MRNA (messenger ribonucleic acid) vaccine and application thereof in preparing medicine for treating tumors

Abstract

The invention provides an mRNA (messenger ribonucleic acid) vaccine and application of the mRNA vaccine in preparation of a medicine for treating tumors. The vaccine consists of ionizable lipid, cholesterol, phospholipid, a PEG (Polyethylene Glycol) derivative and mRNA (Messenger Ribonucleic Acid) containing a gene sequence for coding norovirus S protein fusion tumor antigen or a gene sequence for coding tumor antigen fusion ferritin. According to the mRNA vaccine designed by the invention, the antigen expression quantity is increased, a nanocrystallization antigen structure is formed to promote antigen processing and presentation, strong anti-tumor cell immune response is stimulated, and a tumor local microenvironment is effectively remodeled, so that a remarkable tumor inhibition effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of mRNA vaccines, and in particular relates to mRNA vaccines and their application in the preparation of drugs for treating tumors. Background Art

[0002] Cancer has always been a difficult problem in the world. It seriously endangers human health and affects the development of the global economy. Every year, a large amount of money is invested in cancer research. Although certain results have been achieved, there are still many problems that need to be further solved. Cervical cancer is a malignant tumor that is common in women and has a serious impact on women's life safety. According to relevant reports, there are more than 500,000 new cases of cervical cancer and more than 270,000 deaths each year in the world. HPV infection is the main factor for cervical cancer. Studies have shown that 99.7% of cervical cancer patients are infected with HPV. It is worth noting that the average age of cervical cancer has been gradually decreasing in recent years, with a trend of younger age. To date, surgery is still the main treatment for solid tumors. Despite the continuous advancement and updating of surgical techniques and equipment, 30% to 40% of patients still have tumor recurrence or metastasis within 5 years. The main reason is the residual tumor cells at the surgical margin and the temporary immunosuppression during wound healing.

[0003] In addition to surgical resection, existing cancer treatment methods include chemotherapy, radiotherapy, and immunotherapy to remove cancer cells from the body. Radiotherapy and chemotherapy have low targeting and serious side effects, causing great pain to patients; while immunotherapy is the only method in modern science that can completely remove cancer cells. It has great advantages over traditional treatment methods and was named the top of the top ten scientific breakthroughs by Science magazine in 2013, and has broad prospects.

[0004] Tumor vaccines are a type of immunotherapy, the principle of which is to inject tumor antigens into tumor patients in various forms to overcome the immunosuppression caused by the tumor, activate the patient's own immune system, induce the body to produce cellular and humoral immune responses, and thus control or eliminate the tumor. Compared with other immunotherapies, tumor vaccines have incomparable advantages.

[0005] Tumor vaccines can be divided into four categories according to the formulation method, namely tumor cell vaccines, vaccines based on viruses and bacteria, peptide protein vaccines and nucleic acid vaccines. Nucleic acid vaccines are the third generation of vaccines after inactivated vaccines, live attenuated vaccines and genetically engineered recombinant protein vaccines, and are divided into DNA vaccines and RNA vaccines. Compared with other types of tumor vaccines, RNA vaccine molecules do not need to enter the nucleus and can be directly translated into proteins after entering the cytoplasm; RNA vaccines are non-genome-integrating, non-infectious and well-tolerated vaccines; and RNA is transiently expressed in cells, thus allowing repeated vaccination; in addition, the coding units in the RNA open reading frame can be flexibly selected.

[0006] At present, traditional RNA cancer vaccines generally use a mode of encoding and expressing a single antigen or expressing multiple antigen epitopes. However, the anti-tumor specific T cell immune response effect stimulated is limited by factors such as insufficient antigen expression, low antigen processing and presentation efficiency, and short duration of immune response, making it difficult to achieve the best therapeutic effect. Therefore, proposing a new antigen design concept is crucial to obtain mRNA vaccines with higher immune efficacy. Summary of the invention

[0007] In order to develop a potent and long-lasting anti-tumor immune response vaccine, the present invention proposes a new mRNA vaccine and its preparation.

[0008] In the first aspect of the present invention, an mRNA vaccine is provided, which is composed of ionizable lipids, cholesterol, phospholipids, PEG derivatives and mRNA containing a gene sequence encoding a Norovirus S protein fusion tumor antigen or a gene sequence encoding a ferritin fusion tumor antigen.

[0009] In one embodiment of the present invention, the tumor antigen is selected from at least one tumor antigen of colon cancer, liver cancer, pancreatic cancer, breast cancer, bladder cancer, fibrosarcoma, kidney cancer, leukemia, lung cancer, leukemia, lymphoma, melanoma, myeloma, neuroblastoma, plasmacytoma and prostate cancer.

[0010] In a preferred embodiment of the present invention, the tumor antigen is the full-length HPV-E7 protein, and its amino acid sequence is shown in SEQ ID NO:1.

[0011] In another embodiment of the present invention, the amino acid sequence of the Norovirus S protein fused with the HPV-E7 full-length protein is as shown in SEQ ID NO:2.

[0012] In a preferred embodiment of the present invention, the amino acid sequence of the ferritin fusion HPV-E7 full-length protein is as shown in SEQ ID NO:3.

[0013] In another embodiment of the present invention, the mRNA consists of a T7 transcription promoter, a 5'UTR, a gene sequence encoding the full-length HPV-E7 protein, a 3'UTR, and a polyA tail.

[0014] In a specific embodiment of the present invention, the mRNA is composed of a T7 transcription promoter, a 5'UTR derived from a human hemoglobin α2 subunit, a gene sequence encoding a norovirus S protein fusion tumor antigen or a gene sequence encoding a ferritin fusion tumor antigen, a linker, a gene sequence of the tumor antigen, a 3'UTR derived from a non-coding sequence encoding a mitochondrial 12S ribosomal RNA base, and a polyA tail;

[0015] Wherein, the nucleic acid sequence of the 5'UTR is shown in SEQ ID NO:4;

[0016] The nucleic acid sequence of the 3'UTR is shown in SE Q ID NO:5;

[0017] The nucleic acid sequence of the polyA tail is shown in SEQ ID NO:6.

[0018] In a specific embodiment of the present invention, the amino acid sequence encoded by the connecting arm is GSGSGS.

[0019] In a preferred embodiment of the present invention, the mRNA further comprises a polyC tail having a nucleic acid sequence as shown in SEQ ID NO:7.

[0020] The second aspect of the present invention provides a method for preparing the above-mentioned mRNA vaccine, comprising the following steps:

[0021] (1) Plasmid construction:

[0022] Using pVAX1 as a plasmid vector, loading a gene sequence encoding a norovirus S protein fused with a tumor antigen or a gene sequence encoding a tumor antigen fused with ferritin, to construct a recombinant plasmid;

[0023] (2) Plasmid linearization:

[0024] After the recombinant plasmid was digested with restriction endonucleases, the linearized plasmid was purified and recovered;

[0025] (3) In vitro transcription synthesis of mRNA molecules:

[0026] The linearized plasmid of step (2) is mixed with dNTPs, RNA polymerase, cap analog and buffer, and after the reaction, DNase I is added to digest the DNA template to obtain the mRNA of the tumor antigen;

[0027] (4) Combined lipid nanoparticles:

[0028] mRNA encoding tumor antigens is mixed with ionizable lipids, cholesterol, phospholipids, PEG derivatives, and anhydrous ethanol to prepare lipid nanoparticles encapsulating mRNA encoding tumor antigens, thereby obtaining an mRNA vaccine.

[0029] In one embodiment of the present invention, in step (2), the restriction endonuclease is Esp3 I, and the reaction conditions are 37° C. for 1 h to digest the recombinant plasmid.

[0030] In one embodiment of the present invention, in step (3), the reaction conditions for digesting the DNA template with DNase I are 37° C. for 3 hours.

[0031] The third aspect of the present invention also provides an application of the above-mentioned mRNA vaccine in the preparation of drugs for treating tumors.

[0032] In one embodiment of the present invention, the tumor drug further comprises a pharmaceutically acceptable carrier or excipient.

[0033] In another embodiment of the present invention, the mRNA vaccine is a lipid nanoparticle with a particle size of 100 to 200 nm.

[0034] In the fourth aspect of the present invention, in the application of the above-mentioned mRNA vaccine in the treatment of tumors, the amount of nucleic acid injected per dose is 10 μg.

[0035] In one embodiment of the present invention, the mRNA vaccine is administered by intramuscular injection.

[0036] The present invention has successfully developed a new mRNA vaccine design model. The present invention is based on the mRNA form of tumor antigens fused with Norovirus S protein and ferritin. Compared with the simple expression of tumor antigens, the fusion of Norovirus S protein or ferritin gene sequence forms tumor antigens in the form of nanoparticles, and can increase the expression level of tumor antigens, among which the expression level of tumor antigens fused with Norovirus S protein gene sequence is most significantly increased.

[0037] The mRNA vaccine designed in the present invention promotes antigen processing and presentation by increasing antigen expression and forming a nanosized antigen structure, thereby stimulating a powerful anti-tumor cell immune response and effectively reshaping the local tumor microenvironment, thereby achieving a significant tumor inhibitory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0039] Figure 1This is a schematic diagram of the pVAX1-luciferase, pVAX1-E7, pVAX1-E7-ferritin, and pVAX1-Norovirus-E7 plasmid elements in Example 1.

[0040] Figure 2 This is a diagram of the preparation, expression, identification and characterization results of mRNA in Example 1;

[0041] in, Figure 2 A is the agarose gel electrophoresis of plasmid linearization (1 is the original plasmid, 1# is the linearized plasmid), Figure 2 B is agarose gel electrophoresis of RNA bands after in vitro transcription and purification; Figure 2 C is Western blot detection of protein expression of E7 molecules; Figure 2 D is Dotblot detection of protein expression of E7 molecules (1 to 3 are repeated spots); Figure 2 E is the chemiluminescence method to detect the expression of Luciferase at different time points; Figure 2 F is the formation of E7-Fe and Nov-S-E7 nanoparticles observed by transmission electron microscopy (scale bar is 100 nm); Figure 2 G is a graph showing the particle size measurement results of LNP-mRNA; Figure 2 H is the morphology and size of LNP-mRNA observed by transmission electron microscopy (scale bar is 200 nm);

[0042] Data are expressed as mean ± standard deviation (SD). (E) Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0043] Figure 3 Evaluation of the anti-tumor immune effect of the mRNA vaccine based on the E7 antigen in Example 3;

[0044] in, Figure 3 A is a diagram of the vaccine immunization program for mice; Figure 3 B is the tumor growth curve of mice; Figure 3 C is a tumor picture of mice, and the red circle represents the elimination of the tumor; Figure 3 D is a statistical graph of mouse tumor weight; Figure 3 E is a statistical graph of mouse spleen weight; Figure 3 F is the proportion of CD3+CD8+T cells in the spleen analyzed by flow cytometry; Figure 3 G is the proportion of CD3+CD4+T cells in the spleen analyzed by flow cytometry; Figure 3H is the proportion of CD69b+NK1.1+NK cells in the spleen analyzed by flow cytometry; Figure 3 I is the proportion of MDSC (CD11b+Gr1+) in tumors analyzed by flow cytometry; Figure 3 J is the proportion of Treg (CD25+CD4+Foxp3+) in spleen analyzed by flow cytometry; Figure 3 K and Figure 3 L are pictures and statistical quantification of IFN-γ secretion by infiltrating lymphocytes in spleen tissue by ELISPOT analysis; Figure 3 M is the proportion of CD3+CD8+T cells in lymph nodes analyzed by flow cytometry; Figure 3 N and Figure 3 O are pictures and statistical quantification of IFN-γ secretion by infiltrating lymphocytes in spleen tissue by ELISPOT analysis;

[0045] Data are expressed as mean ± standard deviation (SD). Except for (B), which was analyzed by two-way ANOVA, all other data were analyzed by one-way ANOVA with Tukey multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0046] Figure 4 This is an evaluation of the spleen systemic immune effect of E7 and Nov S-E7 antigen mRNA vaccines based on different doses in Example 4;

[0047] in, Figure 4 A is a diagram of the vaccine immunization program for mice; Figure 4 B is the tumor growth curve of mice; Figure 4 C is a tumor picture of mice, and the red circle represents the elimination of the tumor; Figure 4 D is a statistical graph of mouse tumor weight; Figure 4 E is a statistical graph of mouse spleen weight; Figure 4 F is the proportion of CD3+CD8+T cells in the spleen analyzed by flow cytometry; Figure 4 G is the flow cytometry analysis of the proportion of IFN-γ+ cells in CD3+CD8+T cells in the spleen and the representative flow cytometry graph; Figure 4 H is the picture and statistical quantification of IFN-γ secretion by infiltrating lymphocytes in spleen tissue by ELISPOT analysis; Figure 4 I is the proportion of CD3+CD4+T cells in the spleen analyzed by flow cytometry; Figure 4 J is the proportion of CD69b+NK1.1+NK cells in the spleen analyzed by flow cytometry; Figure 4 K is the proportion of MDSC (CD11b+Gr1+) in tumors analyzed by flow cytometry;

[0048] Data are expressed as mean ± standard deviation (SD). Except for (B), which was analyzed by two-way ANOVA, all other data were analyzed by one-way ANOVA with Tukey multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0049] Figure 5 The evaluation of the immune effect of tumor-draining lymph nodes of E7 and Nov S-E7 antigen mRNA vaccines based on different doses in Example 4;

[0050] in, Figure 5 A and 5B are representative flow cytometric images and statistical quantification of CD3+CD4+ T cells in lymph nodes by flow cytometry analysis, respectively; Figure 5 C and 5D are representative flow cytometric images and statistical quantification of CD3+CD8+ T cells in lymph nodes, respectively; Figure 5 E and 5F are representative flow cytometric images and statistical quantification of IFN-γ+ cells in CD3+CD8+ T cells in lymph nodes, respectively; Figure 5 G and 5H are pictures and statistical quantification of IFN-γ secretion by infiltrating lymphocytes in lymph nodes by ELISPOT analysis, respectively;

[0051] Data are expressed as mean ± standard deviation (SD) and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons. The significant differences are as follows: ns, not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. DETAILED DESCRIPTION

[0052] In the following examples, unless otherwise specified, all experimental instruments and raw materials involved are commercially available.

[0053] definition

[0054] Administration refers to the method of delivering the composition to a subject or patient. The administration method can be selected to target (for example, specifically deliver) a specific area or system of the body. For example, administration can be parenteral (for example, subcutaneous, intradermal, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection, and any suitable infusion technique), oral, percutaneous or intradermal, intradermal, rectal, intravaginal, local (for example, by powders, ointments, creams, gels, lotions and / or drops), mucosa, nasal cavity, oral cavity, intestinal, vitreous, tumor, sublingual, intranasal; by intratracheal instillation, bronchial instillation and / or inhalation; as oral spray and / or powder, nasal spray and / or aerosol, and / or by portal vein catheter.

[0055] dNTPs refers to dATP, dCTP, dGTP, and dUTP.

[0056] Cap analogs refer to non-polymerizable dinucleotides that have a cap function because it promotes translation or localization and / or prevents degradation of RNA molecules when incorporated into the 5' end of an RNA molecule. Non-polymerizable means that the cap analog will only be incorporated at the 5' end because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template-dependent RNA polymerase. Cap analogs include, but are not limited to: a chemical structure selected from m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G) or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and its tetraphosphate derivatives).

[0057] Pharmaceutically acceptable means that the pharmaceutical composition can be administered to a subject without causing adverse physiological reactions that prevent the administration of the pharmaceutical composition. For example, "pharmaceutically acceptable excipients" refer to excipients useful in preparing generally safe, non-toxic, and desirable pharmaceutical compositions. Preferably, examples of these excipients or diluents include, but are not limited to, water, saline, Ringer's solution, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, polyalkylene glycols such as polypropylene glycol, triglycerides, 5% human serum albumin, and liposomes and non-aqueous vehicles, such as fixed oils, may also be used.

[0058] Pharmaceutically acceptable carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and delayed absorption agents, etc. that are physiologically compatible. Specific examples may be one or more of water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents such as sugars, polyols (such as mannitol, sorbitol) or sodium chloride are included in the pharmaceutical composition. Of course, pharmaceutically acceptable carriers may also include trace amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, to extend the shelf life or efficacy of the antibody.

[0059] The cell lines used in the present invention are shown in the table below.

[0060] Table 1

[0061] Cell lines name source TC-1 Mouse cervical cancer cells Chinese Academy of Sciences Cell Bank 293T Human embryonic kidney adherent cells Chinese Academy of Sciences Cell Bank 293F Human embryonic kidney suspension cells Chinese Academy of Sciences Cell Bank

[0062] Culture method: TC-1 cells were cultured with RPMI-1640 complete medium containing 10% fetal bovine serum, and 293T cells were cultured with DMEM complete medium containing 10% fetal bovine serum. The complete medium was supplemented with 1% penicillin-streptomycin and cultured in a 37°C constant temperature cell culture incubator containing 5% CO2. 293F cells were cultured in a shaker flask with OPM-293CD05 Medium medium at 220 rpm on a 37°C constant temperature shaker containing 8% CO2.

[0063] The experimental instruments used in the present invention are shown in the table below.

[0064] Table 2

[0065] instrument model source Transmission electron microscopy SU7000 Hitachi Flow cytometry CytoFLEXLX BeckmanCoulter Particle size analyzer ZetasizerNanoZS Malvern Instruments Ltd. Microfluidics INanoTML / Myanna (Shanghai) Instrument Technology Co., Ltd. ELISPOT detector / AIDDiognostika GmbH

[0066] The raw materials used in the present invention are shown in the following table.

[0067] Table 3

[0068]

[0069]

[0070] mRNA cancer vaccines stimulate and regulate immune response effects by delivering mRNA encoding tumor antigens or immunomodulatory molecules. Specifically, mRNA is taken up by dendritic cells (DCs) at the vaccine injection site, and activated DCs reach the draining lymph nodes, where they are translated and processed into antigenic peptides MHC class I or class II complexes and presented to the cell surface through two pathways. One is that the antigens in the cells are broken down into peptides by the protease complex, which are transported to the cell surface through MHC class I molecules and presented to CD8+T cells. The activated CD8+T cells have cytotoxicity and can specifically kill target cells; the other is that the secreted antigens are degraded in the body through endocytosis, and are transported to the cell surface through MHC class II molecules and presented to CD4+T cells. CD4+T cells activate phagocytes through inflammatory cytokines to enhance their ability to kill and engulf target cells, and can also assist B cells in activating antibodies to exert humoral immunity. At the same time, after the extracellular proteins are presented to B cells, B cells can also act as antigen-presenting cells to activate CD4+T cells in turn. In addition, the co-stimulatory signal molecules CD80 / CD86 and OX40 ligand (OX40L) bind to the receptors CD28 and OX40 on T cells respectively as the second signal; the cytokines IFN-I, IL-12, and IL-1 bind to the cytokine receptors on T cells as the third signal, inducing the activation and proliferation of T cells. Finally, the secretion of effector molecules such as IFN-γ, tumor necrosis factor (TNF), perforin, and granzyme exerts a tumor-killing effect.

[0071] The inventors have found through a large number of experiments that by constructing mRNA encoding a specific protein fusion antigen, the mRNA can be translated and expressed in eukaryotic cells to form round nanoparticles, which can improve the immune effect of the prepared mRNA vaccine. In particular, when the specific protein is the Norovirus S protein, compared with the mRNA vaccine that only encodes the antigen, the dosage of the nucleic acid used is only 1 / 10 of that, which is sufficient to obtain a significant tumor suppression effect and produce the same anti-tumor immune effect. This is of great significance to the research and development of mRNA vaccines. By reducing the dosage of nucleic acid injected by the vaccine, not only can the production cost of the vaccine be reduced, but also the potential side effects that may be caused by the mRNA vaccine can be reduced. In addition, the mRNA vaccine is easy to prepare and the finished product can be obtained in just one day.

[0072] Example 1 Preparation and characterization of mRNA vaccines based on different antigens

[0073] Follow the steps below to prepare LNP-mRNA vaccines:

[0074] (1) Plasmid construction: pVAX1 was used as the plasmid vector to directly synthesize Figure 1The gene sequence shown is specifically composed of a T7 transcription promoter, a 5'UTR luciferase gene sequence or an E7 gene sequence derived from human hemoglobin α2 subunit (HBA2), a Norovirus S domain fused HPV-E7 (Norovirus S-E7) gene sequence, or a ferritin fused HPV-E7 (E7-ferritin) gene sequence, a 3'UTR derived from a non-coding sequence of the mitochondrial 12S ribosomal RNA base, and a poly (A) tail and a poly (C) tail. The linearization restriction site is Esp3 I or EcoR I, and the mRNA gene sequence containing only a poly (A) tail can be obtained by selecting the restriction site;

[0075] Among them, the amino acid sequence corresponding to the Luciferase gene sequence or the E7 gene sequence or the Norovirus S-E7 gene sequence or the E7-ferritin gene sequence is shown in the following table;

[0076] Table 4

[0077]

[0078]

[0079] The nucleic acid sequences of each component in the mRNA gene sequence are shown in the following table.

[0080] Table 4

[0081]

[0082] (2) Plasmid linearization: Figure 2 As shown, the plasmid was digested with restriction endonuclease Esp3 I at 37°C for 1h, and 1% agarose gel electrophoresis was used to identify whether the plasmid template was linearized. Lane 1 is the band of the original plasmid, and lane 1# is the linearized plasmid lane after digestion. The results show that the plasmid template is completely linearized after digestion ( Figure 2 A); then use a DNA purification recovery kit to recover the linearized plasmid template and determine the DNA concentration for later use.

[0083] (3) In vitro transcription to synthesize mRNA molecules: Figure 2 As shown, the required substrate, template, enzyme, cap analog and buffer were added according to the instructions of the in vitro transcription kit, and DNase I was added to digest the DNA template after reacting at 37°C for 3 hours; the synthesized mRNA molecules were identified by 1% agarose gel electrophoresis, and lane 1 is the well of the synthesized mRNA molecule, which is displayed as a single band ( Figure 2 B) Then, use RNA purification and recovery kit to recover mRNA molecules and measure their concentration for later use.

[0084] (4) The mRNA obtained above was introduced into human embryonic kidney epithelial cell line (293T) using lipo6000 transfection reagent at a dose of 2 μg. After 24 hours of expression, the cells were collected and Western blot and Dotblot (1, 2, and 3 are repeated wells) were used to quantitatively detect the expression of HPV-E7 protein. The results showed that the expression levels of E7, E7-Fe, and Nov S-E7 proteins increased in sequence ( Figure 2 C&D). At the same time, the cell supernatant expressing the firefly luciferase gene was collected at 24h and 48h, and the chemiluminescence detection showed that the expression level increased with time ( Figure 2 E).

[0085] (5) Using 250 mL of 1x10 6 / mL of 293F suspension cells were transfected to express a large amount of E7-Fe and Nov S-E7 proteins, and the cells were collected by centrifugation at 1200g for 5 minutes. The cells were resuspended with 5mL of 50mmol Tris-Hcl, placed at -80℃ and repeatedly frozen and thawed three times, and centrifuged at 1200g for 5 minutes to collect the supernatant. 30%, 25%, 20%, 15%, 10%, and 5% sucrose gradient solutions of different concentrations were prepared and placed in centrifuge tubes. The collected cell supernatant was spread on the top layer and centrifuged at 40000rpm in an ultracentrifuge for 4h, with an increase speed of 8 and a decrease speed of 5. After centrifugation, obvious stratification can be seen. After taking out the stratification, the formed nanoparticles were observed under an electron microscope, with a scale of 100nm ( Figure 2 F).

[0086] (6) Lipid nanoparticles (LNP) encapsulate mRNA molecules: Figure 2 As shown, the LNP solution was prepared according to Pfizer's LNP formula and ratio, and the mRNA solution and LNP solution were prepared according to the microfluidic packaging instructions of Maianna to prepare LNP-mRNA; the particle size was uniformly distributed at about 100 to 200 nm ( Figure 2 G); electron microscopy showed round nanoparticles, with a scale of 200 nm ( Figure 2 H).

[0087] Example 2

[0088] Basically the same as Example 1, except that the polyC tail is not included.

[0089] Example 3 Evaluation of the anti-tumor immune effect of mRNA vaccine based on E7 antigen

[0090] The antitumor effect of mRNA vaccines based on fusion expression of Norovirus S protein or ferritin nanostructured E7 antigen was tested in tumor-bearing mice.

[0091] Specifically, TC-1 cells were transplanted subcutaneously and the tumor volume grew to about 50-100 mm. 3At the same time, LNP-mRNA vaccine was injected into the hind leg muscle of mice, with a total amount of 10μg. PBS and LNP-LUC (Luciferase) were set as control groups, and immunization was performed 3 times with an interval of 3 days. The immunization process is shown in the figure ( Figure 3 A). The anti-tumor ability was evaluated by measuring the tumor volume of mice. The tumor volume was significantly suppressed after the third injection of LNP-mRNA vaccine, and cure began to appear on the 33rd day ( Figure 3 B). On the 42nd day, the tumor tissues of mice were collected and weighed. The results showed that the LNP-Nov S-E7 (Norovirus S-E7) vaccine group had the best anti-tumor effect, with 90% of the tumors regressing. In addition, the anti-tumor effect of the LNP-E7-Fe (E7-ferritin) vaccine group was also better than that of the LNP-E7 vaccine group ( Figure 3 C&D).

[0092] It is speculated that the fusion expression of Norovirus S protein or ferritin may change the gene sequence, improve the translation efficiency and thus increase the expression of E7 antigen, while changing the structural form of the antigen protein. The formed nanoparticles help cross-presentation of antigens, further promoting the stimulation of anti-tumor immune efficacy.

[0093] The spleen weight of each group was weighed and counted, and it was found that the smaller the tumor, the smaller the spleen ( Figure 3 E). Next, flow cytometry analysis of lymphocytes in the spleen showed that the E7 antigen vaccine injection could significantly increase the proportion of CD8+T cells (CD3+CD8+) and CD4+T cells (CD3+CD4+). Figure 3 F&G), of which the Nov S-E7 vaccine group had the most obvious upregulation, and there were statistical differences compared with other antigen groups. At the same time, we also detected NK cells (CD49b+NK1.1+), of which only the Nov S-E7 vaccine group had statistical differences ( Figure 3 H). In addition, immunosuppressive cells were also analyzed by flow cytometry. The proportions of MDSC (CD11b+Gr1+) and Treg (CD25+CD4+Foxp3+) cells in the E7 antigen vaccine group were downregulated ( Figure 3 I&J).

[0094] E7 protein is a specific tumor antigen for HPV-induced cervical cancer. 49-57 After peptide stimulation of lymphocytes, ELISPOT test was performed to take pictures and count the number of IFN-γ spots. The number of spots in the vaccine group with E7 antigen increased significantly, among which the number of spots in the Nov S-E7 vaccine group increased most significantly, and there was a statistically significant difference ( Figure 3 K&L).

[0095] When the mRNA vaccine is injected into mice intramuscularly, it will first be taken up by the APC cells at the injection site to express the antigen, and then it will be transported to the lymph nodes for antigen expression. The antigen molecules expressed by these two pathways are mainly presented to T cells by the APC antigens in the lymph nodes. Therefore, the flow cytometric analysis of the immune response of lymphocytes to T cells in the lymph nodes showed that the proportion of CD3+CD8+T cells in the Nov S-E7 vaccine group increased the most ( Figure 3 M). Similarly, similar results were verified again in the IFN-γ-ELISPOT test, in which the Nov S-E7 vaccine group had the largest number of spots and the most significant statistical difference ( Figure 3 N&O). In summary, the mRNA vaccine group expressing the Norovirus S protein fused with the E7 antigen has the best anti-tumor effect and the strongest immune effect.

[0096] Example 4 Comparison of the anti-tumor immune effects of different doses of E7 and Nov S-E7 antigen mRNA vaccines

[0097] Further experiments were designed to demonstrate the potent anti-tumor effect of Norovirus S protein fused to express E7 antigen by comparing different vaccination doses.

[0098] Specifically, LNP-E7 was used at doses of 1 μg and 10 μg, and LNP-Nov S-E7 was used at doses of 1 μg, 5 μg, and 10 μg. In the TC-1 tumor-bearing mouse model, when the tumor volume grew to about 50-100 mm 3 Different LNP-mRNA vaccines were injected into the hind leg muscles of mice. PBS was used as the control group, and immunization was performed 3 times with an interval of 3 days. The immunization process is shown in the figure ( Figure 4 A). The tumor volume of mice was measured to evaluate the anti-tumor ability. The results showed that the tumor volume was significantly suppressed after the third injection of LNP-mRNA vaccine, and the cure began on the 27th day. The tumor tissues of mice were collected and weighed on the 36th day. The Nov S-E7-10μg vaccine group had the best anti-tumor effect, with 30% tumor regression; the Nov S-E7-1μg and 5μg vaccine groups had 10% tumor regression; therefore, the higher the antigen dose, the better the tumor inhibition effect.

[0099] However, there was no statistical difference among the three groups of LNP-Nov S-E7 vaccine at 1μg, 5μg, and 10μg, and the 1μg dose of Nov S-E7 vaccine achieved the same anti-tumor effect as the 10μg dose of LNP-E7 vaccine ( Figure 4 B, C&D). Weighing and counting the spleen weights of each group, we found that the smaller the tumor, the smaller the spleen ( Figure 4 E).

[0100] In addition to directly monitoring the effect of inhibiting tumor growth, the following also evaluated the corresponding immunological indicators. Specifically, the CTL epitope E7 of HPV 16 was used. 49-57 After peptide stimulation of lymphocytes in the spleen and lymph nodes, flow cytometry was used to analyze CD3+CD8+T cells. The results showed that the proportion of CD3+CD8+T cells increased after E7 antigen vaccine injection, among which the NovS-E7 vaccine 10μg dose group had the most obvious increase, and the statistical difference was the most significant ( Figure 4 F). The same conclusion was reached by analyzing cells expressing IFN-γ ( Figure 4 G) Obviously, this E7 49-57 The specific immune response was confirmed again in the IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay ( Figure 4 H). Further analysis showed that the E7 antigen vaccine could significantly increase CD4+T cells (CD3+CD4+) after injection, with the Nov S-E7 vaccine 10μg dose group showing the most significant increase and the most significant statistical difference ( Figure 4 I). For NK cells (CD49b+NK1.1+), only the LNP-Nov S-E7 vaccine 10μg dose group had the most obvious increase in proportion, with a statistically significant difference ( Figure 4 J). Further analysis found that the 1μg dose of LNP-Nov S-E7 vaccine achieved the same efficacy as the 10μg dose of LNP-E7 vaccine in stimulating anti-tumor immunity. In addition, immunosuppressive cells were also analyzed by flow cytometry. The proportion of MDSC (CD11b+Gr1+) cells in the E7 antigen vaccine group was downregulated, and except for the E7 antigen 1μg dose vaccine group, the rest showed statistical differences ( Figure 4 K).

[0101] In addition, flow cytometric analysis of lymphocytes in tumor-draining lymph nodes showed that the E7 antigen vaccine injection could significantly increase the proportion of CD4+T cells (CD3+CD4+) and CD8+T cells (CD3+CD8+). Similarly, the Nov S-E7 vaccine 10μg dose group had the most obvious increase, and the statistical difference was the most significant ( Figure 5 A, B, C & D). On this basis, flow cytometry and ELISPOT were performed to analyze the expression of IFN-γ in CD8+T cells, and the conclusions were consistent with the above ( Figure 5 E, F, G & H).

[0102] In summary, it is speculated that the optimized design of tumor antigens fused with Norovirus S protein and expressed with E7 protein has achieved the desired ideal effect. For LNP-mRNA vaccines, such antigen design has increased the expression amount. By evaluating the immune effect through vaccination with different doses of vaccines, it is proved that reducing the antigen dose by 10 times can still achieve a good tumor inhibition effect and produce the same anti-tumor immune effect. Reducing the injection dose of each vaccine means reducing the production cost and reducing the potential side effects that may be caused by mRNA vaccines.

Claims

1. An mRNA vaccine, characterized in that The vaccine is composed of ionizable lipids, cholesterol, phospholipids, PEG derivatives and mRNA containing a gene sequence encoding a Norovirus S protein fused tumor antigen or a gene sequence encoding a ferritin fused tumor antigen.

2. The mRNA vaccine according to claim 1, characterized in that The tumor antigen is selected from at least one tumor antigen of colon cancer, liver cancer, pancreatic cancer, breast cancer, bladder cancer, fibrosarcoma, kidney cancer, leukemia, lung cancer, leukolymphoma, lymphoma, melanoma, myeloma, neuroblastoma, plasmacytoma and prostate cancer.

3. The mRNA vaccine according to claim 1, characterized in that The tumor antigen is the full-length HPV-E7 protein, and its amino acid sequence is shown in SEQ ID NO:

1.

4. The mRNA vaccine according to claim 3, characterized in that The amino acid sequence of the Norovirus S protein fused with the HPV-E7 full-length protein is shown in SEQ ID NO:

2.

5. The mRNA vaccine according to claim 3, characterized in that The amino acid sequence of the ferritin-fused tumor antigen is shown in SEQ ID NO:

3.

6. The mRNA vaccine according to claim 1, characterized in that The mRNA containing a gene sequence encoding a norovirus S protein fused tumor antigen or a gene sequence encoding a tumor antigen fused ferritin is composed of a T7 transcription promoter, a 5'UTR, a gene sequence encoding a norovirus S protein fused tumor antigen or a gene sequence encoding a ferritin fused tumor antigen, a 3'UTR, and a polyA tail; Wherein, the nucleic acid sequence of the 5'UTR is shown in SEQ ID NO:4; The nucleic acid sequence of the 3'UTR is shown in SE Q ID NO:5; The nucleic acid sequence of the polyA tail is shown in SEQ ID NO:

6.

7. The mRNA vaccine according to claim 6, characterized in that The mRNA also includes a polyC tail as shown in the nucleic acid sequence of SEQ ID NO:

7.

8. The mRNA vaccine according to claim 1, characterized in that The preparation method of the mRNA vaccine comprises the following steps: (1) Plasmid construction: Using pVAX1 as a plasmid vector, loading a gene sequence encoding a norovirus S protein fused tumor antigen or a gene sequence encoding a ferritin fused tumor antigen, to construct a recombinant plasmid; (2) Plasmid linearization: After the recombinant plasmid was digested with restriction endonucleases, the linearized plasmid was purified and recovered; (3) In vitro transcription synthesis of mRNA molecules: The linearized plasmid of step (2) is mixed with dNTPs, RNA polymerase, cap analog and buffer, and after the reaction, DNase I is added to digest the DNA template to obtain mRNA encoding the tumor antigen; (4) Combined lipid nanoparticles: mRNA encoding tumor antigens is mixed with ionizable lipids, cholesterol, phospholipids, PEG derivatives, and anhydrous ethanol to prepare lipid nanoparticles encapsulating mRNA encoding tumor antigens and having a particle size of 100 to 200 nm, thereby obtaining an mRNA vaccine.

9. Use of the mRNA vaccine according to any one of claims 1 to 8 in the preparation of drugs for treating tumors.

10. The use according to claim 9, characterized in that The tumor drug also includes a pharmaceutically acceptable carrier or excipient.

Images