Anti-tumor mRNA vaccine and preparation method and application thereof

Abstract

The application discloses a novel anti-tumor mRNA vaccine and a preparation method and application thereof, and belongs to the technical field of biological medicines. The anti-tumor mRNA vaccine comprises a first composition and a second composition. The first composition comprises an immune adjuvant and a first active ingredient, wherein the first active ingredient comprises a first antigen for inducing the body to produce an antibody. The second composition comprises a pharmaceutical carrier and a second active ingredient, wherein the second active ingredient comprises an mRNA molecule for encoding a membrane-anchored antigen recognized by the antibody, and the mRNA molecule comprises, from 5' end to 3' end, a signal peptide coding sequence, a second antigen coding sequence and a membrane-anchored protein coding sequence. The second composition is in an intratumoral or paratumoral local administration dosage form. The application labels tumor cells with antigens, and relies on the body to induce an anti-tumor attack by means of a specific antibody which is extensive and strong, and exhibits a strong and effective tumor inhibition effect in multiple solid tumor models.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a novel anti-tumor mRNA vaccine and a preparation method and application thereof. BACKGROUND

[0002] Tumor vaccine is an important tool for the body to form a protective immune response and achieve specific clearance of tumor tissue. Its active ingredients include four key components: tumor antigen, preparation, immune adjuvant and delivery carrier. The production of anti-tumor immune response of tumor therapeutic vaccine needs to be taken up, processed and presented to T and B lymphocytes by antigen-presenting cells (APC). With the in-depth study of tumor immunity, it is found that there are various defects in antigen processing and presentation in tumor hosts (Sellars, 2022; Lin, 2022).

[0003] Tumor cells can enhance their ability to evade immune surveillance and attack through various modification and change mechanisms. Among them, tumor cells can directly escape the recognition and destruction of the immune system by rapid mutation or loss of antigens, thereby causing off-target of antigen-specific immune attack (Beatty, 2015). Although the use of gene sequencing and other means to obtain tumor neoantigen polypeptide epitopes has shown great potential in improving vaccine efficacy, the specificity and immunogenicity of polypeptide antigen epitopes greatly limit the efficacy of the vaccine (Chu, 2018). Therefore, it is urgent to develop new combination vaccine strategies to cope with the challenges of immune escape due to low neoantigen load, rapid loss of antigen epitopes, and difficulty in inducing tumor-specific T cell response in the process of tumor immunity.

[0004] mRNA as a new treatment mode instead of DNA and recombinant protein has received more and more attention in the field of cancer immunotherapy (Beck, 2021). Because of its high protein expression rate and no potential risk of inserting the host cell genome. It also has the dual role of antigen presentation and self-adjuvant, which is more likely to activate the innate immune pathway, so it has broad application value in anti-tumor vaccines. At present, lipid nanoparticles (Lipid Nanoparticle, LNP) as the mainstream mRNA delivery system can effectively encapsulate and protect nucleic acids into the cell, and be released and translated into functional proteins (Hou, 2021).

[0005] The current anti-tumor mRNA vaccine encodes tumor-specific or tumor-related antigens or tumor neoantigens. The tumor-related antigens are part of normal cells, and there are problems such as weak immunogenicity or difficulty in stimulating the body to produce strong and effective anti-tumor immune response. The vaccine encoding tumor neoantigens has many technical barriers in the early neoantigen analysis and identification and the later personalized vaccine production process, and the treatment cost is very high. Therefore, how to effectively enhance the anti-tumor immune response of the mRNA vaccine and shorten the time cost is a technical problem that needs to be solved by those skilled in the art. SUMMARY

[0006] The purpose of the present application is to provide a new anti-tumor drug. The immune antigen is locally administered intratumorally or paratumorally in the form of mRNA drug to achieve antigen labeling of tumor cells. With the help of specific antibodies produced by the body in the early stage of vaccination, the antigen is recognized and induced to attack the body specifically, and finally the tumor tissue is cleared.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] The present application provides an anti-tumor mRNA vaccine, comprising a first composition and a second composition,

[0009] The first composition comprises an immune adjuvant and a first active ingredient, and the first active ingredient comprises a first antigen that induces the body to produce antibodies;

[0010] The second composition comprises a pharmaceutical carrier and a second active ingredient, and the second active ingredient comprises an mRNA molecule encoding a membrane-anchored antigen recognized by the antibody. The mRNA molecule comprises, in order from 5' end to 3' end, a signal peptide coding sequence, a second antigen coding sequence and a membrane-anchoring protein coding sequence. The second composition is in the form of intratumoral or paratumoral local administration.

[0011] The anti-tumor mRNA vaccine provided by the present application is composed of two parts of the first composition and the second composition. The first composition is used to induce the body to produce specific antibodies in immune response. The mRNA molecule in the second composition encodes a membrane-anchored antigen, which can be specifically recognized by the antibodies induced by the first composition. The antigen is used to label tumor cells, and the immune system attacks tumor cells in the process of recognizing and combining the antigen, and finally achieves the effect of clearing tumor tissue.

[0012] The first composition in the present application can use the vaccines widely used on the market at present, which can induce the body to produce strong and effective specific antibody response, such as hepatitis B vaccine, new coronavirus vaccine, etc. However, the present application is not limited thereto.

[0013] The use of the already existing antibody response with broad specificity in most individuals to achieve the purpose of anti-tumor can shorten the time cost and win the window period for tumor surgical treatment. The hepatitis B vaccine and the new coronavirus vaccine are currently more mature vaccines, which have the advantages of wide vaccination population, long-lasting and effective antibody response in the body.

[0014] As a preferred, the first antigen and the second antigen are hepatitis B virus S protein, and the second antigen coding sequence is shown as SEQ ID NO. 1.

[0015] As a preferred, the first antigen and the second antigen are novel coronavirus S protein receptor binding domain RBD, and the second antigen coding sequence is shown as SEQ ID NO. 2.

[0016] The mRNA molecule includes three parts, which are signal peptide coding sequence, second antigen coding sequence and membrane anchor protein coding sequence. The second antigen is the antibody specific binding region, in order to ensure the expression of the second antigen on the tumor cell membrane to facilitate the recognition of the immune system, the signal peptide is modified at the N-terminus of the second antigen to guide the transmembrane transfer of the second antigen to the extracellular; the membrane anchor protein is modified at the C-terminus of the second antigen to assist the anchoring of the second antigen on the cell membrane.

[0017] As a preferred, the signal peptide coding sequence is shown as SEQ ID NO. 3.

[0018] As a preferred, the membrane anchor protein coding sequence is shown as SEQ ID NO. 4.

[0019] As a preferred, the mRNA molecule has a capping modification. The mRNA molecule has an m7G-PPPNm structure at the 5' end.

[0020] The second composition in the application is an intratumoral local administration or a peritumoral local administration dosage form. Through the intratumoral or peritumoral local administration, the mRNA molecule is taken up by tumor cells and expressed to achieve the labeling of tumor cells. Studies have shown that this administration method has biological safety. Compared with the secreted antigen, the mRNA molecule provided by the application encodes a membrane-anchored antigen, which is not easy to enter the systemic circulation, and greatly reduces the toxicity.

[0021] As a preferred, the pharmaceutical carrier in the second composition is a lipid nanoparticle (LNP), and the mRNA molecule is encapsulated in the lipid nanoparticle. Studies have shown that the use of a lipid nanoparticle as a mRNA molecule delivery carrier helps to improve the expression efficiency of the mRNA molecule in tumor cells.

[0022] Preferably, the first composition and the second composition exist independently and are not mixed. When applying the anti-tumor mRNA vaccine provided by this invention, the first composition should be administered first, followed by the second composition. If the recipient has previously received a vaccine with the same components as the first antigen, the second composition can be administered directly. The first composition can be administered via intramuscular injection, and the second composition can be administered locally within or near the tumor. Preferably, the interval between the administration of the first and second compositions is at least four days.

[0023] The present invention also provides a method for preparing the aforementioned anti-tumor mRNA vaccine, comprising the following steps:

[0024] (1) An immune adjuvant is mixed with a first active ingredient to prepare a first composition;

[0025] (2) DNA fragments encoding signal peptide, second antigen and membrane anchoring protein were amplified by polymerase chain reaction. Using these fragments as templates, homologous arms were added by polymerase chain reaction to carry out chain extension reaction to obtain fusion genes. The fusion genes were then ligated into an in vitro transcription vector to obtain recombinant plasmids. After enzyme digestion, in vitro transcription linearization templates were obtained. The mRNA molecules were then obtained through in vitro transcription, modification and purification. Finally, the mRNA molecules were loaded onto a pharmaceutical vector to obtain the second composition.

[0026] Both the first and second compositions are individually packaged.

[0027] Preferably, in step (2), the mRNA molecule is encapsulated using lipid nanoparticles (LNPs). The preparation process includes: diluting the mRNA molecule in a citrate-sodium citrate buffer solution with a pH of 4.5, adding a lipid ethanol solution and mixing well, repeatedly blowing and dialysis to obtain lipid nanoparticles loaded with mRNA; the lipid ethanol solution contains SM102, DSPC, CHO-HP and PEG2000 in a molar ratio of 50:10:38.5:1.5.

[0028] The present invention also provides the application of the aforementioned anti-tumor mRNA vaccine in the preparation of tumor therapeutic drugs.

[0029] The tumor is a solid tumor, including but not limited to: melanoma, colon cancer, breast cancer, liver cancer, etc.

[0030] Animal experiments of this invention show that the application of mRNA-LNP vaccine can provide a strong anti-tumor immune response in the corresponding protein vaccine immunization model, and has a significant inhibitory effect on tumor growth in melanoma and colon cancer model mice.

[0031] The beneficial effects of this invention are as follows:

[0032] The application provides a vaccine strategy for anti-tumor attack by means of body-specific antibodies, "old drugs for new use" such as hepatitis B or new crown antigen vaccines, and realizes antigen labeling of tumor cells by intratumoral or peritumoral local administration in the form of mRNA drugs, antigen anchoring on the surface of tumor cells is more conducive to inducing the recognition of the body's immune system, and finally achieves the effect of clearing tumor cells by relying on the body's extensive and strong specific antibodies to induce the body's specific attack. The novel mRNA vaccine provided by the application exhibits strong and effective tumor inhibition effect in multiple solid tumor models, has strong universality, high immunogenicity, wide application range, and can be used as a neoadjuvant therapy to improve the efficacy in combination with clinical surgery and other anti-tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The map of the in vitro transcription template plasmid T7-HBsAg-S.

[0034] Figure 2 The map of the in vitro transcription template plasmid T7-SARS-CoV-2 SRBD.

[0035] Figure 3 The expression map of mRNA-LNP in tumor cells B16F10.

[0036] Figure 4 The protein vaccine induced hepatitis B-specific antibody level in a colon cancer model.

[0037] Figure 5 The average and individual tumor growth curves of mice in a colon cancer model treated with a locally applied mRNA vaccine encoding hepatitis B S protein, wherein the first row of pictures is the average tumor growth curve, and the second row of pictures is the individual tumor growth curve of the control group, the LNP group and the mRNA-LNP group.

[0038] Figure 6 The average and individual tumor growth curves of mice in a colon cancer model treated with a locally applied mRNA vaccine encoding new crown S RBD, wherein the first row of pictures is the average tumor growth curve and the individual tumor weight, and the second row of pictures is the individual tumor growth curve of the control group, the LNP group and the mRNA-LNP group.

[0039] Figure 7 The flow cytometry analysis results of the anti-tumor immune response level of mice in a colon cancer model.

[0040] Figure 8 The biological safety evaluation of LNP and LNP / mRNA administration in a colon cancer model, wherein A is each tissue section, B is the ALT result in serum, and C is the AST result in serum. DETAILED DESCRIPTION

[0041] The application will be further described in connection with the following specific examples. The following examples are only used to illustrate the application, and are not used to limit the application scope. Modifications or replacements of the application methods, steps or conditions without departing from the spirit and essence of the application shall fall within the scope of the application.

[0042] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0043] Example 1, in vitro transcription linear vector construction of HBsAg-S mRNA and SARS-CoV-2 SRBD mRNA

[0044] The mRNA vaccine encodes hepatitis B virus S protein or novel coronavirus S protein receptor binding region RBD, and the recombinant plasmid is obtained by the following steps using conventional molecular biology techniques.

[0045] (1) Obtain the antigen fragment of interest

[0046] Obtain the HBsAg-S and SARS-CoV-2 SRBD nucleotide sequences through NCBI, design F / R primers, and perform polymerase chain amplification with HBV1.3-mer WT replicon plasmid and puc57-SARS-CoV-2 S plasmid as templates, respectively, to obtain the target fragments HBsAg-S and SARS-CoV-2 SRBD;

[0047] HBsAg-S:

[0048] Upstream primer F1: 5'-ATGGAGAACATCACATCAGGA-3';

[0049] Downstream primer R1: 5'-AATGTATACCCAAAGACAAA-3';

[0050] SARS-CoV-2 SRBD:

[0051] Upstream primer F2: 5'-AGAGTCCAACCAACAGAATC-3';

[0052] Downstream primer R2: 5'-GAAATTGACACATTTGTTTT-3;

[0053] The reaction conditions are as follows: pre-denaturation at 94℃ for 5 min, amplification at 94℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 30 s, 35 cycles of reaction, and finally extension at 72℃ for 10 min;

[0054] The HBsAg-S and SARS-CoV-2 SRBD antigen fragments were obtained, with lengths of 678 bp and 669 bp, respectively, and nucleotide sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 8, respectively.

[0055] (2) Fusion gene acquisition

[0056] The membrane-anchoring protein GPI and its transmembrane signal peptide SP nucleotide sequences were obtained through NCBI, and a Myc tag protein was added after the signal peptide SP for subsequent immunofluorescence. A stop codon TAA was added at the end of the membrane-anchoring GPI sequence. The gene synthesis was performed by Beijing Qikexin Biotechnology Co., Ltd. The nucleotide sequences are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively. The signal peptide SP, the target antigen HBsAg-S or SARS-CoV-2 SRBD, and the membrane-anchoring GPI were subjected to a chain extension reaction through homologous arms to obtain the fusion genes SP-HBsAg-S-GPI and SP-SARS-CoV-2 SRBD-GPI, which are hereinafter referred to as H1 and S1, respectively. The fusion genes have lengths of 915 bp and 906 bp, respectively, and nucleotide sequences as shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively.

[0057] (3) Recombinant plasmid construction

[0058] The fusion gene H1 and S1 fragments obtained by concatenating the target antigen and the membrane-anchoring sequence were connected to the linearized template vector Cloning Kit for mRNA Template (Takara, Cat: 6143) through homologous recombination, and were transformed into DH5α competent cells. The correct single colony was picked to obtain the in vitro transcription template plasmids T7-HBsAg-S and T7-SARS-CoV-2 SRBD. The plasmid maps are shown in Figure 1 and Figure 2 The sequences were correct after sequencing. The encoded amino acid sequences are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively.

[0059] (4) Linearized template acquisition

[0060] The obtained recombinant plasmid was digested with restriction enzyme HindIII at 37°C overnight. Then, the linear fragments were separated by 1.5% gel electrophoresis, and the enzyme digestion products were recovered by a DNA recovery kit. Finally, the DNA concentration was determined by Nanodrop, and the linearized template was obtained for subsequent in vitro mRNA transcription.

[0061] Example 2, in vitro mRNA transcription, modification, and purification

[0062] HBsAg-S mRNA and SARS-CoV-2 RBD mRNA products were purified by the following steps using RNA Purification Kit (TransGen Biotech, Cat: ER701-01) respectively.

[0063] (1) The following reagents were added into 200 uL microcentrifuge tubes according to Table 1 respectively on ice.

[0064] Table 1

[0065] Component Amount Final concentration T7 RNA polymerase Mix 2 μL 10 x Reaction buffer 2 μL 1× ATP solution (100 mM) 2 μL 5 mM GTP solution (100 mM) 2 μL 5 mM CTP solution (100 mM) 2 μL 5 mM UTP solution (100 mM) 1.5 μL 3.75 mM N1-Me-Pseudo UTP Solution (100 mM) 0.5 μL 1.25 mM Linearized template x μL (1 μg) 50 ng / μL RNase-Free ddH2O y μL Total 20 μL

[0066] After centrifugation, the above reaction tubes were incubated at 37 °C for 3 hours. Subsequently, to remove the template DNA, 2 uL DNase I and 2 uL DNase buffer (10x) (Vazyme, Cat: DD4104) were added into the above reaction tubes and incubated at 37 °C for 30 minutes.

[0067] (2) The in vitro transcribed HBsAg-S mRNA and SARS-CoV-2 RBD mRNA products were purified by the following steps using RNA Purification Kit (TransGen Biotech, Cat: ER701-01) respectively. RNA Purification Kit (TransGen Biotech, Cat: ER701-01) respectively.

[0068] The in vitro transcription product was supplemented with nuclease-free water to a volume of 100 μΐ^ and transferred to a 1.5 mL centrifuge tube. After vortexing, 350 μΐ^ of BB12 (containing 1% β-mercaptoethanol) was added. After vortexing again, 900 μΐ^ of absolute ethanol was added. The mixture was added to the column in two portions, centrifuged at 12000 x g for 1 min, and the flow-through was discarded. 500 μΐ^ of WB12 was added, centrifuged at 12000 x g for 1 min, and the flow-through was discarded. The above steps were repeated. The residual ethanol was removed by centrifugation at 12000 x g for 2 min. The column was transferred to a new 1.5 mL nuclease-free centrifuge tube, and 30 μΐ^ of nuclease-free water was added to the column, which was incubated at room temperature for 2 min and centrifuged at 12000 x g for 1 min. The concentration and purity of the purified in vitro transcription mRNA product were determined using a Nanodrop, and the quality was determined by 1.5% agarose gel electrophoresis.

[0069] (3) The purified mRNA product was subjected to one-step enzymatic capping. First, the mRNA product was incubated at 65°C for 10 min to completely open the secondary structure at the 5' end. Subsequently, the following reagents were added to 1.5 mL centrifuge tubes on ice according to Table 2.

[0070] Table 2

[0071] Component Amount Final concentration 10 x Capping buffer 2 μL 1× Vaccinia Capping Enzyme (10 U / μL) 1 μL 0.5 U / μL mRNA Cap 2'-O-Methyltransferase (50 U / μL) 1 μL 2.5 U / μL GTP solution (10 mM) 1 μL 0.5 mM SAM solution (4 mM) 1 μL 0.2 mM Denatured Cap0 RNA 10 μg 500 ng / μL RNase-Free ddH2O x μL Total 20 μL

[0072] After thorough mixing and centrifugation, the reaction tube was incubated at 37°C for 1.5 h. Subsequently, the capped mRNA product was purified using the column purification method described above, and the concentration and purity of the capped and purified mRNA were determined using a Nanodrop, and the quality was determined by 1.5% agarose gel electrophoresis.

[0073] Example 3, Preparation of lipid nanoparticles loaded with antigen-encoding mRNA

[0074] (1) Lipid ethanol solution was prepared. SM102 (Cat: 02010), distearoylphosphatidylcholine DSPC (Cat: S01005), high-purity cholesterol CHO-HP (Cat: 57-88-5), and DMG-PEG2000 (Cat: 02005) purchased from AvantiPolar Lipids (Shanghai) Co., Ltd. were dissolved in absolute ethanol, and a lipid ethanol solution was prepared according to the molar percentages in Table 3.

[0075] Table 3

[0076]

[0077]

[0078] (2) The purified mRNA was diluted with citric acid-sodium citrate buffer (pH = 4.5) and stored for later use. The above lipid ethanol solution was mixed with the mRNA dilution solution and blown repeatedly.

[0079] (3) 120 μL of the above mixture was placed in a Slide-A-Lyzer dialysis caddy (10K MWCO, 0.1 mL) in a centrifuge tube filled with PBS solution and placed on a 4°C shaker overnight. The mRNA-loaded lipid nanoparticles were obtained. TM

[0080] (4) 2 x 10 5 mouse melanoma cells B16F10 were prepared using the mRNA-LNP encoding red fluorescent protein mcherry (myc fragment replaced by mcherry fragment) prepared by the above method, and free mRNA and DPBS were added as controls. After 48 hours of cell transfection, 4% paraformaldehyde was used for fixation, and DPBS was used for washing 3 times, and then DAPI was added for 10 minutes for nuclear staining. The red fluorescence was observed by laser scanning confocal microscope (LSCM). The results showed that the red fluorescence could be successfully expressed in tumor cells by using mRNA-LNP, and no fluorescence was observed in the control group, indicating that the mRNA-LNP prepared as above can be used to express specific antigen proteins in tumor cells, as shown in Figure 3

[0081] Example 4: Evaluation of the antitumor effect of peritumoral injection of mRNA-LNP on the corresponding vaccine immunized mice in solid tumor model

[0082] (1) Experimental animals and solid tumor tumor model:

[0083] 6-week-old female C57 mice and Balb / c mice purchased from Hangzhou Patsy Biological Technology Co., Ltd. were used to construct melanoma and colon cancer models. They were purchased one week before the experiment and allowed to drink and eat freely. Mouse melanoma cells B16F10 were cultured in DMEM medium containing 10% fetal bovine serum, 1% double antibody, and mouse colon cancer cells CT26 were cultured in RPMI1640 medium containing 10% fetal bovine serum, 1% double antibody. The culture conditions were 37°C, 5% CO2. According to 5 x 10 5 6 B16F10 cells and 1 x 10

[0084] (2) Protein immunization:

[0085] ​​​The mice were immunized with the corresponding protein vaccine about 6 days after tumor implantation. Recombinant hepatitis B virus surface antigen (Sangon Biotech, Cat: D111147-0001) and SARS-CoV-2 S-RBD prokaryotic protein (Sangon Biotech, Cat: C500304-0001) were dissolved in ddH2O, and immune adjuvant CpG was added to prepare the corresponding hepatitis B and COVID-19 protein vaccines. The corresponding hepatitis B and COVID-19 antigens were subcutaneously inoculated into the left groin of the mice at 20 μg per mouse to induce protein vaccine immunization. The mice were taken blood from the orbit 1 day before protein immunization, 3 days after immunization, and 15 days after immunization, and the serum was obtained by centrifugation and frozen for later use for in vivo antibody titer determination.

[0086] (3) mRNA-LNP administration:

[0087] 8-10 days after tumor implantation, when the tumor volume of the mice grew to 50-100 mm 3 , the mice were randomly divided into 3 groups, 8 mice in each group. The first group was the control group, and normal saline was administered intratumorally or peritumorally; the second group was the LNP group, and lipid nanoparticles without loaded mRNA were administered intratumorally or peritumorally; the third group was the mRNA-LNP group, and mRNA-LNP loaded with coding HBsAg-S (peritumorally) or SARS-CoV-2 RBD (intratumorally) was administered intratumorally or peritumorally as the experimental group. The dose was 10 μg mRNA per mouse. The body weight and tumor volume of the mice in each group were recorded every 2 days, and the formula was tumor volume = 0.5 x length x width 2 . After the mice were sacrificed by cervical dislocation 28 days after tumor implantation, the main organs of the mice in each group, including the heart, liver, spleen, lung, and kidney, were taken for paraffin tissue section, and then H&E staining was used to analyze the pathological conditions of the main organs.

[0088] (4) Detection of antibody levels in protein immunized mice in vivo:

[0089] The antibody titer in mice was determined by indirect method Elisa. Elisa kit (Solarbio, Cat: SEKF105) was used. High-binding ELISA plates were coated with 500 ng / mL of hepatitis B virus surface antigen HBsAg at 4°C overnight. The plates were washed with washing solution three times and blocked with blocking solution for 2 h. Mouse serum was gradient diluted at a ratio of 1:20, 1:50, 1:100, 1:500, 1:1000, 1:2000, 1:5000, 1:10000, 1:50000. The diluted serum was added to the Elisa plate and incubated at 37°C for 2 hours. After washing 3 times with washing solution, 1:5000 diluted HRP anti-IgG (H+L) antibody was incubated at 37°C for 1 hour, the plate was washed three times again, and then incubated with 100 μL color developing solution for 30 minutes. Finally, the reaction was stopped with 0.16M sulfuric acid solution. The absorbance at 450 nm was determined by the enzyme label instrument. The antibody titer was defined as the maximum dilution factor of the serum recognizing the antigen.

[0090] (5) Anti-tumor immune response level detection:

[0091] After the mice were sacrificed, the tumors of the mice in each experimental group were taken, cut into pieces with scissors, digested with tissue digestion solution, and then red into tumor single cell suspension. The cells were labeled with mouse PE-Cy7 anti-CD3 and Pacific blue-anti-CD8a antibodies, and the CD3 + / CD8 + T cells were measured. + / CD4 + / Foxp3 + Treg cell proportion.

[0092] The inguinal lymph nodes of the mice in each group were also taken, digested with tissue digestion solution, and then centrifuged to obtain single cell suspension. The surface markers were labeled with mouse PE-anti-CD11c, FITC-anti-CD80, and APC-anti-CD86 antibodies, and the CD11c + / CD80 + , CD11c + / CD86 + DC proportion was analyzed by flow cytometry.

[0093] The spleens of the mice in each group were also extracted for in vitro culture, and 1×10 7One or more spleen cells per well were added to a 12-well plate, and 20 μg of the above-mentioned antigen protein was added to each well. The plates were incubated at 37°C for 48 hours. At hour 43, brefeldin A (1:1000) was added and incubated for 5 hours. Subsequently, mouse surface and intracellular markers were labeled using mouse PE / Cy7-anti-CD3, Pacific Bule-anti-CD8a, FITC-anti-CD4, PE-anti-IFN-γ, and APC-anti-IL-4 antibodies, respectively. Flow cytometry analysis was then used to determine the CD3 marker. + / CD8 + / IFN-γ + CD3 + / CD4 + / IFN-γ + CD3 + / CD4 + / IL-4 + The proportion of T cells.

[0094] (6) Results Analysis

[0095] The results of ELISA antibody level determination showed that ( Figure 4 High levels of antibodies were produced 3 days after protein immunization, and the antibody titer reached 50,000 15 days after immunization. This indicates that the protein vaccine induces effective specific antibodies in mice.

[0096] Analysis of tumor growth curves in each group of mice showed that, following immunization with hepatitis B or COVID-19 vaccines, subcutaneous or peritumoral injection of mRNA-LNP encoding the antigen significantly inhibited tumor growth in a colon cancer model. Figure 5 , Figure 6 ).

[0097] Flow cytometry data showed that mRNA-LNP increased intratumoral CD3. + / CD4 + CD3 + / CD8 + T cell infiltration ( Figure 7 Simultaneously, it effectively induced the maturation of dendritic cells in lymph nodes and the spleen antigen-specific T cell immune response, and formed a lasting immune memory to protect the body. Figure 7 ).

[0098] HE pathological sections of major mouse organs and serum ALT / AST levels assessed the biosafety of this strategy. Figure 8 ).

[0099] Therefore, this novel mRNA-LNP administration strategy, administered near or within the tumor, can effectively inhibit solid tumors by labeling tumor cells with antigens and relying on the body's specific antibodies in a specific vaccine immunization model.

[0100] The above description is only preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Use of an anti-tumor mRNA vaccine in the preparation of a medicament for the treatment of colon cancer, characterized in that, The anti-tumor mRNA vaccine is composed of a first composition and a second composition, The first composition comprises an immune adjuvant CpG and a first active ingredient containing a first antigen inducing the body immune system to produce antibodies; The second composition is composed of a pharmaceutical carrier and a second active ingredient, wherein the pharmaceutical carrier is a lipid nanoparticle, and the second active ingredient is an mRNA molecule encoding a membrane-anchored antigen recognized by the antibodies, the mRNA molecule comprising, from 5' end to 3' end, a signal peptide coding sequence, a second antigen coding sequence and a membrane-anchoring protein coding sequence; the mRNA molecule is encapsulated in the lipid nanoparticle; The first antigen and the second antigen are hepatitis B virus S proteins, and the hepatitis B virus S protein coding sequence is shown in SEQ ID NO. 1; the signal peptide coding sequence is shown in SEQ ID NO. 3; and the membrane-anchoring protein coding sequence is shown in SEQ ID NO. 4; The first composition and the second composition are independently present and do not mix with each other; the second composition is in an intratumoral or peritumoral local administration dosage form; the anti-tumor mRNA vaccine is applied by first administering the first composition and then administering the second composition; the interval between the administration of the first composition and the second composition is more than 4 days.

2. Use according to claim 1, wherein The mRNA molecule has a capping modification.

3. The use according to claim 1, wherein The preparation method of the anti-tumor mRNA vaccine comprises the following steps: (1) mixing an immune adjuvant with a first active ingredient to prepare a first composition; (2) amplifying DNA fragments encoding signal peptides, second antigens and membrane-anchoring proteins by polymerase chain reaction, using the DNA fragments as templates, performing a chain extension reaction by polymerase chain reaction with homologous arms, obtaining a fusion gene; connecting the fusion gene into an mRNA in vitro transcription vector to obtain a recombinant plasmid, and obtaining an in vitro transcription linear template after enzyme digestion; then preparing the mRNA molecule by in vitro transcription, modification and purification; and finally loading the mRNA molecule on a pharmaceutical carrier to prepare a second composition. The mRNA molecule has a capping modification. The preparation method of the anti-tumor mRNA vaccine comprises the following steps: (1) mixing an immune adjuvant with a first active ingredient to prepare a first composition; (2) amplifying DNA fragments encoding signal peptides, second antigens and membrane-anchoring proteins by polymerase chain reaction, using the DNA fragments as templates, performing a chain extension reaction by polymerase chain reaction with homologous arms, obtaining a fusion gene; connecting the fusion gene into an mRNA in vitro transcription vector to obtain a recombinant plasmid, and obtaining an in vitro transcription linear template after enzyme digestion; then preparing the mRNA molecule by in vitro transcription, modification and purification; and finally loading the mRNA molecule on a pharmaceutical carrier to prepare a second composition.

Images