vaccine as well as preparation method and application thereof

Abstract

The invention discloses a broad-spectrum multivalent tumor cell vaccine as well as a preparation method and application thereof. The tumor cell vaccine consists of inactivated tumor cells with protective shell layers, lipidosome and functional molecules, the liposome covers inactivated tumor cells with protective shell layers, and the functional molecules are modified on the surface of the liposome. The vaccine can be used for treating and preventing heterologous and different subtypes of tumors; the hardness of the cells can be regulated and controlled by regulating and controlling the shell layers of the vaccine cells, so that the phagocytosis and vaccine cell presentation capabilities of immune cells on the vaccine cells are regulated and controlled; a functional molecule capable of specifically or non-specifically enhancing or changing immune response of an organism to an antigen can be modified, dendritic cell differentiation can be activated, and the antigen can be ingested through receptor-mediated endocytosis, macrophagocytosis and phagocytosis of the dendritic cells; the cellular morphology of the vaccine can be kept stable under different conditions, and the vaccine has good clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of tumor prevention and treatment, and particularly to a broad-spectrum multivalent tumor cell vaccine and a preparation method and application thereof. Background Art

[0002] Tumor immunotherapy refers to the use of vaccines or genetically engineered vaccines prepared from processed tumor cells or cell extracts for immunization to stimulate or enhance the specific tumor immune response of tumor patients, which can prevent tumor growth, spread and recurrence. However, due to the heterogeneity of tumors during the succession process, and the differences in tumor-specific antigens and tumor-associated antigens in different patients, many immunotherapies cannot be promoted.

[0003] Tumor cell vaccines are usually made of tumor-related proteins and peptides extracted from tumor tissue, dead cancer cells, cancer cell proteins or fragments, or immune system cells. They identify, attack and eliminate residual and recurrent tumor cells by activating and enhancing the body's immune system. At present, this therapy has some limitations that cannot be ignored. The most important limitation to its clinical application is the lack of tumor cell sources in clinical treatment, and the loss of some tumor-specific antigens during traditional inactivation and preservation.

[0004] Therefore, the development of vaccines that are low-cost, have a low preparation threshold, a simple preparation process, and can achieve stable long-term storage is an important direction of current cancer treatment research.

[0005] This study uses cell structure strengthening technology to enable vaccine cells to have good structural enhancement, so that they can maintain the integrity of tumor cell antigen proteins during the freeze-thaw inactivation process, thereby effectively solving the problem of poor immunogenicity due to the loss of cell vaccine antigens during traditional inactivation technology. At the same time, the structural strengthening technology can regulate the mechanical hardness of vaccine cells, thereby regulating the phagocytic rate of dendritic cells DCs to vaccine cells. Cancer whole cell vaccines can provide tumor-specific antigens and tumor-associated antigens to induce anti-tumor immune responses in the body. At the same time, the present invention can regulate the phagocytic presentation efficiency of immune cells, such as dendritic cells, by regulating the type and hardness of the structural strengthening shell. This study aims to design a tumor whole cell vaccine that can not only achieve personalized targeted precision immunotherapy, but also achieve broad-spectrum multivalent preventive treatment of different tumor subtypes. In therapeutic vaccines, by inoculating tumor whole cell vaccines, the proliferation and differentiation of DCs in the body are activated, thereby stimulating or enhancing the specific tumor immune response of tumor patients, so as to achieve the purpose of treating tumors through autoimmune reactions. When used as a preventive vaccine, it can also achieve the purpose of improving immune memory and preventing tumors. By freezing and thawing inactivation of tumor cells, tumor cells lose their tumorigenicity. At the same time, in order to enhance the activation and response of vaccines or antigens to the immune system, this study functionally modified the surface of vaccine cells. The vaccine was coated with cationic liposomes, and the immune adjuvant was loaded onto the inactivated tumor cells through electrostatic forces to enhance the body's immune response to the antigen. The preparation method of this invention is simple, short, and low-cost, and can provide a new solution for achieving personalized precision treatment and broad-spectrum treatment and prevention of tumors. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a broad-spectrum multivalent tumor cell vaccine.

[0007] The second object of the present invention is to provide a method for preparing the above vaccine.

[0008] The third object of the present invention is to provide the application of the above vaccine.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A tumor cell vaccine consists of inactivated tumor cells with a protective shell, liposomes and functional molecules; the liposomes encapsulate the inactivated tumor cells with a protective shell, and the functional molecules are modified on the surface of the liposomes.

[0011] Furthermore, the inactivation is carried out by freezing the tumor cells with a protective shell and then thawing them at low temperature; further, the freezing is performed at -198°C for more than 6 hours, and the low-temperature thawing is performed at 0-8°C for 6-24 hours.

[0012] Furthermore, the shell layer includes at least one of a nanoshell layer, a polymer shell layer or a metal phenolic network shell layer.

[0013] Furthermore, the nanoshell layer includes but is not limited to at least one of a noble metal nanoparticle shell layer, a silicon nanoshell layer, a titanium nanoshell layer, a manganese nanoshell layer, a calcium nanolayer, a cerium nanolayer, an organic nanolayer, and an organic-inorganic hybrid nanoshell layer;

[0014] The polymer shell layer includes but is not limited to at least one of a polydopamine shell layer, a pyrogallic acid shell layer, and a polyelectrolyte shell layer;

[0015] The phenolic network in the metal phenolic network shell layer includes but is not limited to at least one of tannic acid, epicatechin (EC), catechin, epicatechin-3-gallate, epigallocatechin, epigallocatechin gallate, gallic acid and ellagic acid.

[0016] The metal in the metal phenolic network shell layer includes but is not limited to Mn 2+ 、Gd、MnO2、Ag + 、Ti 4+ , Cu 2+ , Fe 2+ , Fe 3 + 、Zn 2+ At least one of the elements and the corresponding metal element, metal compound or metal ion of the element.

[0017] Furthermore, the shell layer includes a silicon nanoshell layer, a polydopamine shell layer and an iron tannic acid metal phenolic network shell layer.

[0018] The shell layer includes but is not limited to the following functions and features: improving the structural strength of vaccine cells, protecting the immune antigens of tumor cells, and ensuring the immunogenicity of the vaccine. The cells have good stress resistance rigidity, can be degraded in the body, and are non-toxic to organs such as the heart, liver, spleen, and lungs. They can be lysed by immune cells in the body through endophagy or macrophagy, releasing the cell's own immune antigens.

[0019] Furthermore, the tumor cells include but are not limited to: at least one of melanoma cells, breast cancer cells, glioma cells, colorectal cancer cells, ovarian cancer cells, lung cancer cells, gastric cancer cells and liver cancer cells.

[0020] Furthermore, the sources of the tumor cells include but are not limited to: tumor cells extracted from tumors resected from individual tumor patients, and allogeneic tumor cells of vaccine recipients cultured and extracted from relevant research institutions.

[0021] Furthermore, the liposomes include but are not limited to at least one of cationic liposomes, cholesterol, phosphatidylethanolamine liposomes, phosphatidyl amine base, glycerophospholipid liposomes, serine phospholipids and phosphatidic acid; further, the cationic liposomes include but are not limited to 2,3-bis (n-octyloxy) propyl trimethylammonium bromide (DOSPA, Dioleoyloxypropyl-Trimethylammonium Methanesulfonate), 1,2-dioleoyl-3-trimethylammonium bromide (DOTAP, Dioleoyloxy-3-Trimethylammonium-Propane), GL67 (DiooleoylDimethylaminoethyl Carbamoyl Phosphatidylcholine, DODAC, DiooleoylDimethylaminoethyl-β-Alanine, DODMA, Cholesterol, Diooleoyl At least one of acetylated phosphatidylglycerol, DOAPG), 1,2-dioleoyl-3-dimethylaminopropane (DODMA), 1,2-dioleoyl-3-(β-aminoethyl)-aminopropane (DOBAQ, 1,2-Dioleoyl-3-(β-Aminoethyl)-Aminopropane) and 1,2-distearoyl-3-trimethylammonium bromide (18:0DAP, 1,2-Distearoyl-3-Trimethylammonium-Propane); the phosphatidylethanolamine liposomes include but are not limited to 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, Diol At least one of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE); the phosphatidyl amide base includes but is not limited to at least one of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);The glycerophospholipid liposomes include but are not limited to at least one of 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DLPG) and 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol (DPPG); the serine phospholipids include but are not limited to at least one of 1,2-distearoyl-sn-glycerol-3-phosphoserine (DSPS) and 1,2-dioleoyl-sn-glycerol-3-phosphoserine (DOPS); the phosphatidic acid includes but is not limited to at least one of 1,2-dimyristoyl-sn-glycerol-3-phosphate (DMPA); further, the liposomes include 5 to 20% by mole of cationic liposomes and 80 to 95% by mole of cholesterol. ;

[0022] Furthermore, the liposomes are prepared into a film by a rotary evaporation method for coating, and the specific steps are as follows: dissolving the liposomes in an organic solvent and mixing them evenly; removing the solvent by rotary evaporation to obtain a lipid film, adding a phosphate buffer to the lipid film, and ultrasonically treating it to form a uniform liposome suspension; mixing the suspension with inactivated tumor cells with a protective shell layer, and incubating them together to achieve liposome coating of the inactivated tumor cells with a protective shell layer.

[0023] Furthermore, the functional molecule may be a substance that specifically or non-specifically enhances or changes the body's immune response to an antigen, including but not limited to: at least one of an immune adjuvant, a targeted molecule, and a labeled molecule; further, the immune adjuvant includes but is not limited to an adjuvant of a pathogen-associated molecular pattern (PAMP), and the targeted molecule and the labeled molecule include but are not limited to targeted and / or labeled DNA or RNA; further still, the adjuvant of the pathogen-associated molecular pattern (PAMP) includes but is not limited to at least one of a TLR agonist and MPL, and the targeted and / or labeled DNA or RNA includes but is not limited to CpG oligonucleotide (CpG ODN).

[0024] The method for preparing the above tumor cell vaccine comprises the following steps:

[0025] (1) allowing tumor cells to react with a shell protection reaction solution to form a shell layer on the surface of the tumor cells, and removing the supernatant after the reaction is completed, washing, and obtaining tumor cells with a protective shell layer;

[0026] (2) suspending the tumor cells with the protective shell in a buffer and freezing them in liquid nitrogen, followed by low-temperature thawing;

[0027] (3) mixing the tumor cells with the protective shell layer after remelting in step (2) with the liposome suspension, and incubating them together;

[0028] (4) Add modified functional molecules, mix well, and incubate.

[0029] Furthermore, the shell protection reaction solution in step (1) comprises:

[0030] Reagents for forming silicon nanoshells, reagents for forming calcium nanoshells, reagents for forming polydopamine shells, reagents for forming pyrogallic acid shells, reagents for forming polyelectrolyte shells, and reagents for forming metal phenolic network shells;

[0031] Furthermore, the reagent for forming the silicon nanoshell layer includes at least one of methyltrimethoxysilane, methyldiethoxysilane, ethyltriethoxysilane, methyltriethoxysilane, tetraethoxysilane, tetramethoxysilane, propyltriethoxysilane, 3-aminopropyltriethoxysilane, propyltrimethoxysilane, 3-aminopropyltrimethoxysilane, phenyltrimethoxysilane, phenyltriethoxysilane, benzyltrimethoxysilane, benzyltriethoxysilane, 3-mercaptopropyltriethoxysilane, 3-mercaptopropyltrimethoxysilane, chlorotriethoxysilane, octyltrimethoxysilane, cyclohexylmethyldimethoxysilane, vinyltrimethoxysilane and isobutyltriethoxysilane, and further includes tetraethoxysilane;

[0032] Furthermore, the reagent for forming the calcium nanoshell layer includes sodium phosphate and calcium chloride;

[0033] Furthermore, the reagent for forming the polydopamine shell layer includes dopamine hydrochloride;

[0034] Furthermore, the reagent for forming the pyrogallic acid shell layer includes pyrogallic acid;

[0035] Furthermore, the reagent for forming the polyelectrolyte shell layer includes at least one of a positively charged polyelectrolyte solution and a negatively charged polyelectrolyte solution; the positively charged polyelectrolyte solution includes a PEI solution; and the negatively charged polyelectrolyte solution includes a PAA solution.

[0036] Furthermore, the reagent for forming the metal phenolic network shell layer includes a metal salt and an organic ligand; the metal salt includes Mn 2+ 、Ag + 、Ti 4+ , Cu 2+ , Fe 2+ , Fe 3+、Zn 2+ The organic ligand comprises at least one of tannic acid, epicatechin (EC), catechin, epicatechin-3-gallate, epigallocatechin, epigallocatechin gallate, gallic acid and ellagic acid;

[0037] Furthermore, the concentration of the shell protection reaction solution in step (1) is 5 mM to 100 mM.

[0038] Furthermore, the tumor cells and the shell protection reaction solution in step (1) are mixed at a rate of 2×10 6 ~4×10 6 The ratio of tumor cells to 1 mL of shell protection reaction solution was reacted.

[0039] Furthermore, the shell protection reaction solution described in step (1) comprises: a tetraethoxysilane solution with a concentration of 100 mM, a dopamine hydrochloride solution with a concentration of 2 mg / mL, and a Fe-TA reaction solution; the Fe-TA reaction solution is composed of the following components: a 5% by volume FeCl3·6H2O aqueous solution with a concentration of 1.25 mg / mL, a 5% by volume tannic acid (TA) aqueous solution with a concentration of 5 mg / mL, and the remainder is a 20 mM 3-morpholinepropanesulfonic acid (MOPS) aqueous solution.

[0040] Furthermore, the reaction in step (1) is carried out at 4-8°C for 1-60 min; further, at 4°C for 1-60 min.

[0041] Furthermore, the freezing in step (2) is performed at -198°C for more than 6 hours.

[0042] Furthermore, the low-temperature thawing in step (2) is thawing at 0-8°C for 6-24h.

[0043] Furthermore, the co-incubation in step (3) is ultrasonic co-incubation for 1 to 2 minutes.

[0044] Furthermore, the liposome suspension in step (3) is prepared as follows: dissolving the liposome in an organic solvent and mixing uniformly; removing the solvent by rotary evaporation to obtain a lipid film, adding phosphate buffer to the lipid film, and ultrasonically treating to form a uniform liposome suspension.

[0045] Furthermore, the co-incubation in step (4) is for a total of 20 to 40 minutes.

[0046] The use of the above vaccine in the preparation of drugs for preventing and / or treating tumors.

[0047] Compared with the prior art, the present invention has the following advantages and effects:

[0048] 1. Broad spectrum and multivalent efficacy: The tumor vaccine cell source can be a polyclonal cell line or a monoclonal cell line, and the vaccine can treat and prevent allogeneic homologous and different subtypes of tumors. For example, a vaccine prepared from polyclonal melanoma subtype B16-F4 cells can treat B16-F10 tumors, and its efficacy is better than that of monoclonal melanoma subtype B16-F10.

[0049] 2. The vaccine cell shell can be constructed through structural reinforcement shell construction technology, thereby regulating the hardness of the cells and further regulating the ability of immune cells to phagocytize and present vaccine cells.

[0050] 3. Able to modify functional molecules that specifically or non-specifically enhance or change the body's immune response to antigens, taking CpG-ODN adjuvant as an example.

[0051] 4. Vaccines can activate dendritic cell differentiation, take up antigens through dendritic cell receptor-mediated endocytosis, macropinocytosis and phagocytosis, degrade antigens in cells and process them into antigenic polypeptide fragments, which are then expressed on the cell surface in the form of antigenic peptide-MHC complexes, thereby activating the specific immune effect of the body.

[0052] 5. The protective shell layer can make the cells have good stress resistance, rigidity and stability. When the cells treated with the protective shell layer are placed in clean water, the cells still maintain a good shape and do not break.

[0053] 6. The protective shell can protect the immune antigens of tumor cells and ensure the immunogenicity of the vaccine. Through SDS-page and mass spectrometry analysis, it was found that the protective shell can effectively protect the types and quantities of whole cell proteins.

[0054] 7. Vaccine cells can be rapidly phagocytosed and degraded in the body, with low cytotoxicity. They are also non-toxic to the heart, liver, spleen, lungs and other organs.

[0055] 8. After dehydration, the vaccine cells can be quickly rehydrated at room temperature and still maintain good cell morphology after rehydration. It is easy to store, can overcome the problem of difficult storage of traditional vaccines, and has good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is a schematic diagram of the vaccine cell preparation of the present invention.

[0057] Figure 2 It is a schematic diagram of the principle of the vaccine cell immunotherapy of the present invention.

[0058] Figure 3 This is a staining image of the Live-Si-30min cells prepared in Example 1, wherein the red color is the cell membrane stained with dil, the blue color is the cell nucleus stained with Hoechst 33342, and the green color is the protective shell stained with FITC.

[0059] Figure 4 This is a scanning electron micrograph of breast cancer 4T1 cells.

[0060] Figure 5 This is a scanning electron micrograph of Live-Fe-TA-1min cells.

[0061] Figure 6 This is a scanning electron micrograph of Live-PDA-45min cells.

[0062] Figure 7 These are microscopic images of Live-Si-30min cells, Live-Si-60min cells and Live-Si-12h cells after cryopreservation and thawing; (A) is a microscopic image of Live-Si-30min cells after cryopreservation and thawing, (B) is a microscopic image of Live-Si-60min cells after cryopreservation and thawing, and (C) is a microscopic image of Live-Si-12h cells after cryopreservation and thawing.

[0063] Figure 8 These are microscopic images of Live-Fe-TA-1min cells and Live-Fe-TA-5min cells after cryopreservation and thawing; (A) is a microscopic image of Live-Fe-TA-1min cells after cryopreservation and thawing, and (B) is a microscopic image of Live-Fe-TA-5min cells after cryopreservation and thawing.

[0064] Fig. 9 These are microscopic images of Live-PDA-30min cells and Live-PDA-45min cells after cryopreservation and thawing; (A) is a microscopic image of Live-PDA-30min cells after cryopreservation and thawing, and (B) is a microscopic image of Live-PDA-45min cells after cryopreservation and thawing.

[0065] Fig.10 This is the experimental result of rehydration after freeze drying.

[0066] Fig.11 This is the result of SDS-page protein detection.

[0067] Fig.12This is the result of Pearson correlation analysis.

[0068] Fig.13 This is a graph showing the protein content of the supernatant solution after reconstitution.

[0069] Fig.14 This is a transmission electron micrograph of a cell section before freezing.

[0070] Fig.15 This is a transmission electron micrograph of a frozen cell section.

[0071] Fig.16 This is the result chart of cell size after being placed in clean water.

[0072] Fig.17 This is a graph of the hardness results of the Young's modulus test.

[0073] Fig.18 This is a graph showing the phagocytic rate of the tested cells by BMDC cells.

[0074] Fig.19 This is the correlation diagram between the cell Young's modulus and the phagocytic rate of dendritic cells.

[0075] Fig. 20 This is a graph showing the results of a vaccine cell safety experiment.

[0076] Fig.21 It is the electrical potential diagram of vaccine cells.

[0077] Fig. 22 This is a graph showing the results of a cytotoxicity experiment on vaccine cells after cationic liposome coating and adjuvant modification.

[0078] Fig.23 This is the staining result of dendritic cells phagocytizing and taking up vaccine cells; the red color is the DC cell membrane stained with dil, the blue color is the DC cell nucleus stained with Hoechst33342, and the green color is the vaccine cell protective shell stained with FITC.

[0079] Fig.24 This is a graph showing the results of an experiment to verify the non-tumorigenicity of vaccine cells.

[0080] Fig.25 This is the in vivo fluorescence imaging result of the therapeutic effect of vaccine cells on B16-LUC tumors.

[0081] Fig.26 is a flow chart of mouse modeling and treatment.

[0082] Fig. 27 This is a photograph of the tumor growth in the PBS control group on the 13th day.

[0083] Fig.28This is a photograph of the tumor growth on the 13th day in the B16-F10 vaccine group.

[0084] Fig.29 This is a photograph of the tumor growth on the 13th day in the B16-F4 vaccine group. DETAILED DESCRIPTION

[0086] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. Figure 1 The principle of treatment is as shown in Figure 2 shown.

[0087] Example 1

[0088] Tetramethoxysilane (TMOS) solution (TMOS concentration was 100 mM) was reacted with melanoma B16-F10 at 4°C for 30 min or 60 min to obtain Live-Si-30min cells and Live-Si-60min cells, respectively; the ratio of TMOS solution to melanoma B16-F10 was 1 mL / 2×10 6 cell.

[0089] To observe the formation of the silicon protective shell, a TMOS solution (TMOS concentration was 100 mM) containing 1 mg / mL fluorescein isothiocyanate-modified 3-aminopropyltriethoxysilane (FITC-APTES) was reacted with melanoma B16-F10 at 4°C for 30 min, and the cells were stained with Dil and Hoechst3342. Images were collected using Leica TCS SP8. The results are shown in Figure 2. Figure 3 As shown, red is the cell membrane stained with Dil, blue is the cell nucleus stained with Hoechst 33342, and green is the silicon protective shell labeled with FITC. It can be seen that the Live-Si-30 cells are successfully coated with the protective shell.

[0090] Example 2

[0091] 2×10 64T1 breast cancer cells, 1mL of reaction solution (including 50μL of 1.25mg / mL FeCl3·6H2O aqueous solution, 50μL of 5mg / mL tannic acid (TA) aqueous solution, and the remainder of 20mM 3-morpholinepropanesulfonic acid (MOPS) aqueous solution), were reacted at 4℃ for 1min, 3min or 5min respectively; they were respectively used as 1min reaction group, 3min reaction group and 5min reaction group, and Live-Fe-TA-1min cells, Live-Fe-TA-3min cells and Live-Fe-TA-5min cells were obtained respectively. Unreacted breast cancer 4T1 cells were used as the live cell group. Images were taken using a high-resolution field emission scanning electron microscope, and the results are shown in the figure. Figure 4 and Figure 5 As shown, compared with the living cell group (such as Figure 4 As shown in Figure 2, the tumor cells in the 1 min reaction group in Example 2 were successfully coated with the shell layer, and the original fibrous tentacles on the surface of the living cells were covered by the shell layer (as shown in Figure 2). Figure 5 shown).

[0092] Example 3

[0093] 3×10 6 Melanoma B16-OVA cells were reacted with 1 mL of 2 mg / mL dopamine hydrochloride solution at 4°C for 30 min, 45 min or 1 h, respectively; they were used as the 30 min reaction group, 45 min reaction group and 1 h reaction group, and Live-PDA-30 min cells, Live-PDA-45 min cells and Live-PDA-1 h cells were obtained, respectively. Unreacted melanoma B16-OVA cells were used as the live cell group. Images were taken using a high-resolution field emission scanning electron microscope. The results are shown in Figure 2. Figure 6 As shown, compared with the living cell group (there is no photo of the melanoma B16-OVA cell group for comparison), the 45 min reaction group in Example 3 was successfully coated with the polydopamine shell layer, and the original fibrous antennae on the surface of the living cells were covered by the shell layer.

[0094] Example 4

[0095] The Live-Si-30min cells, Live-Si-60min cells, Live-Fe-TA-1min cells, Live-Fe-TA-3min cells, Live-Fe-TA-5min cells, Live-PDA-30min cells, Live-PDA-45min cells and Live-PDA-1h cells obtained in the reactions of Examples 1-3 were suspended in buffer solutions, respectively, and frozen at -196°C for 24 hours to obtain frozen cells.

[0096] The frozen cells were then taken out from the cryogenic conditions and slowly thawed at 0-8°C (named LN-Si-30min cells, LN-Si-60min cells, LN-Fe-TA-1min cells, LN-Fe-TA-3min cells, LN-Fe-TA-5min cells, LN-PDA-30min, LN-PDA-45min cells and LN-PDA-1h cells, respectively). The images were taken using Leica TCS SP8. The results are shown in the figure. Figures 7 to 9 As shown in the figure, the cell morphology did not change before and after freezing, indicating that the shell coating strategy can effectively protect the cells from damage caused by ice crystals during the freezing and thawing process.

[0097] Example 5

[0098] The cells frozen in Example 4 were taken out and rehydrated (placed in water for 1 min). Fig.10 As shown, the cells can return to their original state in a short time, indicating that the vaccine can be stored stably for a long time and has good clinical application and promotion capabilities.

[0099] Example 6

[0100] The protective shell has a better protein preservation effect than traditional methods and can maintain the protein antigen inside the shell from leaking. The total protein of the cells was extracted using the Tissue or cell total protein extraction kit C510003. 200,000 cells were collected (the experimental group was LN-Si-30min cells or LN-Si-60min cells; the Live cell group used melanoma B16-F10 living cells as a control, and the LNC group used melanoma B16-F10 living cells frozen and thawed under the same conditions as this implementation as a control) and added to the pre-cooled lysis buffer. The cells were then ultrasonically disrupted for a total of 5 times, each time for 30 seconds. Centrifuge at 14000rpm, four degrees Celsius for 15 minutes, and the supernatant was taken as the total protein extract. There were 3 biological replicates in each group. The total protein retention of the cells was determined by SDS-page, and the results are shown in the following figure. Fig.11 As shown in the figure, the protective shell coating and cryopreservation can well preserve the protein during the freeze-thaw inactivation of the whole cell vaccine. Subsequently, the protein information in different experimental groups was further verified by proteomics methods. The obtained data was analyzed by Pearson correlation. The results are as follows Fig.12As shown, it can be verified that the vaccine groups coated with protective shells and frozen (LN-Si-30min cells, LN-Si-60min cells) have a stronger correlation with the proteins in the live cell group than the vaccines that are directly frozen without protective shells (LNC group), and are closer to the original tumor cell antigens in abundance and type. This shows that this technology can well protect the immunogenic antigens of vaccine cells from being reduced during the preparation process, thereby solving the problem of poor effect caused by immunogenicity loss due to preparation technology during the production and preparation of current tumor vaccines.

[0101] Example 7

[0102] Based on the samples in Example 4, the protein content in the supernatant solution after reconstitution was measured. The LN-Si-30min cells, LN-Fe-TA-1min cells and LN-PDA-30min cells with the shortest reaction time (theoretically the thinnest shell layer) were used as examples. TM BCA Protein Assay Kits (Thermo) was used to measure the protein concentration of the supernatant of the above-mentioned reconstituted cells according to its instruction manual. The results are as follows: Fig.13 As shown, there is no leakage of basic proteins in the three groups of LN-Si-30min cells, LN-Fe-TA-1min cells and LN-PDA-30min cells, indicating that this invention can effectively prevent the loss of vaccine antigens during the freezing process through a variety of shell encapsulation strategies. It solves the problem of reduced immunogenicity caused by protein leakage during vaccine freezing and thawing in traditional technical means. Among them, the LNC group is a control group of melanoma B16-F10 living cells that were directly frozen and thawed without shell protection.

[0103] Example 8

[0104] Collect 2×10 6Live-Si-30min cells prepared by the method of Example 1 and LN-Si-30min cells after redissolution in Example 4 (freshly and quickly collected); fixation treatment: the collected cells were placed in a 1.5mL centrifuge tube, 2.5% glutaraldehyde solution (fixative) was added, the centrifuge tube was filled up, the sample was completely immersed in the fixative, and placed at 4°C. After overnight, the fixative was poured out, and the sample was rinsed three times with 0.1M, pH 7.0 phosphate buffer, each time for 15 minutes; dehydration treatment: the sample was dehydrated with ethanol solutions of gradient concentrations (including six concentrations of volume fractions of 30%, 50%, 70%, 80%, 90% and 95%), each concentration was treated for 15 minutes, and then treated with 100% ethanol for 20 minutes; finally, it was transferred to pure acetone for 20 minutes. Infiltration treatment: treat the sample with a mixture of embedding agent epoxy resin and acetone (V / V=1 / 1) for 1 hour; treat the sample with a mixture of embedding agent epoxy resin and acetone (V / V=3 / 1) for 3 hours; treat the sample with pure embedding agent epoxy resin overnight. Embedding treatment: embed the sample that has undergone infiltration treatment and heat it at 70°C overnight to obtain the embedded sample. The sample is sliced ​​in a LEICA EMUC7 ultrathin slicer to obtain 70-90nm slices. The slices are stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 5-10 minutes each, and can be observed in a transmission electron microscope. Hitachi-HT7700 was used to take biological TEM photos, and the results are as follows. Fig.14 and Fig.15 As shown, the intracellular structure of the reconstituted LN-Si-30min cells was broken after freezing, but no leakage occurred due to the protective effect of the shell layer.

[0105] Example 9

[0106] Based on the samples LN-Si-30min cells, LN-Si-60min cells, LN-Fe-TA-1min cells, LN-Fe-TA-3min cells, LN-Fe-TA-5min cells, LN-PDA-45min cells and Live group cells (melanoma B16-F10 live cells were directly frozen and thawed) in Example 4, the cells were placed in clean water for 24 hours and then the cell size (diameter) was measured. The results are as follows: Fig.16 As shown in the figure, the cells can still maintain a stable shape and size. This shows that the protective shell has a certain rigidity and has the mechanical strength to resist ice crystals, so that the cell components will not leak into the external environment, and can also remain stable under hypotonic conditions.

[0107] Embodiment 10:

[0108] The Young's modulus of the samples LN-Si-30min cells, LN-Si-60min cells, LN-Fe-TA-1min cells, LN-PDA-45min cells and Live group cells (melanoma B16-F10 live cells were directly frozen and thawed) in Example 4 were measured using an atomic force microscope. The results are as follows Fig.17 As shown, the shell coating strategy can regulate the hardness of vaccine cells.

[0109] Embodiment 11

[0110] LN-Si-30min cells, LN-Si-60min cells, LN-Fe-TA-1min cells, LN-PDA-45min cells and Live group cells (melanoma B16-F10 live cells were directly frozen and thawed) were fluorescently labeled (LN-Si added FITC-ATES in the reaction of forming the shell layer in Example 1, LN-PDA and LN-Fe-TA added 0.1mL FITC in the reaction of forming the shell layer in Examples 2 and 2; melanoma B16-F10 live cells were fluorescently labeled by CellTrace TM CFSE dye labeling, 1×10 4 The ratio of cells / 1μl CFSE PBS solution was used for staining and labeling at room temperature for 30 minutes), C57BL / 6 mouse BMDC cells were extracted and labeled with BV42-anti-mouse CD11c Antibody antibody, and the labeled BMDC cells were co-incubated with the labeled LN-Si-30min cells, LN-Si-60min cells, LN-Fe-TA-1min cells, LN-PDA-45min cells and Live group cells for 3h, and the phagocytic rate of immune cell BMDC on shell cells of different hardness was compared. The specific steps are as follows:

[0111] (1) Mice (8-12 weeks) were killed by cervical dislocation and their tibiae were removed;

[0112] (2) Disinfection of tibia: transfer the tibia to a culture dish containing 70% ice ethanol and soak for 5 minutes;

[0113] (3) Extract bone marrow aseptically and transfer the tibia to sterile-filtered RPMI 1640 medium. Rinse the bone marrow and place it in a sterile culture dish.

[0114] (4) Resuspend the bone marrow cells in RPMI 1640 medium containing 10% FBS, 20 ng / mL GM-CSF, and 10 ng / mL IL-4, transfer the cells to a centrifuge tube, and gently pipette into a single-cell suspension;

[0115] (5) Day 0 Cells were centrifuged and counted, and plate culture was performed at 1-2 × 10 cells per ml of culture medium. 6 cells;

[0116] (6) Day 3 medium change: remove 50% of the culture medium and non-adherent cells, and add primary DC culture medium again; change the medium every two days thereafter;

[0117] (7) On Day 6, transfer the culture medium (including non-adherent DCs) to a 50-mL centrifuge tube a, and keep the adherent cells in the original culture dish moist; add PBS solution containing 3 mM EDTA to the dish and let it stand for 5 min to digest the cells; gently rinse the adherent cells several times and collect them in a 50-mL centrifuge tube a;

[0118] (8) Count the cells and re-plate them into new dishes (the new dishes can be: 6-well plates / 12-well plates / 10 cm culture dishes);

[0119] (9) Day 10-11, DC cells labeled with BV421-CD11c were co-incubated with FITC-labeled vaccine cells at a ratio of 1:1 for 3 h;

[0120] (10) BD LSR fortessa flow cytometer was used to count the phagocytic amount in different experimental groups.

[0121] The results are as follows Fig.18 As shown, the LN Si group with higher hardness has a higher phagocytosis rate. Therefore, the subsequent experimental verification preferably uses the LN Si group as the effect verification of the vaccine.

[0122] The correlation between the Young's modulus of Example 10 and the engulfment rate of Example 11 was statistically analyzed, and the results are as follows: Fig.19 shown.

[0123] Example 12

[0124] To verify the safety of vaccine cells in vitro, LN-Si-30min cells and LN-Si-60min cells were selected as the experimental group, and melanoma B16-F10 live cells (Live cells) and melanoma B16-F10 cells stored at 4°C for 24h (4°C) were used as controls. Cell viability was detected using the CCK8 cell activity detection kit. The specific steps are as follows: 1. Inoculate the cell suspension in a 96-well plate, with 100 μl of RPMI-1640 medium containing 10% FBS in each well, and inoculate 1×10 4 2. After the cells were cultured for 0, 24, 48, and 72 hours, 10 μl of CCK8 solution was added to each well of the 96-well plate and the culture plate was incubated in the incubator for 1 hour. The absorbance at 450 nm was measured using an ELISA reader. The results are shown in Figure 2. Fig. 20 As shown, living cells and cells stored at 4°C for 24 h have high proliferation activity, while LN-Si-30min cells and LN-Si-60min cells thawed after cryopreservation have no proliferation ability.

[0125] Example 13

[0126] LN-Si-30min cells were coated with 5% cationic liposomes, 10% cationic liposomes and 20% cationic liposomes by volume respectively. The specific steps are as follows: dissolve the required liposomes in an appropriate amount of organic solvent and mix them evenly to obtain a liposome solution. The liposomes used in this embodiment are 1,2-dipalmitoyl-sn-glycero-3-phosphamide, 1-cyclic phosphorylcholine (DOTAP) and cholesterol, and the organic solvent is methanol; transfer the liposome solution to a round-bottom flask of a rotary evaporator, and use the rotary evaporator to rotary evaporate under reduced pressure and water bath heating (40°C) to remove the solvent and form a uniform lipid film. This embodiment obtains. Add pH 7.4 phosphate buffer to the lipid film, and use an ultrasonic processor to perform ultrasonic treatment for 5 minutes to form a uniform liposome suspension. This embodiment obtains a PBS suspension of DOTAP and cholesterol with a molar ratio of DOTAP to cholesterol of 5:95, 10:90 and 20:80 (the total content of liposomes in the suspension is 1 mg / mL). 3×10 6 LN-Si-30min cells were incubated with PBS suspensions of DOTAP and cholesterol at molar ratios of 5:95, 10:90, and 20:80, respectively, and ultrasonicated for 1 min. The potential of the liposome film was measured using zeta potential. 6 LN-Si-30min cells coated with 5% cationic liposomes, 10% cationic liposomes and 20% cationic liposomes were mixed with 5 μg of immune adjuvant CpG-ODN 1826 and incubated for 30 minutes to achieve adjuvant loading. The cells were then centrifuged to remove the supernatant and resuspended in phosphate buffer to measure the potential. The results are shown in Fig.21 As shown in the figure, cationic liposomes of different concentrations can successfully coat the negatively charged cell membrane, changing the membrane potential from negative to positive. After loading the immune adjuvant, the potential returns to negative. This shows that after the vaccine cells are injected into the body, they will not have the strong toxicity of positive charge.

[0127] Embodiment 14

[0128] The vaccine safety and performance of the vaccine cells coated with cationic liposomes of different concentrations and the vaccine cells coated and modified with adjuvants in Example 13 were verified. 5000 B16-F10 cells were implanted in 96-well plates and cultured in RPMI 1640 medium containing 10% FBS and 1% double antibody for 24 hours of adherence and proliferation. Vaccine cells coated with cationic liposomes of different concentrations and B16-F10 vaccine cells loaded with CpG adjuvant were added to each well for administration. The dosage was: vaccine cells: tumor cells = 2:1, cultured in RPMI 1640 medium containing 10% FBS and 1% double antibody for 36 hours; 5 biological replicates per group; live melanoma B16-F10 cells were added to the Live cell group for administration as a control. CellTiter-Lumi TM The luminescence method was used to test the cytotoxicity of the vaccine. Fig. 22 As shown, after coating or coating and adjuvant modification, 5% and 20% concentrations of cationic liposomes are not strongly toxic to cells, while high concentrations of 30% cationic liposomes are relatively toxic, but cell viability is not significantly reduced. Therefore, it can be inferred that the vaccine cells are safe. At the same time, it can be inferred that when this vaccine is used for tumor treatment, it does not rely on the toxicity of the vaccine cells themselves, but activates the patient's own immune system to achieve tumor immunotherapy.

[0129] Embodiment 15

[0130] In vitro validation of vaccine effectiveness: Vaccine cells (using a siliconized layer labeled with FITC green fluorescence) were injected into BMDCs cultured in vitro (the cell membrane and cell nucleus were stained with DiI and Hoechst stains, respectively).

[0131] The specific BMDC extraction, staining, and co-incubation procedures are as follows:

[0132] 1. Kill mice (8-12 weeks) by cervical dislocation and remove tibiae;

[0133] 2. Disinfect the tibia: move the tibia to a culture dish containing 70% ice ethanol and soak for 5 minutes;

[0134] 3. Extract bone marrow aseptically and transfer the tibia to sterile-filtered RPMI 1640 medium; rinse the bone marrow and place it in a sterile culture dish;

[0135] 4. Resuspend the bone marrow cells in RPMI 1640 medium containing 10% FBS, 20 ng / mL GM-CSF, and 10 ng / mL IL-4, transfer the cells to a centrifuge tube, and gently pipette into a single-cell suspension;

[0136] 5. Day 0 cells were centrifuged and counted, and plate culture was performed, with 1-2×10 cells per ml of culture medium.6 cells;

[0137] 6. Day 3 medium change: remove 50% of the primary culture medium and non-adherent cells, and add primary DC culture medium again; change the medium every two days thereafter;

[0138] 7. On Day 6, transfer the culture medium (including non-adherent DCs) to a 50 ml centrifuge tube a, and keep the adherent cells in the original culture dish moist; add PBS solution containing 3 mM EDTA to the dish and let it stand for 5 min to digest the cells; gently rinse the adherent cells several times and collect them into a 50 ml centrifuge tube a;

[0139] 8. Count the cells and re-plate them into new dishes (the new dishes can be: 6-well plates / 12-well plates / 10 cm culture dishes);

[0140] 9. Day 10-11, the cell membrane and nucleus were stained with DiI dye and Hoechst 33342 dye respectively, and the stained DCs were co-incubated with FITC-labeled Si vaccine cells (LN-Si-30min cells; FITC labeling method was the same as in Example 1) at a ratio of 1:1 for 6 hours.

[0141] 10. Images were acquired using a Leica TCS SP8.

[0142] The results are as follows Fig.23 As shown, the vaccine cells can effectively activate BMDC cells and be phagocytosed to produce antigens.

[0143] Example 16

[0144] In vivo validation of vaccine safety. 2×10 5 Melanoma cells B16-OVA-GFP or LN-Si-30min cells, the results are as follows Fig.24 As shown, the survival rate of mice injected with vaccine cells remained 100% after 60 days of culture, and the cells coated with a protective shell and frozen were non-tumorigenic and could be safely injected into the body.

[0145] Embodiment 17

[0146] Validation of the effectiveness of vaccine treatment for metastatic tumors. In C57BL / 6 female mice, 2×10 4 Melanoma B16-LUC cells were treated with medication on days 3, 7, and 14. The specific medications were as follows: blank control group (PBS group): injected with 100 μL PBS; vaccine group (LN-Si-30min-5μg-CpG group): injected with 100 μL containing 3×10 6PBS containing 10 vaccine cells, wherein the vaccine cells are 3×10 B16-F10 cells prepared by the steps of Examples 1, 4 and 13. 6 20% cationic liposome-coated, 5μg-CpG-ODN-modified LN-Si-30min cells. The experimental group with traditional non-structure enhanced shell coating and direct cryopreservation inactivation (LNC-5μg-CpG group): Inject 100μL containing 3×10 6 PBS of LNC cells modified with CpG-ODN, LNC cells modified with CpG-ODN were passaged 3×10 6 B16-F10 was mixed with 5 μg of immune adjuvant CpG-ODN and incubated for 30 minutes. The distribution and size of tumors on the 7th, 11th and 18th days were monitored by small animal in vivo imaging. Fig.25 As shown, the shell-coated vaccine can effectively inhibit the occurrence of metastatic tumors, while the vaccine prepared by the traditional method cannot inhibit lung metastasis when given the same amount of vaccine cells. The previous experiment verified that this is because the traditional preparation method will cause protein antigen loss. Therefore, it was verified at the animal experiment level that the effect of the present invention is far better than the inactivated whole-cell vaccine prepared by the traditional method.

[0147] Embodiment 18

[0148] The effectiveness of multivalent vaccines in treating orthotopic tumors and different subtypes of tumors was verified. In C57BL / 6 female mice, 5×10 of the most aggressive melanoma tumors were implanted subcutaneously. 5 B16-F10 cells were expressed in the cells. Successful subcutaneous tumor formation was observed on the back of the right upper limb in both the treatment group and the blank control group on day 3 before treatment.

[0149] Subsequently, the experimental group was injected with the vaccine on the right upper abdomen on the 3rd, 7th, and 11th day after tumor implantation. The dose was 3×10 6 The specific administration was as follows: blank control group (PBS group): injected with 100 μL PBS; vaccine group 1 (B16-F4-10 μg-CpG): injected with 100 μL containing 3×10 6 PBS containing vaccine cells 1, wherein vaccine cells 1 are 20% cationic liposome-coated, 10 μg-CpG-ODN-modified LN-Si-30min cells prepared from B16-F4 by the steps of Examples 1, 4 and 13 (wherein the immune adjuvant CpG-ODN in Example 13 is increased to 10 μg); vaccine group 2 (B16-F10-10 μg-CpG): 100 μL containing 3×10 6PBS containing vaccine cells 2, wherein vaccine cells 2 are 20% cationic liposome-coated, 10μg-CpG-ODN-modified LN-Si-30min cells prepared by B16-F10 through the steps of Example 1, Example 4 and Example 13 (wherein the immune adjuvant CpG-ODN in Example 13 is increased to 10μg). The growth of the in situ tumor volume and changes in the animal weight were monitored. The results are shown in Figures 26 to 29 As shown, the results show that the vaccine prepared by B16-F10 cells can effectively inhibit the in situ B6-F10 tumor. At the same time, the vaccine prepared by B16-F4 cells also has a good effect in treating the more aggressive B6-F10 melanoma subtype. This means that To a certain extent, vaccines can solve the problem of tumor heterogeneity, which causes mutations and leads to poor efficacy of the originally designed vaccines. At the same time, tumors of different patients also have some different specific antigens, indicating that the vaccine that can treat heterogeneity has the potential for broad-spectrum promotion. This solves the problem that vaccines prepared by traditional methods cannot cure metastatic tumors and heterogeneous tumors.

[0150] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A tumor cell vaccine, characterized in that: The invention is composed of inactivated tumor cells with a protective shell, liposomes and functional molecules; the liposomes encapsulate the inactivated tumor cells with a protective shell, and the functional molecules are modified on the surface of the liposomes.

2. The tumor cell vaccine according to claim 1, characterized in that The inactivated tumor cells with protective shells are inactivated by freezing the tumor cells with protective shells and then thawing at low temperature; The shell layer comprises at least one of a nano shell layer, a polymer shell layer or a metal phenolic network shell layer.

3. The tumor cell vaccine according to claim 2, characterized in that The cryopreservation is performed at -198°C for more than 6 hours; The low-temperature rethawing is rethawing at 0-8°C for 6-24h; The nanoshell layer includes at least one of a noble metal nanoparticle shell layer, a silicon nanoshell layer, a titanium nanoshell layer, a manganese nanoshell layer, a calcium nanolayer, a cerium nanolayer, an organic nanolayer, and an organic-inorganic hybrid nanoshell layer; The polymer shell layer comprises at least one of a polydopamine shell layer, a pyrogallic acid shell layer and a polyelectrolyte shell layer.

4. The tumor cell vaccine according to claim 2, characterized in that The phenolic network in the metal phenolic network shell layer includes but is not limited to at least one of tannic acid, epicatechin, catechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, gallic acid, and retrogallic acid; The metal in the metal phenolic network shell layer includes but is not limited to Mn 2+ 、Gd、MnO2、Ag + 、Ti 4+ , Cu 2+ , Fe 2+ , Fe 3+ 、Zn 2+ At least one of the elements and the corresponding metal element, metal compound or metal ion of the element.

5. The tumor cell vaccine according to claim 1, characterized in that The tumor cells include but are not limited to: at least one of melanoma cells, breast cancer cells, glioma cells, colorectal cancer cells, ovarian cancer cells, lung cancer cells, gastric cancer cells and liver cancer cells.

6. The tumor cell vaccine according to claim 1, characterized in that The liposomes include at least one of cationic liposomes, cholesterol, phosphatidylethanolamine liposomes, phosphatidyl amide bases, glycerophospholipid liposomes, serine phospholipids and phosphatidic acid; The functional molecules include at least one of immune adjuvants, targeting molecules and labeling molecules.

7. The tumor cell vaccine according to claim 6, characterized in that The cationic liposomes include at least one of DOSPA, DOTAP, GL67, MVL5, DODMA, DOBAQ and 18:0DAP; The phosphatidylethanolamine liposomes include at least one of DOPE, DPPE and DSPE; The phosphatidyl alcohol base comprises at least one of DOPC and DSPC; The glycerophospholipid liposomes include at least one of DLPG and DPPG; The serine phospholipids include at least one of DSPS and DOPS; The phosphatidic acid includes at least one of DMPA; The immune adjuvants include but are not limited to pathogen-associated molecular pattern adjuvants; The targeting molecules and labeling molecules include but are not limited to targeting and / or labeling DNA or RNA.

8. The method for preparing a tumor cell vaccine according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) allowing tumor cells to react with a shell protection reaction solution to form a shell layer on the surface of the tumor cells, and removing the supernatant after the reaction is completed, washing, and obtaining tumor cells with a protective shell layer; (2) suspending the tumor cells with the protective shell in a buffer and freezing them in liquid nitrogen, followed by low-temperature thawing; (3) Encapsulating the reconstituted tumor cells with the protective shell layer in step (2) with liposomes, and then modifying the functional molecules on the surface of the liposomes.

9. The method according to claim 8, characterized in that The shell protection reaction solution in step (1) comprises a reagent for forming a silicon nanoshell, a reagent for forming a calcium nanoshell, a reagent for forming a polydopamine shell, a reagent for forming a pyrogallic acid shell, a reagent for forming a polyelectrolyte shell, and a reagent for forming a metal phenolic network shell; The concentration of the shell protection reaction solution in step (1) is 5 mM to 100 mM; The tumor cells and shell protection reaction solution described in step (1) were mixed at a rate of 2×10 6 ~4×10 6 The ratio of tumor cells to 1 mL of shell protection reaction solution; The reaction in step (1) is carried out at 4 to 8°C for 1 to 45 minutes; The freezing in step (2) is freezing at -198°C for more than 6 hours; The co-incubation in step (3) is ultrasonic co-incubation for 1 to 2 minutes; The co-incubation in step (4) is for 20 to 40 minutes.

10. Use of the tumor cell vaccine according to any one of claims 1 to 7 in the preparation of a drug for preventing and / or treating tumors.

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