Cell membrane-derived tumor vaccine with dendritic cell regulation function and application thereof

Abstract

The invention belongs to the technical field of tumor vaccines, and relates to a cell membrane-derived tumor vaccine with a dendritic cell regulation function and application thereof. The tumor vaccine comprises a tumor cell membrane shell which is subjected to induction treatment to generate immunogenic death, and a cationic carrier core which entraps small interfering RNA (siRNA) and an immunologic adjuvant. According to the preparation method disclosed by the invention, a tumor cell membrane of calreticulin eversion is obtained in a drug induction manner, so that the immunogenicity of the tumor vaccine is improved, and the uptake ability of the dendritic cells to the tumor vaccine is improved; meanwhile, the cation core loaded with siRNA can down-regulate up-regulated negative feedback regulatory factors after the dendritic cells are activated by antigens, inhibit a negative feedback pathway for immunoregulation of the dendritic cells, and enhance the in-vivo immune activation effect of the tumor vaccine.

Description

Technical Field

[0001] The invention belongs to the technical field of tumor vaccines and relates to a cell membrane-derived tumor vaccine with dendritic cell regulation function and application thereof. Background Art

[0002] The body's immune system plays a key role in the development of tumors. With the development of immunology and tumor biology, tumor immunotherapy has achieved great success in clinical treatment. It has not only changed the treatment model of tumors, but also overturned the traditional concept of tumor treatment, bringing new hope for curing tumors and becoming one of the most promising development directions in the field of tumor treatment. Unlike conventional treatments such as surgery, radiotherapy and chemotherapy, tumor immunotherapy mainly activates the body's innate immune system to kill tumor cells and inhibit the development and metastasis of tumors. Among them, tumor vaccines have great application potential in the field of tumor immunotherapy because of their characteristics of "efficient presentation, specific killing, and lasting memory". As an active immunotherapy, tumor vaccines increase the exposure of tumor antigens to immune cells, thereby activating tumor antigen-specific T cell responses, significantly improving the immune system's ability to recognize and eliminate tumor cells, and at the same time establishing immune memory to effectively prevent tumor recurrence, thereby achieving the purpose of treating malignant tumors.

[0003] Tumor vaccines induce effective immune responses in the body in four steps: (1) Tumor antigens are taken up, processed and presented by antigen presenting cells (APCs); (2) Appropriate stimulation signals cause APCs to differentiate and migrate to lymph nodes, presenting antigens to T cells through the major histocompatibility complex (MHC) pathway; (3) T cells are activated by binding of co-stimulatory molecules such as CD80 and CD86 to molecules such as CD28 and CD27 on the surface of T cells; (4) Proinflammatory factors such as IFN-γ and IL-12 are secreted to induce T cells to proliferate and differentiate into CTLs and migrate to the tumor site, thereby inducing cell apoptosis. Among them, dendritic cells (DCs) are the most important antigen presenting cells in the body, closely related to immune activation and immune tolerance, and are at the center of initiating, regulating, and maintaining immune responses. However, after being activated by antigens, DCs will trigger their own negative feedback regulation, activate inhibitory receptor-mediated signaling pathways, and then induce the tolerance phenotype of DCs, hinder the antigen presentation process of DCs, resulting in the obstruction of T cell proliferation and activation, and weakening the intensity of the triggered immune response.

[0004] In addition, clinical studies have found that traditional tumor vaccine therapies that target a single antigen, such as tumor-specific proteins, peptides, and deoxyribonucleic acid (DNA) fragments, have limited therapeutic effects. Whole tumor cell vaccines can provide a wide range of autologous antigens and are considered a promising strategy. However, due to the presence of a large number of housekeeping genes, nucleic acids, and lipids, the clinical effects of whole tumor cell vaccines are not significant. Therefore, it is urgent to explore and develop a tumor vaccine with high immunogenicity, strong antigen presentation ability, safety, and efficiency. Summary of the invention

[0005] Purpose of the invention:

[0006] In view of the problems of low immunogenicity and weak antigen presentation ability of the above-mentioned tumor vaccine, the present invention provides a cell membrane-derived tumor vaccine with dendritic cell regulation function and its preparation method and application. The vaccine uses drug-induced method to obtain tumor cell membranes that have undergone immunogenic death as the shell of the tumor vaccine, selects cationic carriers to encapsulate RNA and immune adjuvants as the core through electrostatic and coordination effects, and wraps the extracted cell membranes on the outside of the nano core through electrostatic interaction, forming a nano vaccine with a "core-shell" structure.

[0007] Technical solution:

[0008] A cell membrane-derived tumor vaccine with dendritic cell regulation function, characterized in that the tumor vaccine comprises an inner core and an outer shell; wherein: the inner core comprises a cationic carrier, a nucleic acid molecule and an immune adjuvant; and the outer shell is a tumor cell membrane.

[0009] The tumor vaccine is characterized in that the shell is a cell membrane in which the immunogenic death inducing agent induces immunogenic death of tumor cells and CRT translocation.

[0010] The tumor vaccine is characterized in that the tumor cell membrane is selected from any one or more of 4T1, B16-F10, Hepa1-6, ID8, RM-1, LLC, MFC, CT26 and Caov-3.

[0011] The tumor vaccine is characterized in that the immunogenic death inducing agent is any one of adriamycin, doxorubicin, mitoxantrone, oxaliplatin, cyclophosphamide and bleomycin.

[0012] The tumor vaccine is characterized in that the nucleic acid molecule in the inner core is a small interfering RNA, and the small interfering RNA includes any one of siSOCS1, siPD-L1, siTIM-3, and siIDO1; the cationic material includes any one of polyethyleneimine, polylysine, polyamidoamine, deacetylated chitosan, gelatin, cationic polypeptide, cationic polyester, cationic polyphosphate, polyvinyl pyridinium salt, and poly (dimethylamino) ethyl methacrylate; and the immune adjuvant is any one of manganese chloride, aluminum phosphate, CPG, and cGAMP.

[0013] The tumor vaccine is characterized in that the nucleic acid molecule is a small interfering RNA, preferably, the small interfering RNA is siSOCS1, and its nucleotide sequence is: sense strand: '5-CUCCGUGACUACCUGAGUU-3', antisense strand: 5'-AACUCAGGUAGUCACGGAG-3';

[0014] The tumor vaccine is characterized in that the mass ratio of the shell to the core is 0.5:1-3:1; the mass ratio of the cationic material to the small interfering RNA is 18:1-24:1.

[0015] The tumor vaccine is characterized in that the outer shell is a cell membrane that induces immunogenic death of 4T1 and CRT translocation by 3 μg / mL doxorubicin; the inner core is a nanoparticle formed by polylysine encapsulating siSOCS1 and manganese chloride, wherein the mass ratio of polylysine to siSOCS1 is 18:1; and the mass ratio of the outer shell to the inner core is 3:1.

[0016] The tumor vaccine is used in the preparation of drugs for preventing or treating tumors.

[0017] The application is characterized in that the tumor is any one of breast cancer, colorectal cancer, melanoma, esophageal cancer, prostate cancer, liver cancer, bile duct cancer, bladder cancer, and ovarian cancer.

[0018] In some embodiments of the present invention, the incubation time of the immunogenic death-inducing agent and the cells ranges from 6 to 48 hours.

[0019] Principle of the present invention:

[0020] Calreticulin (CRT) translocation refers to the phenomenon that calreticulin is transferred from the endoplasmic reticulum cavity where it is usually located to other parts of the cell, such as the cell membrane, cytoplasm or nucleus; on the surface of immunogenic dead cells or cells treated with some drugs (such as immunogenic death inducers, such as doxorubicin, doxorubicin, mitoxantrone, oxaliplatin, cyclophosphamide, bleomycin), the expression of calreticulin increases and translocation occurs. It can act as an "eat me" signal, binding to the receptor CD91 / LRP1 on the surface of immune cells such as macrophages and dendritic cells, enhancing the immune response of the immune system to these cells, thereby promoting the phagocytosis and clearance of tumor cells or apoptotic cells by immune cells.

[0021] Suppressor of cytokine signaling-1 (SOCS1) is the most important negative feedback regulatory factor in DCs cells. When DCs cells take up antigens and process them, their SOCS1 expression is significantly upregulated, blocking JAK / STAT-mediated cytokine-induced gene transcription, inhibiting the secretion of proinflammatory cytokines, and thus hindering the maturation of DCs cells and antigen presentation, resulting in the proliferation and activation of T cells being blocked. The SiRNA (siSOCS1) designed by the present invention can silence SOCS1, downregulate the negative feedback effect after DCs activation, increase antigen presentation, and thus promote the proliferation and activation of T cells.

[0022] Fig.11 As shown in A, the present invention induces immunogenic death of tumor cells by immunogenic death inducing agents (such as doxorubicin), causes CRT translocation, and breaks the cells to separate and obtain CRT translocated tumor cell membranes. Cationic carriers encapsulate SiRNA (siSOCS1) and immune adjuvants (such as manganese chloride) through electrostatic interaction and coordination to form nanoparticle cores. The negatively charged CRT translocated tumor cell membrane is wrapped in the cationic nanocore through electrostatic adsorption to form a tumor vaccine, which has dendritic cell regulation function.

[0023] like Fig.11 As shown in Figure B, after the tumor vaccine of the present invention is subcutaneously injected, DCs will take up the tumor vaccine due to the translocated CRT cell membrane, thereby activating DCs and initiating an immune response (such as proliferation and activation of T cells). At the same time, the nucleic acid drug siSOCS1 that enters the inner core of DCs weakens the negative feedback regulation pathway of DCs after immune activation, further enhancing the antigen presentation ability of DCs, inducing a stronger immune response, and having a stronger anti-tumor immune effect.

[0024] Beneficial effects:

[0025] The tumor antigen in the vaccine of the present invention is the tumor cell membrane of CRT translocation after immunogenic death. The cell membrane has a high proportion of tumor antigens, which can overcome the problem of single antigen and low immunogenicity of traditional tumor vaccines, and can avoid the interference of intracellular protein housekeeping genes, nucleic acids and lipids in tumor cells and tumor cell lysate vaccines and immune escape caused by down-regulation of some antigen expression in tumors, and has stronger immune activation ability. On this basis, the siRNA encapsulated in the kernel can effectively silence the expression of negative feedback conditional factors in DCs, further promote the maturation and antigen presentation of DCs, and induce a more powerful anti-tumor immune response. This invention also provides a new design idea for the research of tumor vaccines.

[0026] Specifically:

[0027] 1) The tumor cell membrane has complete tumor-associated antigens, including a complete array of cell membrane protein antigens, which can trigger a personalized immune response to the corresponding tumor, while avoiding the interference of cytoplasmic proteins and immune escape caused by downregulation of some antigen expression in the tumor. The tumor cell membrane induced by the drug in the present invention has stronger immunogenicity, and its surface translocated calreticulin (CRT) can interact with receptors on the surface of DCs to send out "eat me" signals, promote the phagocytosis of cell membrane antigens, and thus activate innate immunity and adaptive immunity.

[0028] 2) The tumor vaccine core is loaded with RNA and immune adjuvants that can silence the immunosuppressive molecule SOCS1 expressed by DCs, which can block the immunosuppressive signals after DCs activation and further enhance the anti-tumor immune response.

[0029] 3) The tumor cell shell can shield the positive charge of the cationic core, reduce the toxic side effects on cells, and protect the RNA carried by the core from being degraded by abundant intracellular enzymes, thereby improving the stability and safety of the vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the expression level of CRT on the cell surface after different concentrations of doxorubicin hydrochloride induce immunogenic death of 4T1 cells in Example 1;

[0031] Figure 2 The agarose gel electrophoresis results in Example 2;

[0032] Figure 3 is the particle size of the nano core at different mass ratios in Example 2;

[0033] Figure 4 is the potential size of the nano core at different mass ratios in Example 2;

[0034] Figure 5is the particle size of the tumor vaccine with different nuclear membrane mass ratios in Example 3;

[0035] Figure 6 The particle size changes of tumor vaccines with different nuclear membrane mass ratios in Example 3 within 7 days;

[0036] Figure 7 Protein characterization of tumor cells, cell membranes and tumor vaccines in Example 4;

[0037] Figure 8 This is the situation of tumor vaccine being taken up by DCs in Example 5;

[0038] Fig. 9 The gene silencing effect of the tumor vaccine on DCs in Example 6;

[0039] Fig.10 This is the promoting effect of the tumor vaccine on DCs in Example 7.

[0040] Fig.11 Schematic diagram of the structure (A) and mechanism of action (B) of the tumor vaccine of the present invention. DETAILED DESCRIPTION

[0041] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions made to the embodiments described below are also within the scope of the present invention. Therefore, the equalization and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

[0042] The experimental materials, experimental reagents and instruments used in the examples of the present invention can all be commercially available.

[0043] Terminology: CRT: calreticulin; PBM siRNA : cationic core encapsulating RNA and immune adjuvants; DCs: dendritic cells; TM: normal tumor cell membrane

[0044] Example 1 Induction of immunogenic dead tumor cell membrane (i.e., obtaining CRT translocated tumor cell membrane) and verification

[0045] In order to induce immunogenic death of tumor cells, the concentration of the immunogenic death inducer doxorubicin incubated with 4T1 cells was first screened. Mouse breast cancer 4T1 cells were cultured in 1640 medium containing 10% fetal bovine serum and 1% double antibody and placed at 37°C and 5% CO 2 4T1 cells were cultured in a saturated humidity incubator at a rate of 1×10 5The cells were seeded at a density of 100 cells / well in a 12-well plate. After overnight attachment, doxorubicin (DOX) at concentrations of 1 μg / mL, 2 μg / mL, 3 μg / mL, and 4 μg / mL were added for treatment for 24 h. The cells were digested with trypsin, centrifuged and resuspended, and FITC-anti-calreticulinantibody was added for staining in the dark for 30 min. Then, the CRT expression level of 4T1 cells was examined by flow cytometry.

[0046] After incubation with 4 μg / mL doxorubicin for 24 hours, 4T1 cells were collected and resuspended in PBS solution, centrifuged at 12000 rpm for 5 minutes, and washed 3 times. The cells were resuspended in PBS containing PMSF, a protease inhibitor, precooled at 4°C. The cell suspension was subjected to ultrasonic mechanical treatment for 4 minutes (power 30%) under ice bath conditions, centrifuged at 4°C, 1500g for 15 minutes, and the supernatant after centrifugation was collected. The collected supernatant was then centrifuged again at 4°C, 18000g for 1 hour, the supernatant was discarded, the precipitated cell debris was collected, and stored at -80°C for further use.

[0047] result:

[0048] like Figure 1 As shown, when the concentration of doxorubicin reached 3 μg / mL, the expression level of CRT was 44.6%. When the concentration was further increased to 4 μg / mL, there was no significant difference in the expression level of CRT. Considering the toxicity to the cell membrane, a concentration of 3 μg / mL was selected to induce immunogenic death of 4T1 cells.

[0049] Example 2 Cationic core PBM encapsulating RNA and immune adjuvant siRNA Construction

[0050] In this example, 3,4-dihydroxybenzaldehyde is modified on low molecular weight polylysine (PLL) as a core cationic carrier (PB) to encapsulate RNA and immune adjuvant (manganese chloride). The specific preparation method is as follows:

[0051] (1) Add 1 μL of diethyl pyrocarbonate (DEPC) to 1 mL of ultrapure water and sterilize to obtain nuclease-free water;

[0052] (2) dissolving 5 nmol siRNA in 250 μL of the nuclease-free water described in step (1) to obtain a siRNA solution with a concentration of 20 nmol / mL;

[0053] (3) dissolving 3.33 mg of manganese chloride in 1 mL of the nuclease-free water in step (1) to obtain a 3.33 mg / mL manganese chloride solution, filtering with a 0.22 μm filter membrane, taking 30 μL of the manganese chloride solution and adding 270 μL of nuclease-free water to obtain a manganese chloride solution with a concentration of 0.33 mg / mL;

[0054] (4) mixing the siRNA solution described in step (2) with the manganese chloride solution described in step (3) in a ratio of 1:1 and incubating for 5 minutes;

[0055] (5) Dissolve 5 mg of PB in 1 mL of the nuclease-free water described in step (1) to obtain a 5 mg / mL PB solution;

[0056] (6) The PB solution described in step (5) and the mixed solution of siRNA and manganese chloride described in step (4) were compounded at different mass ratios (10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1), vortexed for 30 seconds, and incubated at room temperature for 30 minutes to prepare a cationic core PBM. siRNA .

[0057] The compression ability of PB on siRNA was investigated by agarose gel electrophoresis blocking experiment. The specific operation method is as follows:

[0058] Preparation of 1% agarose gel: weigh 2g agarose and add it to 200mL 1×TAE, heat it to dissolve completely, and add 20μL Gel Red nucleic acid dye to the solution after it cools down slightly and shakes gently to avoid bubbles. Pour the gel solution slowly into the gel tank while it is hot, and soak it in 1×TAE electrophoresis buffer after it cools to form a gel.

[0059] Take the nano-cores of each ratio described in step (6), add an appropriate amount of 10× Loading Buffer, mix well, load the sample into each well of the agarose gel, and perform electrophoresis. The electrophoresis conditions are: 1×TAE buffer, voltage 120V, time 20min. After the electrophoresis, use a gel imaging system to analyze the compression of siRNA by PB of different masses. Use a Malvern Zetasizer to measure the nano-core PBM prepared at different mass ratios at 25°C. siRNA The particle size and Zeta potential are used to determine the optimal formulation of the gene nanocore.

[0060] result:

[0061] like Figure 2 As shown in Figure 2, as the mass of PB increases, the adsorption capacity of siRNA gradually increases. When the mass ratio of PB to siRNA reaches 18, free siRNA is almost undetectable in the electrophoresis band, indicating that PB has completely wrapped siRNA to form an inner core PBM. siRNA .

[0062] like Figure 3 , 4As shown in the figure, as the mass ratio of PB to siRNA increases, the particle size of the formed nanocomplex gradually decreases, and the Zeta potential value changes from negative to positive and gradually increases, indicating that as the mass of PB increases, its ability to compress siRNA also increases significantly. When the mass ratio of PB to siRNA reaches 18:1, PBM siRNA The particle size of the nanocore is 129.23±3.03nm and the Zeta potential is 18.54±0.53mV. In order to avoid the toxic side effects caused by cationic carrier materials to the greatest extent, 18:1 was selected as PBM. siRNA The optimal mass ratio of PB and siRNA in the nanocore was used for preparation.

[0063] Example 3 Construction of a cell membrane-derived tumor vaccine with dendritic cell regulatory function

[0064] This embodiment provides a method for constructing a cell membrane-derived tumor vaccine with dendritic cell regulation function, and the specific steps are as follows:

[0065] (1) resuspending the CRT translocated cell membrane extracted in Example 1 with the nuclease-free water described in step (1) of Example 2 to obtain a CRT cell membrane suspension;

[0066] (2) repeatedly extruding the CRT cell membrane suspension described in step (1) through polycarbonate membranes with pore sizes of 1000 nm, 800 nm, and 400 nm in sequence using a microliposome extruder 11 times to obtain CRT cell membrane vesicles;

[0067] (3) using a BCA kit to measure the protein content of the CRT cell membrane vesicles in step (2);

[0068] (4) The CRT cell membrane vesicles quantified in step (3) were mixed with the inner core PBM described in Example 2. siRNA The mixture was mixed at different mass ratios (0.5:1, 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 6:1), incubated at room temperature for 30 min, and ultrasonicated on ice for 10 min to obtain the tumor vaccine CRT / PBM. siRNA .

[0069] (5) The particle size and Zeta potential of the sample were measured at 25° C. using a Malvern Zetasizer, and the particle size of the sample was measured again on the 3rd, 5th, and 7th day.

[0070] result:

[0071] like Figure 5 As shown, CRT cell membrane and PBM siRNAThe particle size was small and had no significant difference when the mass ratio was in the range of 0.5:1 to 3:1. The particle size gradually increased with the increase of CRT cell membrane mass. Figure 6 As shown, within 7 days, CRT cell membrane and PBM siRNA The particle size stability was best when the mass ratio was 2:1. At this time, the potential was reversed from the positive potential of the core to the negative potential of the cell membrane, indicating that the cell membrane was successfully wrapped on the surface of the nanocore. The tumor vaccine with this ratio was placed in a 30kDa ultrafiltration tube and centrifuged at 4500rpm for 20min. The free drug content in the lower filtrate was detected to calculate the encapsulation rate. The CRT cell membrane and PBM were measured. siRNA When the mass ratio of siRNA and Mn is 2:1 2+ The encapsulation efficiency of CRT cell membrane and PBM was greater than 90%. siRNA The vaccine was prepared at a mass ratio of 2:1 for subsequent experiments.

[0072] Example 4 Investigation of tumor vaccine surface antigen retention

[0073] In this example, the antigen retention of the prepared tumor vaccine was investigated by SDS-PAGE gel electrophoresis. The specific operation method is as follows: (1) preparing a 10% SDS-PAGE protein gel;

[0074] (2) Add PMSF, a protease inhibitor, to the RIPA lysis buffer and lyse tumor cells, immunogenic dead tumor cells, CRT cell membranes, and tumor vaccines (CRT / PBM) on ice. siRNA );

[0075] (3) centrifuging the lysate in step (2) at 12000 g at 4°C for 5 min and collecting the supernatant;

[0076] (4) using a BCA protein detection kit to quantify the protein concentration of the sample supernatant described in step (3);

[0077] (5) mixing the quantified protein sample in step (4) with 5× protein electrophoresis loading buffer and boiling for 5 minutes;

[0078] (6) adding the solution of step (5) to the 10% SDS-PAGE protein gel of step (1), with 30 μg of sample loaded per well;

[0079] (7) Perform electrophoretic separation of proteins at a voltage of 80 V. After 30 min, adjust the voltage to 120 V and wait until the protein electrophoresis reaches the bottom of the gel before turning off the power supply.

[0080] (8) staining the separated gel in step (7) with 0.75× Coomassie Brilliant Blue stain for 1 h;

[0081] (9) The stained gel in step (8) was placed in 90° C. hot water for 6 h for decolorization and then photographed and observed using a gel imaging device.

[0082] result:

[0083] like Figure 7 As shown in Figure 2, cells that underwent immunogenic death expressed more abundant proteins than normal cells, and protein bands on the CRT cell membrane and CRT / PBM were observed. siRNA The protein bands were basically consistent, indicating that the cell membrane was successfully wrapped onto the inner core surface, and that the cell membrane protein had good protein retention on the nanoparticles, retaining its immunogenicity, which was beneficial to the subsequent activation of the immune response.

[0084] Example 5 Investigation of the ability of DCs to take up tumor vaccines

[0085] This example uses a tumor vaccine prepared with a common cell membrane as a control to examine the uptake capacity of DCs for the tumor vaccine. The specific operation method is as follows:

[0086] (1) Extract and separate the common tumor cell membrane TM and CRT translocated cell membrane as described in Example 1, and prepare nanocore PBM using Cy5 fluorescently labeled siRNA as described in Example 2 siRNA , further, TM / PBM was prepared as described in Example 3 siRNA and CRT / PBM siRNA (2) Dendritic cells (DCs) growing in the logarithmic phase were selected, washed with pH 7.4 phosphate buffer, and digested with 0.25% trypsin-EDTA digestion solution. The cells were collected, centrifuged at 400 g, and resuspended at 1×10 5 The cells were seeded at a density of 1 / well at the bottom of a 12-well cell culture plate;

[0087] (3) After the cells have adhered overnight, the culture medium in the wells was aspirated and the PBM described in step (1) was added. siRNA 、TM / PBM siRNA 、CRT / PBM siRNA and a negative control (named "Control" in this example) were incubated at 37°C and 5% CO 2 The culture medium was discarded, the cells were washed three times with pH 7.4 phosphate buffer, 0.25% trypsin-EDTA digestion solution was added to digest the cells, the cells were collected, centrifuged at 400 g and resuspended, and then the cellular uptake of the nanosystem was quantitatively investigated using flow cytometry.

[0088] result:

[0089] like Figure 8 As shown, due to PBM siRNAPositively charged, less stable, DCs have poor uptake capacity, TM / PBM siRNA The surface lacks receptors that interact with DCs, has low immunogenicity, and the fluorescence intensity after DCs uptake is weaker than CRT / PBM siRNA , while CRT / PBM siRNA It has high immunogenicity, and the CRT on its surface can interact with the receptors on the surface of DCs to send out an "eat me" signal and promote the phagocytosis of DCs. Therefore, the fluorescence intensity of this group is stronger after being taken up by DCs.

[0090] Example 6 Investigation of the gene silencing effect of tumor vaccines on DCs

[0091] This example uses a tumor vaccine prepared with a common cell membrane as a control to examine the gene silencing effect of the tumor vaccine on the uptake capacity of DCs. The specific operation method is as follows:

[0092] (1) The common tumor cell membrane TM and CRT translocation cell membrane were extracted and separated as described in Example 1, and nanocore PBM was prepared using siSOCS1 (sense chain: '5-CUCCGUGACUACCUGAGUU-3', antisense chain: 5'-AACUCAGGUAGUCACGGAG-3') and negative control siNC as described in Example 2 siSOCS1 and PBM siNC , further, TM / PBM was prepared as described in Example 3 siSOCS1 、TM / PBM siNC 、CRT / PBM siSOCS1 and CRT / PBM siNC ;

[0093] (2) Dendritic cells (DCs) growing in the logarithmic phase were selected and washed with phosphate buffer (pH 7.4). The cells were digested with 0.25% trypsin-EDTA digestion solution, collected, centrifuged at 400 g, and resuspended. 5 The cells were seeded at a density of 100 cells / well at the bottom of a 6-well cell culture plate;

[0094] (3) After the cells have adhered overnight, the culture medium in the wells was aspirated and the PBM described in step (1) was added. siSOCS1 、PBM siNC 、TM / PBM siSOCS1 、TM / PBM siNC 、CRT / PBM siSOCS1 、CRT / PBM siNC and a negative control (named "Control" in this example) were incubated at 37°C and 5% CO 2 After incubation for 12 h under the same conditions, the culture medium was discarded and replaced with 1640 medium containing 10% fetal bovine serum and cultured for 48 h.

[0095] (4) Aspirate the culture medium, wash three times with pH 7.4 phosphate buffer, and add 1 mL of Trizol to each well to extract RNA from each group of cells;

[0096] (5) Collect the lysate and transfer it to an enzyme-free tube. Add 200 μL of chloroform to each tube, place on ice for 10 min, centrifuge at 12,000 g for 15 min at 4°C, and collect 500 μL of the upper aqueous phase.

[0097] (6) Add isopropanol in equal proportions to the collected aqueous phase, place on ice for 15 min, centrifuge at 12000 g for 15 min at 4°C, discard the supernatant, add 1 mL of freshly prepared 75% (v / v) ethanol solution, centrifuge at 7500 g for 5 min at 4°C, discard the supernatant, and evaporate the ethanol at room temperature;

[0098] (7) After the pellet is dried, add 30 μL of nuclease-free water to resuspend it and place it at 55°C for 10 min to dissolve the RNA;

[0099] (8) Using a nucleic acid quantifier to detect the concentration of RNA extracted from each sample, and determining the purity of RNA by measuring the ratio of OD260 nm to OD280 nm of the sample;

[0100] (9) Take 500 ng RNA from each sample, add 4 μL 4g DNA wiper mix to remove genomic DNA contamination, add nuclease-free water to a total volume of 16 μL, gently pipette evenly, incubate in a 42°C water bath for 2 min, then add 4 μL 5HiScriptⅡqRT Super MixⅡ reverse transcriptase, place the sample in a gradient PCR instrument, react at 50°C for 15 min, and reverse transcribe at 85°C for 5 s to obtain cDNA for each sample.

[0101] (10) Dilute the cDNA sample obtained by reverse transcription 2.5 times with DEPC water for qRT-PCR reaction. Add 2 μL cDNA template, 10 μL 2ChamQ SYBR qPCR Master Mix, 0.4 μL each of 50ROX Reference DyeⅠ fluorescent probe, forward primer (10 μM), and reverse primer (10 μM) to the PCR plate, add DEPC to a total volume of 20 μL, cover each well of the PCR plate with a sealing film, place it in the fully automatic fluorescent quantitative PCR system, and perform qRT-PCR reaction according to the following steps:

[0102] Pre-denaturation: 95℃ for 30s (1.6℃ / s)

[0103] Cyclic amplification: 95℃ reaction for 10s-60℃ reaction for 30s-acquire signal

[0104] Melting curve: 95℃ reaction for 15s—60℃ reaction for 60s—95℃ reaction for 15s—acquire signal

[0105] (11) The Ct value was obtained according to the amplification curve, and the T and T values ​​were calculated by the following formula to analyze the SOCS1 mRNA level in each group (GAPDH was used as the internal reference gene):

[0106] ΔT = Ct sample - Ct GAPDH

[0107] ΔΔT=ΔT-ΔT Control

[0108] mRNA expression level (%) = 2 -ΔΔT ×100

[0109] result:

[0110] like Fig. 9 As shown, due to the DCs to PBM siRNA The uptake capacity of PBM is poor siSOCS1 and PBM siNC There was no significant difference in the level of SOCS1 mRNA between the TM / PBM group and the Control group. siNC In the control group, the expression of immunosuppressive molecule SOCS1 increased due to the activation of DCs by tumor cell membrane antigens, and the TM / PBM siSOCS1 Group and TM / PBM siNC The level of SOCS1 mRNA was significantly lower than that of the control group, indicating that the encapsulated siSOCS1 can effectively achieve gene silencing. siNC The level of SOCS1 mRNA in the CRT group was significantly higher than that in the Control group, which may be due to the stronger immunogenicity of the CRT cell membrane, higher efficiency of DCs uptake, and stronger immune activation, which further increased the expression of immunosuppressive molecules. siSOCS1 Compared with CRT / PBM siNC The expression of SOCS1 mRNA in the group was significantly downregulated, indicating that the tumor vaccine can effectively achieve gene silencing while activating the DCs immune response, reducing the expression of the immunosuppressive molecule SOCS1, which is conducive to inducing a stronger anti-tumor immune response.

[0111] Example 7 Investigation of the ability of tumor vaccines to promote DCs maturation

[0112] This example uses a tumor vaccine prepared with a common cell membrane as a control to examine the ability of the tumor vaccine to promote DCs maturation. The specific operation method is as follows:

[0113] (1) Extract and separate the common tumor cell membrane TM and CRT translocated cell membrane as described in Example 1, and use siSOCS1 and negative control siNC to prepare nanocore PBM as described in Example 2 siSOCS1 and PBM siNC , further, TM / PBM was prepared as described in Example 3 siSOCS1 、CRT / PBM siSOCS1 and CRT / PBM siNC ;

[0114] (2) Dendritic cells (DCs) growing in the logarithmic phase were selected and washed with phosphate buffer (pH 7.4). The cells were digested with 0.25% trypsin-EDTA digestion solution, collected, centrifuged at 400 g, and resuspended at 1 × 10 5 The cells were seeded at a density of 1 / well at the bottom of a 12-well cell culture plate;

[0115] (12) After the cells adhered overnight, the culture medium in the wells was aspirated and the TM, CRT, and PBM described in step (1) were added. siSOCS1 、TM / PBM siSOCS1 、CRT / PBM siSOCS1 and a negative control (named "Control" in this example) were incubated at 37°C and 5% CO 2 After incubation for 12 h under the same conditions, the culture medium was discarded and replaced with 1640 medium containing 10% fetal bovine serum and cultured for 36 h.

[0116] (3) Aspirate the culture medium, wash with pH 7.4 phosphate buffer, digest the cells with 0.25% trypsin-EDTA digestion solution, collect the cells, centrifuge at 400 g and resuspend;

[0117] (4) Wash once with pH 7.4 phosphate buffer, resuspend, add 1 μL FITC anti-CD80 antibody and 1 μL PE anti-CD86 antibody, and incubate in the dark for 30 min;

[0118] (5) The cells were washed once with pH 7.4 phosphate buffer, resuspended and the expression of CD80 and CD86 on the surface of DCs was detected by flow cytometry to examine the maturity of DCs.

[0119] result:

[0120] like Fig.10 As shown, due to the DCs to PBM siRNA The uptake capacity of PBM is poor siSOCS1 There was no significant difference in the proportion of CD80 and CD86 positive cells between the TM group and the TM / PBM group.siSOCS1 The proportion of CD80 and CD86 positive cells in the group was slightly higher than that in the control group. siSOCS1 The proportion of CD80 and CD86 positive cells in the TM / PBM group was higher, mainly because tumor cell membrane antigens have an immune activation effect on DCs and can promote the maturation of DCs after being taken up by DCs. siSOCS1 The group reduced the expression of SOCS1 during immune activation and further promoted the maturation of DCs. However, TM has relatively low immunogenicity and lacks relevant receptors on its surface, so its efficiency in being taken up by DCs is not high, and its effect on promoting the maturation of DCs is not significant enough. CRT and CRT / PBM siSOCS1 The proportion of CD80 and CD86 positive cells in the CRT group increased significantly compared with the Control group, indicating that it has a strong promoting effect on the maturation of DCs. This is mainly due to the strong immunogenicity of the CRT cell membrane, which can interact with the receptors on the surface of DCs through the CRT on the surface to send out an "eat me" signal, promoting the phagocytosis of DCs by antigens. siSOCS1 The encapsulated siSOCS1 inhibited the expression of immunosuppressive molecules in DCs, promoting DCs to be more capable and trigger a stronger immune response.

Claims

1. A cell membrane-derived tumor vaccine having dendritic cell regulatory function, characterized in that: The tumor vaccine comprises an inner core and an outer shell; wherein: the inner core comprises a cationic carrier, a nucleic acid molecule and an immune adjuvant; and the outer shell is a tumor cell membrane.

2. The tumor vaccine according to claim 1, characterized in that The shell is a cell membrane in which the immunogenic death inducing agent induces immunogenic death of tumor cells and CRT translocation.

3. The tumor vaccine according to claim 2, characterized in that The tumor cell membrane is selected from any one or more of 4T1, B16-F10, Hepa1-6, ID8, RM-1, LLC, MFC, CT26 and Caov-3.

4. The tumor vaccine according to claim 2, characterized in that: The immunogenic death inducing agent is any one of adriamycin, doxorubicin, mitoxantrone, oxaliplatin, cyclophosphamide and bleomycin.

5. The tumor vaccine according to claim 1, characterized in that The nucleic acid molecule in the inner core is a small interfering RNA, and the small interfering RNA includes any one of siSOCS1, siPD-L1, siTIM-3, and siIDO1; the cationic material includes any one of polyethyleneimine, polylysine, polyamidoamine, deacetylated chitosan, gelatin, cationic polypeptides, cationic polyesters, cationic polyphosphates, polyvinylpyridinium salts, and poly(dimethylamino)ethyl methacrylate; and the immune adjuvant is any one of manganese chloride, aluminum phosphate, CPG, and cGAMP.

6. The tumor vaccine according to claim 5, characterized in that The nucleic acid molecule is a small interfering RNA. Preferably, the small interfering RNA is siSOCS1, and its nucleotide sequence is: sense strand: '5-CUCCGUGACUACCUGAGUU-3', antisense strand: 5'-AACUCAGGUAGUCACGGAG-3'.

7. The tumor vaccine according to claim 6, characterized in that The mass ratio of the shell to the core is 0.5:1 to 3:1; the mass ratio of the cationic material to the small interfering RNA is 18:1 to 24:

1.

8. The tumor vaccine according to claim 7, characterized in that The outer shell is a cell membrane that induces immunogenic death and CRT translocation of 4T1 cells when exposed to 3 μg / mL or more of doxorubicin; the inner core is a nanoparticle formed by polylysine encapsulating siSOCS1 and manganese chloride, wherein the mass ratio of polylysine to siSOCS1 is 18:1; The mass ratio of shell to core is 3:

1.

9. Use of the tumor vaccine according to any one of claims 1 to 8 in the preparation of a drug for preventing or treating anti-tumor.

10. The use according to claim 9, characterized in that: The tumor is any one of breast cancer, colorectal cancer, melanoma, esophageal cancer, prostate cancer, liver cancer, bile duct cancer, bladder cancer, and ovarian cancer.

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