Tumor-retina antigen-based mRNA vaccine and application thereof

Abstract

The invention discloses an mRNA vaccine based on a tumor-retina antigen and application thereof, and relates to the field of breast cancer treatment. The method comprises the following steps: firstly, analyzing transcriptome data and immunohistochemical staining results of a human body, and screening out PDE6G protein which is highly expressed only in normal retina tissues and breast cancer cells of the human body; then, the protein is used as an antigen, a corresponding mRNA vaccine is designed and prepared, and the function and the biological safety of the vaccine are verified on the aspects of cells, tissues and organs. In conclusion, a new target spot and a new mRNA vaccine are provided for treatment of the breast cancer, a theoretical basis is laid for diversified treatment of the breast cancer, and meanwhile, the target spot and the mRNA vaccine have huge application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of breast cancer treatment, in particular to a tumor-retina antigen-based mRNA vaccine and application thereof. BACKGROUND

[0002] Breast cancer is one of the important "killers" that endanger the health of women worldwide. According to the global cancer statistics in 2022, the global incidence of female breast cancer ranks second among all cancers, and the mortality rate also ranks fourth. In addition to surgical treatment, breast cancer also includes chemotherapy, endocrine therapy, and targeted therapy, etc. However, drug resistance after treatment is difficult to avoid. Developing new breast cancer treatment methods to achieve precise and effective treatment and make breast cancer patients achieve better survival benefits is still a problem to be solved.

[0003] Immunotherapy is another way to treat breast cancer, which is a treatment method that regulates or enhances the function of the human immune system to enable it to recognize, attack and eliminate disease-related targets such as tumor cells, pathogens or abnormal autoimmune cells. Its core lies in the use of the specificity, memory and adaptability of the immune system to achieve precise intervention on diseases. Immunotherapy marks a paradigm shift in medicine from "directly killing pathogens" to "enabling the human immune system". As an important treatment strategy in immunotherapy, tumor vaccine has been focused on and widely studied due to its advantages such as actively mobilizing the human anti-tumor immune response, highly specific killing of tumor cells, inducing tumor immune memory, and lower biological toxicity. The main mechanism of tumor vaccine is to maximize the exposure of tumor antigens to the immune system, promote the recognition, processing and presentation of tumor antigens by antigen-presenting cells, effectively "train" T cell-mediated anti-tumor immune response, thereby killing tumor cells and endowing the body with immune memory.

[0004] In recent years, with the rapid development of nucleic acid modification technology and lipid nanoparticle (LNP) delivery system, the stability, effectiveness and safety of mRNA vaccine have been greatly improved. For example, mRNA vaccine as a pandemic vaccine has shown excellent preventive effect and good biological safety during the COVID-19 pandemic. On the other hand, the COVID-19 pandemic has also significantly promoted the research and development of mRNA tumor vaccine. As a carrier of tumor vaccine, mRNA has the advantages of higher immunogenicity, better biological safety, better targeting, and easy production. However, the cost of high-throughput sequencing and individualized vaccine design and production is high, and the target antigens of breast cancer are relatively less than other cancers such as lung cancer and melanoma, which greatly limits the application of mRNA vaccine in the clinical treatment of breast cancer.

[0005] Therefore, screening and identifying new breast cancer target antigens and developing a universal breast cancer mRNA vaccine have great scientific significance and clinical value. SUMMARY

[0006] In view of the above problems, the present application identifies a new target protein PDE6G (phosphodiesterase 6G, cGMP-specific, rod, gamma) for treating breast cancer (BRCA), and combines the target antigen with an LNP delivery system to design and prepare an mRNA vaccine encoding the PDE6G protein, i.e. a PDE6G mRNA-LNP vaccine. The present application proves that the mRNA vaccine can effectively inhibit the growth of breast cancer tumors while ensuring biological safety. In summary, the present application provides a new target for treating breast cancer and a corresponding mRNA vaccine, which provides a certain theoretical basis for the treatment of breast cancer.

[0007] There are many immune-privileged areas in the human body, including the blood-brain barrier (BBB), blood-retinal barrier (BRB), blood-testis barrier, placental barrier, and blood-thymus barrier. Antigen proteins expressed only in the immune-privileged area in the human body have extremely low central immune tolerance and strong immune induction capacity because they are not recognized by the human immune system. Based on this, researchers use antigen proteins only in the immune-privileged area as targets for tumor vaccines to ensure treatment effectiveness while minimizing harm to the body itself. Currently, the most studied tumor-testis antigens in the blood-testis barrier are MAGE-A3 and NY-ESO-1 proteins, which have been confirmed in numerous preclinical studies to be breast cancer vaccine targets. At the same time, the retina is also an immune-privileged organ, ensuring that antigen proteins expressed only in the retina have extremely low central immune tolerance. However, research on tumor-retina antigens (CRAs) is still relatively rare, and even fewer CRAs can be used to treat breast cancer.

[0008] Due to the presence of the blood-retinal barrier, some proteins expressed only in the retina have extremely low central immune tolerance and are abnormally highly expressed in tumors, and these antigen molecules can cause a strong immune response in the body Figure 1This type of antigen is named "tumor-retinal antigen (CRAs)," specifically, the tumor-retinal antigen described in this invention is actually breast cancer-retinal antigen. Studies have shown that CRAs are abnormally highly expressed in many tumors, including breast cancer, and that high expression of CRAs in tumor tissue can induce strong cellular and humoral immunity in the human body, thereby inhibiting tumor progression. Simultaneously, studies have also shown that immune induction can enhance the cellular immune response against CRAs in cancer patients. Therefore, screening for suitable CRAs for breast cancer and designing a CRA-based breast cancer mRNA vaccine holds promise for helping breast cancer patients induce a strong, specific anti-tumor immune response, thereby inhibiting breast cancer development and bringing clinical survival benefits to breast cancer patients.

[0009] PDE6G belongs to the phosphodiesterase (PDE) protein family. Phosphodiesterases (PDEs) hydrolyze intracellular second messengers such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby terminating the biochemical processes mediated by these second messengers. cAMP and cGMP play important regulatory roles in cellular activities, and their concentration regulation is mainly determined by the balance between the synthesis of adenylate cyclase and the hydrolytic activity of phosphodiesterases (PDEs). PDEs are a large, multi-gene family, comprising more than 30 species in 11 types, with different substrate specificities, enzyme kinetics, regulatory characteristics, and cellular and subcellular distribution regions. PDEs share similar structures, all containing both regulatory and catalytic functional regions, and the amino acid sequence similarity of the catalytic region among different types of PDEs is over 75%, demonstrating homology among family members. Although PDEs share high homology, different PDEs exhibit different substrate specificities. For example, PDEs 4, 7, and 8 specifically act on cAMP, while PDEs 5, 6, and 9 selectively act on cGMP.

[0010] PDEs are widely distributed in the human body, and their physiological effects involve multiple research areas. In recent years, PDEs have attracted widespread attention from many scholars as new therapeutic targets, becoming a new research hotspot. Among them, clinical studies of selective PDE4 and PDE5 inhibitors have received particular attention. Conversely, PDE6 itself is confined to the retina and is therefore rarely studied as a target.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] On one hand, the present invention provides a tumor-retinal antigen comprising one or more amino acid residue sequences of PDE6G protein, PDE6D protein and recoveryin protein.

[0013] In order to find new targets for the treatment of breast cancer, this invention initially screened PDE6G protein through bioinformatics analysis; then, the expression profiles of PDE6D protein (which belongs to the same protein subfamily as PDE6G protein) and recoverin protein were analyzed, and the expression profiles of these two proteins were compared with those of PDE6G protein. Only PDE6G protein was highly expressed in normal human retinal tissue and breast cancer tissue. Therefore, PDE6G protein was preferred as a tumor-retinal antigen.

[0014] Furthermore, the antigen contains the amino acid residue sequence of the PDE6G protein; the amino acid residue sequence of the human PDE6G protein is shown in SEQ ID NO:1, and the nucleotide sequence is shown in SEQ ID NO:2; the amino acid residue sequence of the mouse PDE6G protein is shown in SEQ ID NO:15.

[0015] It is important to understand that the purpose of screening new targets is to develop vaccines to prevent and treat breast cancer. Vaccine development is a complex and rigorous process, generally divided into several key stages: the exploratory phase (1-5 years), preclinical research (1-2 years), and clinical trials (divided into three phases, usually 5-10 years). Currently, the vaccine described in this invention is still in the preclinical research stage, that is, testing immunogenicity and toxicity in animal models (preferably mice in this invention) and determining the initial dosage and vaccination regimen. Only in clinical trials will the subjects be humans. Therefore, to avoid species rejection and to more accurately reflect the effect of the antigen, this invention has selected mouse PDE6G protein as the antigen. However, once it enters the clinical trial stage, human PDE6G protein will be selected as the antigen.

[0016] Furthermore, the antigen also includes any one or more of the following elements: a signal peptide, a linker arm, and an element that enhances the immune response.

[0017] To improve the effectiveness of mRNA vaccines, antigens are usually specially designed, including codon optimization, introduction of signal peptides and immune response-enhancing elements, and conformational stabilization.

[0018] The signal peptides include, but are not limited to, classic secretory signal peptides (such as immunoglobulin κ light chain signal peptide, bee venom peptide signal peptide, human serum albumin signal peptide, and insulin signal peptide), virus-derived signal peptides (such as HIV gp160 signal peptide and influenza virus hemagglutinin signal peptide), synthetic / engineered signal peptides, membrane protein signal anchoring sequences, and microbial signal peptides.

[0019] Elements that enhance the immune response include, but are not limited to, tetanus toxoid epitopes (such as P2, P16, P30, etc.), diphtheria toxoid epitopes (such as DT-8), universal Th epitopes (such as TT-830-843, MALP-2), MITD sequences, etc.

[0020] In some preferred embodiments, the antigen, in addition to containing the amino acid residue sequence of the PDE6G protein (SEQ ID NO:15), also includes a signal peptide, a linker arm, and an immune response enhancing element. The signal peptide is the HIV gp160 signal peptide (MRVTAPRTLILLLSGALALTETWAGS, SEQ ID NO:13); the linker arm sequence is GGSGGGGSGG (SEQ ID NO:14), GGGSLGGGGSG (SEQ ID NO:16), GGSGG (SEQ ID NO:18), and GGGSLGGGGSG (SEQ ID NO:20); the immune response enhancing element includes tetanus toxoid CD4. + Epitope P2 (QYIKANSKFIGITEL, SEQ ID NO:17), tetanus toxoid CD4+ epitope P16 (MTNSVDDALINSTKIYSYFPSVISKVNQGAQ, SEQ ID NO:19), and MITD sequence (IVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA, SEQ ID NO:21).

[0021] Furthermore, the antigen comprises an amino acid residue sequence that is 60% or more homologous to the sequences shown in SEQ ID NO:12 and / or SEQ ID NO:15 in the sequence listing; wherein, SEQ ID NO:12 comprises the amino acid residue sequence of the signal peptide, the linker arm enhancing immune response element, and the murine PDE6G protein. It is understood that the sequence of the PDE6G protein in SEQ ID NO:12 can be replaced according to the type of test subject (mouse, monkey, or human) to prepare a more targeted antigen.

[0022] Similarly, since the purpose of this invention is to obtain a target and vaccine that can be used to treat human breast cancer, although this invention uses murine PDE6G antigen for preliminary experiments, human PDE6G protein is required for subsequent clinical applications. The homology between murine PDE6G protein and human murine PDE6G protein is 96.55%.

[0023] On the other hand, the present invention provides the use of PDE6G in the preparation of a breast cancer treatment agent, wherein PDE6G comprises PDE6G protein and / or PDE6G gene; the amino acid residue sequence of the PDE6G protein has 60% or more homology with the sequences shown in SEQ ID NO:12 and / or SEQ ID NO:15 in the sequence listing; and the nucleotide sequence of the PDE6G gene has 60% or more homology with the sequence shown in SEQ ID NO:9 in the sequence listing.

[0024] Furthermore, the tumor is generated by breast cancer cells.

[0025] Furthermore, the breast cancer cells include, but are not limited to, 4T1 cells and / or EMT6 cells.

[0026] In this invention, 4T1 and EMT6 cell lines were used to construct a mouse tumor-bearing model. It should be noted that the effectiveness of the PDE6G antigen and the corresponding mRNA vaccine does not depend on the type of cell line used to construct the model, but rather on whether the cell line can express the PDE6G protein.

[0027] On the other hand, the present invention provides the use of PDE6G in the preparation of reagents that stimulate the body to produce an immune response, wherein the PDE6G includes the PDE6G protein and / or the PDE6G gene.

[0028] Furthermore, the amino acid residue sequence of the PDE6G protein has 60% or more homology with the sequences shown in SEQ ID NO:12 and / or SEQ ID NO:15 in the sequence listing; the nucleotide sequence of the PDE6G gene has 60% or more homology with the sequence shown in SEQ ID NO:9 in the sequence listing.

[0029] Furthermore, the immune response includes the activation of immune cells and the production of immune factors.

[0030] Furthermore, the immune cells include, but are not limited to, CD11c. + Cells, CD80 + CD86 + T cells and CD8 + CD3 + T cells; the immune factors include, but are not limited to, IFN-γ factor.

[0031] On the other hand, the present invention provides an mRNA vaccine comprising an RNA sequence capable of encoding one or more of the following proteins: PDE6G protein, PDE6D protein, and recoveryin protein.

[0032] In the early screening stage of this invention, the tumor-retinal antigen PDE6G was obtained, and the mRNA vaccine was designed and synthesized based on this antigen.

[0033] Furthermore, the vaccine contains an RNA sequence capable of encoding the PDE6G protein.

[0034] Similarly, since the purpose of this invention is to obtain a target and vaccine that can be used to treat human breast cancer, although this invention uses an RNA sequence that can encode murine PDE6G protein for preliminary experiments, in subsequent clinical applications, an RNA sequence that can encode human PDE6G protein is required. The RNA sequence that can encode murine PDE6G protein has 90% homology with the RNA sequence that can encode human PDE6G protein.

[0035] Furthermore, the RNA comprises a nucleotide sequence that is 60% or more homologous to the sequence shown in SEQ ID NO:9.

[0036] Furthermore, the RNA also includes an untranslated region and any one or more sequences in poly A.

[0037] In the design of mRNA vaccines, in order to enhance the efficacy of the vaccine, in addition to optimizing the antigen, 5' cap structures, untranslated region (UTR) fragments, polyadenylated tails (poly A), replicons of self-amplifying mRNA (saRNA), and specific RNA motifs (such as PolyU / UC sequences) can be introduced into the RNA.

[0038] The untranslated regions include the 5'UTR and 3'UTR, which are untranslated fragments introduced at the 5' and 3' ends of the antigen-encoding sequence, respectively. The 5'UTR sequence regulates translation initiation, mRNA binding to ribosomes, and mRNA stability. Sources of the 5'UTR sequence include, but are not limited to, β-globin, human hydroxysteroid dehydrogenase (HSD17B4), and tobacco mosaic virus (TMV) Ω sequences. The 3'UTR sequence regulates mRNA stability, subcellular localization, and translation duration. Sources of the 3'UTR sequence include, but are not limited to, β-globin, α-globin, and human growth hormone.

[0039] In some preferred embodiments, a 5' untranslated region (SEQ ID NO: 8) of human β-globulin was introduced at the 5' end of the RNA sequence encoding mouse PDE6G protein (SEQ ID NO: 9), a 3' untranslated region (SEQ ID NO: 10) of human β-globulin was introduced at the 3' end, and a polyA (SEQ ID NO: 11) structure was added after SEQ ID NO: 10 to obtain the final RNA sequence used in the vaccine (SEQ ID NO: 7); in some specific embodiments, the mRNA was also capped at the 5' end and modified with m1Ψ.

[0040] Furthermore, the RNA sequence shares 60% or more homology with the sequence shown in SEQ ID NO:7.

[0041] Furthermore, the total amount of said RNA is 5 μg to 100 mg.

[0042] This invention has demonstrated through multiple experiments that the effect is better when the test subject is a mouse and the total amount of RNA in the vaccine is 10-20 μg, preferably 10 μg; while humans have a larger weight and volume, and the complexity of their immune system is far greater than that of mice, so the amount of RNA used is also much greater than that used in mice.

[0043] Furthermore, the vaccine also comprises liposomes, which contain one or more of ionizable lipids, neutral phospholipids, cholesterol, and polyethylene glycol (PEG) lipids.

[0044] Liposomes encapsulate the RNA, which not only protects the mRNA and promotes cellular uptake of RNA through endosome escape, but also activates innate immunity (such as the TLR pathway) and enhances adaptive immune responses.

[0045] The ionizable lipids are selected from SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA, with SM-102 being preferred; neutral phospholipids can be divided into phosphatidylcholine and phosphatidylethanolamine, the former including DOPC (dioleoylphosphatidylcholine), DPPC (dispalmitoylphosphatidylcholine), and DSPC (distearylphosphatidylcholine), and the latter including DOPE (dioleoylphosphatidylethanolamine), with DSPC being preferred; polyethylene glycol lipids are selected from DMG-PEG2000, DSPE-PEG2000, DTDA-PEG2000, TPGS, or DSPE-PEG-Mannose, with DMG-PEG2000 being preferred.

[0046] Further, the molar ratio of the ionizable lipid, neutral phospholipid, cholesterol, and polyethylene glycol lipid is (25–50):(1–10):(20–38.5):(0.1–1.5). In some specific embodiments, the ionizable lipid is SM102.

[0047] In some specific embodiments, liposomes for encapsulating mRNA were prepared from ionizable lipid SM102, distearate phosphatidylcholine, cholesterol, and DMG-PEG in a molar ratio of 50:10:38.5:1.5.

[0048] On the other hand, the present invention provides the use of RNA for preparing a breast cancer treatment agent, wherein the sequence of said RNA has 60% or more homology with the sequences shown in sequence listing SEQ ID NO:7 and / or SEQ ID NO:9.

[0049] On the other hand, the present invention provides the use of RNA for preparing reagents that stimulate the body to produce an immune response, wherein the sequence of said RNA has 60% or more homology with the sequences shown in sequence listing SEQ ID NO:7 and / or SEQ ID NO:9.

[0050] Furthermore, the immune response includes the activation of immune cells and the production of immune factors.

[0051] The beneficial effects of this invention include:

[0052] 1. A new antigen for breast cancer treatment, namely PDE6G protein, is provided. This protein is highly expressed only in normal human retinal tissue and breast cancer cells. Therefore, using this protein as an antigen can not only enhance the body's recognition and elimination of breast cancer cells, but also reduce the probability of producing an autoimmune response.

[0053] 2. A PDE6G mRNA vaccine (i.e., PDE6G mRNA-LNP vaccine) based on PDE6G antigen design is provided. This vaccine can not only stimulate the body to produce multiple immune responses against PDE6G antigen and inhibit the growth of breast cancer tumors, but also does not cause damage to the retina, important organs in the body and blood environment, and has high biosafety.

[0054] 3. Regardless of the subtype of breast cancer, as long as the tumor can produce the PDE6G antigen, it can be treated with the mRNA vaccine.

[0055] 4. It provides new options and solutions for breast cancer treatment, reducing the suffering of cancer patients during treatment. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0057] Figure 1 Analysis of the feasibility of tumor-retinal antigen as an antigen target for tumor vaccines: (A) Due to the presence of the blood-retinal barrier, tumor-retinal antigen cannot be recognized by the human immune system. Once exposed to the immune system, it can cause a strong specific immune response; (B) After tumor-retinal antigen is recognized, processed and presented by APCs, APCs migrate to secondary lymphoid structures and present it to T cells. T cells recognize the presented antigen and are activated as tumor-retinal antigen-specific cytotoxic T cells; (C) The activated cytotoxic T cells can specifically recognize and kill tumor cells that highly express tumor-retinal antigen; (D) At the same time, on the one hand, since normal human tissue cells do not express tumor-retinal antigen (except for the retina), tumor-retinal antigen-specific cytotoxic T cells do not attack normally growing cells in the human body. On the other hand, the presence of the blood-retinal barrier prevents tumor-retinal antigen-specific cytotoxic T cells from directly attacking retinal cells. This characteristic also ensures the biosafety of tumor-retinal antigen as a tumor vaccine.

[0058] Figure 2Identification of tumor-retinal antigen PDE6G and construction of its expression profile: (A) Potential tumor-retinal antigen genes with expression levels of 95% or higher in the retina identified from normal human tissue RNA-seq data in the FANTOM5 and GTEx databases, respectively; (B) 32 potential tumor-retinal antigens identified by combining FANTOM5 and GTEx databases; (C) Expression of 32 potential tumor-retinal antigen genes in breast cancer in the TCGA database; (D) Immunohistochemical staining (IHC) scores of PDE6G in normal human tissues in the HPA database; (E) IHC staining map of PDE6G protein in the retina in the HPA database; (F) IHC staining of PDE6G protein in the myocardium, liver, spleen, lung, kidney, and colon in the HPA database. Stained images; (G) Expression levels of PDE6G in normal breast tissue and breast cancer in the TCGA database, Wilcoxon rank-sum test; (H) Expression levels of PDE6G in paired normal breast tissue and breast cancer tissue in the TCGA database, paired-samples t-test; (I) Expression levels of PDE6G protein in four breast cancer subtypes (LumA, LumB, Basal, and Her2) in the TCGA database, analyzed for significant differences using one-way ANOVA; (J) IHC staining results of PDE6G protein in normal breast tissue (n=3) and breast cancer tissue (n=12) in the HPA database, BRCA represents breast cancer; (K) IHC staining results of PDE6G protein in normal breast tissue (n=8) and breast cancer tissue (n=8) from Zhejiang Cancer Hospital; (C, GI) Gene transcription levels are presented in log2(TMP+1) format; ***P<0.001.

[0059] Figure 3 Construction of PDE6D gene expression profiles; (A) Expression patterns of PDE6D gene in various tissues of normal human subjects constructed based on the FANTOM5 database; (B) Expression patterns of PDE6D gene in various tissues of normal human subjects constructed based on the GTEx database; (C) Expression patterns of PDE6D gene in various cells of normal human subjects constructed based on the HPA database; (D) Expression patterns of PDE6D protein in various tissues of normal human subjects constructed based on the HPA database.

[0060] Figure 4Construction of RCRRN gene expression map; (A) Expression pattern of RCRRN gene in various tissues of normal human based on FANTOM5 database; (B) Expression pattern of RCRRN gene in various tissues of normal human based on GTEx database; (C) Expression of RCRRN gene in breast cancer tissue and normal tissue; (D) IHC staining map of breast cancer tissue (n=12) with recoverin protein in HPA database, where BRCA represents breast cancer;

[0061] Figure 5 Correlation analysis of tumor-retinal antigen PDE6G and tumor infiltration immunity; (A) Enrichment scores of 16 immune cells and 13 immune pathways in breast cancer samples with different PDE6G expression levels in the TCGA database calculated based on the ssGSEA algorithm. The median value of log2(TMP+1) (1.680052) was used to define the high and low expression of PDE6G gene in breast cancer, and the differences were verified by Wilcoxon rank-sum test; (B) Pearson correlation analysis of PDE6G gene expression level and scores of 16 immune cells and 13 immune pathways in breast cancer samples in the TCGA database; (C) Correlation analysis of PDE6G gene expression level and CD8 expression level in databases such as TIMER, CIBERSORTx, XCELL, EPIC, and QUANTISEQ. + T cells, CD4 + Pearson correlation analysis of T cells, NK cells, DC cells, macrophages, and B cells; (D) GO pathway clustering analysis of differentially expressed genes in breast cancer with high PDE6G gene expression, using breast cancer with low PDE6G gene expression as the control group, based on differential analysis using the DESeq2 algorithm and the GSEA algorithm; differentially expressed genes in breast cancer with high PDE6G gene expression were enriched in the peptide antigen binding pathway (E) and the MHC protein binding pathway (F); *P<0.05, **P<0.01, ***P<0.001.

[0062] Figure 6Construction and identification of PDE6G mRNA-LNP vaccine; (A) Schematic diagram of mRNA-LNP vaccine construction process; (B) Expression of PDE6G protein in mouse DC2.4 cells after treatment with 0, 0.25, 0.5, 1.0, 2.0, and 4.0 μg / mL Luc mRNA-LNP or PDE6G mRNA-LNP vaccine (n=3), the theoretical size of PDE6G protein is 9.5 kDa; (C) Expression of luciferase at different time points after injection of 10 μg Luc mRNA-LNP vaccine into mice (n=3); (D) Expression of luciferase in mice (n=3) 24 hours after injection of Luc mRNA-LNP vaccine at concentration gradients; (E) Expression of luciferase in inguinal lymph nodes and spleen in mice (n=3) 24 hours after injection of Luc mRNA-LNP vaccine at concentration gradients; (F) Expression of Luc mRNA-LNP vaccine at concentration gradients. Expression of luciferase in the heart, liver, lungs, and kidneys of mice (n=3) 24 hours after injection of mRNA-LNP vaccine; quantitative analysis of fluorescence expression in (G) mice (n=3) and various tissues and organs.

[0063] Figure 7 PDE6G mRNA-LNP vaccine can induce a specific immune response; (A) Mouse immunization flowchart, tumor-bearing mice were immunized with equal doses on days 0, 5, and 10, and the spleens of the mice were taken for flow cytometry analysis on day 18; (B) Flow cytometry analysis results of spleen cells of mice (n=4) 18 days after immunization with PDE6G mRNA-LNP vaccine. From the columns, the flow cytometry scatter plots in the left column are the PBS group, the middle column is the Luc mRNA-LNP group, and the right column is the PDE6G mRNA-LNP group; from the rows, the first row is CD11c + Cells / CD45 + The cell ratio, the second row is CD80 + CD86 + Cells / CD11c - The ratio of cells, the third row is CD8 + CD3 + Cells / CD45 + Cell ratio; (C) Secretion of IFN-γ factor in spleen cells of mice immunized with vaccine by different concentrations of PDE6G peptide library (n=4). The number in the upper left corner of the ELISpot detection result graph above is the result of quantifying the number of secreted factors; The results are presented as mean ± standard deviation, and the Tucky test was used after ANOVA test; *P<0.05, **P<0.01, ***P<0.001.

[0064] Figure 8(A) Antitumor effect and immune induction ability of PDE6G mRNA-LNP vaccine; (B) Mouse immunization flowchart: Mice were injected with different cell lines on day 0, and then tumor-bearing mice were immunized with the same dose of vaccine on days 5, 10 and 15, and the experiment was terminated on day 23; (C) Expression of PDE6G protein in mouse breast cancer EMT-6 and 4T1 cell lines based on Western blot assay, with mouse eyeballs as positive controls; (D) Tumor volume changes in female BALB / c mice (n=6) after inoculation with mouse 4T1 breast cancer cells; (E) Tumor weight of different groups of tumor-bearing mice on day 23 (D) and tumor images (E), n=6; (F) Survival time of tumor-bearing mice treated with PDE6G mRNA-LNP vaccine; (G) CD3 in mouse tumor tissue 18 days after vaccine immunization. + CD4 + and CD8 + Immunofluorescence assay results of T cells; CD8 based on SlideViewer software. + CD3 + T cells (H) and CD4 + CD3 + Quantitative results of T cells (I), n=4; (J) Identification of EMT-6 cell lines overexpressing PDE6G protein at the protein level; (K) Tumor volume changes in female BALB / c mice (n=6) after inoculation with EMT-6 breast cancer cells that do not express PDE6G protein; (L) Tumor volume changes in female BALB / c mice (n=6) after inoculation with EMT-6 breast cancer cells that express PDE6G protein modified by lentivirus; Results are presented as mean ± standard deviation, and the Tucky test was used after ANOVA; *P<0.05, **P<0.01, ***P<0.001.

[0065] Figure 9 Safety assessment of PDE6G mRNA-LNP vaccine; (A) Changes in body weight of mice immunized with PDE6G mRNA-LNP vaccine, n=6; (B) HE staining of heart, liver, spleen, lung and kidney of mice immunized with PDE6G mRNA-LNP vaccine, n=6; (C) Fundus observation and HE staining of retina of mice immunized with PDE6G mRNA-LNP vaccine, n=6; Detection of white blood cell (D), red blood cell (E), platelet (F) levels and serum alanine aminotransferase (ALT) (G), aspartate aminotransferase (AST) (H), urea (I) and creatinine (CR) (J) levels in mice immunized with PDE6G mRNA-LNP vaccine, n=6; (DJ) Results are presented as mean ± standard deviation. ANOVA and Tucky test were performed, n=6; *P<0.05. Detailed Implementation

[0066] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, each experimental group has 3 replicates. Unless otherwise specified, the materials and reagents used are commercially available.

[0068] Example 1: Screening for tumor-retinal antigens

[0069] Although previous studies have identified some tumor-retinal antigens with good immunogenicity, there are still very few tumor-retinal antigens suitable for breast cancer treatment. To obtain new breast cancer targets that can be used for clinical treatment, this embodiment provides a screening process for tumor-retinal antigens highly expressed in breast cancer cells, with the following specific steps:

[0070] 1.1 Download transcriptome sequencing data of normal human tissues from The Genotype-Tissue Expression (GTEx) database and The Functional Annotation of the MAMMALIAN GENOME (FAMTOM) database from THE HUMAN PEOTEIN ATLAS online database (HPA, website: https: / / www.proteinatlas.org / ). The GTEx database contains data of 37 types of normal human tissues, while the FAMTOM database contains data of 60 types of normal human tissues.

[0071] 1.2 Theoretically, tumor-retinal antigens are expressed primarily or only in the retina. In this embodiment, the absolute transcriptional level of a gene in each tissue is converted into a proportional fraction (i.e., the transcriptional level in one tissue / the sum of the levels in all tissues × 100%), and a threshold of 95% is used to screen for genes specifically expressed in the retina. Based on the above screening rules, 50 and 60 retina-biased genes were selected from the GTEx and FAMTOM databases, respectively. Figure 2 A).

[0072] 1.3 The intersection of the retinal biased expression genes screened from two different databases was obtained, yielding 32 intersection genes. Figure 2 B).

[0073] 1.4 Transcriptome (RNA-seq) data from 1,118 breast cancer cases were downloaded from The Cancer Genome Atlas Program online database (TCGA, website: https: / / portal.gdc.cancer.gov / ). The 32 overlapping genes were mapped to the absolute transcriptional levels of breast cancer cell transcriptomes in the TCGA database. It was found that the PDE6G gene (full name: phosphodiesterase 6G, cGMP-specific, rod, gamma) (protein sequence shown in SEQ ID NO:1, nucleotide sequence shown in SEQ ID NO:2) had the highest absolute transcriptional level among the 32 retina-biased expression genes in breast cancer. Figure 2 C) PDE6G protein is preliminarily considered a potential tumor-retinal antigen. PDE6 protein is a core enzyme in visual signal transduction, specifically expressed in rod and cone cells of the retina. Its main function is to hydrolyze cGMP, thereby regulating the cGMP concentration in photoreceptor cells, closing cGMP-gated cation channels, and triggering hyperpolarized electrical signals to be transmitted to the brain.

[0074] 1.5 To further investigate whether PDE6G protein is expressed only in the retina of normal humans, this embodiment extracted immunohistochemical staining (IHC) images of PDE6G protein from 45 normal human tissues from the HPA database. IHC images primarily observed staining intensity (negative, weak, moderate, or strong), the number of stained cells (<25%, 25-75%, or >75%), and the stained area (nucleus and / or cytoplasm / membrane). In IHC, no specific staining, only slight background staining, or no signal was considered as no protein expression; conversely, the darker the color of the stained tissue and the larger the area of ​​the dark region, the higher the expression level of the protein. The results showed that PDE6G protein is highly expressed only in retinal cells, while PDE6G protein was not detected in other normal human tissues. Figure 2 D~ Figure 2 F).

[0075] 1.6 To further explore the possibility of PDE6G protein as a therapeutic target for breast cancer, this embodiment retrieved data from the TCGA and HPA databases that reflect the expression levels of the PDE6G gene / protein in normal breast tissue and breast cancer tissue, respectively. Figure 2 G~ Figure 2 J). The results showed that, compared with normal breast tissue, the transcription level of the PDE6G gene in breast cancer tissue was significantly lower. Figure 2 G) and translation level Figure 2Both J) showed abnormally high expression, with PDE6G gene expression being higher in HER2-positive and triple-negative breast cancer compared to Luminal breast cancer. Figure 2 (I) This may be because, compared to Luminal breast cancer, Her2 and triple-negative breast cancer exhibit more pronounced antigen expression and immune cell infiltration, suggesting that PDE6G antigen is a more suitable target for the treatment of Her2-positive and triple-negative breast cancer. PDE6G expression was not detected in normal breast tissue (n=3) in the HPA database, while in breast cancer tissue (n=12), 5 cases showed positive IHC staining for PDE6G protein. Figure 2 J). In addition, this embodiment also performed IHC staining on 8 cases of breast cancer and 8 cases of normal breast tissue from the Zhejiang Cancer Hospital Biobank. Among them, 5 cases of breast cancer tissue showed positive expression of PDE6G protein (n=8), while the corresponding normal breast tissues all showed negative expression of PDE6G protein (n=8). Figure 2 K).

[0076] The results above reveal that the tumor-retinal antigen PDE6G is expressed only in the immune-exempt region of the retina in normal human tissues, but is expressed at a relatively abnormally high level in breast cancer, suggesting that PDE6G has extremely low central immune tolerance and has great potential to become an antigen target for breast cancer tumor vaccines.

[0077] To investigate whether PDE6G is more suitable for breast cancer vaccine design than other antigen targets and whether there are other better targets, this embodiment analyzes other potential tumor-retinal antigens.

[0078] First, this embodiment analyzed the expression profiles of other members of the PDE6 subfamily, the PDE6D gene (protein sequence shown in SEQ ID NO:3, nucleotide sequence shown in SEQ ID NO:4). This was combined with the FANTOM5 database ( Figure 3 A) and GTEx database ( Figure 3 Transcriptome data from normal tissues (B) show that the PDE6D gene is constitutively expressed, meaning it is not only highly expressed in the retina but also detectable in other normal tissues. Furthermore, single-cell transcriptome data from the HPA database ( Figure 3 C) also confirms that the PDE6D gene is not only expressed in the photoreceptor cells of the retina. Furthermore, IHC staining data from normal tissues in the HPA database ( Figure 3D) further confirmed that the PDE6D protein exhibits low-to-high expression levels in normal tissues outside the retina. These results indicate that although the human PDE6D protein is homologous to the PDE6G protein, its expression pattern differs. Specifically, the PDE6D gene / protein shows high expression levels in normal tissues outside the retina. Therefore, theoretically, the PDE6D protein possesses extremely high central immune tolerance and relatively weak immune induction ability, making it unsuitable as an antigen target for breast cancer vaccines. This demonstrates that not all PDE6G homologous proteins can serve as targets for breast cancer vaccines; the expression pattern of the PDE6 protein largely determines its suitability as a target.

[0079] Next, the differences between PDE6G protein and recoveryin (gene name: RCRRN) (protein sequence see SEQ ID NO:5, nucleotide sequence see SEQ ID NO:6), a tumor-retinal target that has been extensively studied, were further compared. Recoveryin is a calcium-sensing protein, named for its function in promoting the "recovery" response of photoreceptor cells during light adaptation. Specifically, it regulates the termination and adaptation of light signal transduction through a calcium-dependent mechanism to ensure a rapid response of the visual system to changes in light intensity. Current research indicates that, on the one hand, Recoveryin is mainly expressed in rod and cone cells of the retina and is a key regulator of the visual signal transduction pathway; on the other hand, its expression level is also high in lung cancer and clear cell renal cell carcinoma.

[0080] Although the FANTOM5 database ( Figure 4 A) and GTEx database ( Figure 4 Transcriptome data from normal tissues (B) indicate that, under normal conditions, the recoverin gene is specifically expressed in the retina, a result consistent with previous findings. However, transcriptome data from the TCGA database show that the expression level of the RCRRN gene in breast cancer tissue is not higher than that in normal breast tissue (no statistical difference). Figure 4 C), and simultaneously, IHC staining data of breast cancer tissue in the HPA database ( Figure 4 D) This further confirms that the expression of recoverin protein is negative in breast cancer tissue. These results indicate that although recoverin protein may have low immune tolerance, its expression in breast cancer is not upregulated, and may even show a downregulation trend, suggesting that recoverin is not the most suitable antigen target for breast cancer vaccines.

[0081] In summary, whether compared with the PDE6D protein of the same family or with the tumor-retinal antigen target recoverin, which has been extensively studied, the PDE6G protein is a more suitable antigen target for breast cancer vaccines. Therefore, this antigen was used as the research object, and a corresponding mRNA vaccine was designed and its various properties were verified.

[0082] Example 2: Tumor-retinal antigen PDE6G is positively correlated with tumor infiltration immunity.

[0083] Ideal tumor vaccine antigens can induce a strong anti-tumor immune response in the body, which is significantly related to tumor immunity. Therefore, this embodiment explores the correlation between breast cancer-retinal antigen PDE6G and tumor-infiltrating immunity. Tumor-infiltrating immunity refers to the dynamic process by which immune cells infiltrate into tumor tissue and interact with tumor cells and the tumor microenvironment; this process directly determines the immune recognition, clearance, or escape of the tumor and is the core mechanism of anti-tumor immunotherapy. The degree and composition of tumor-infiltrating immunity (such as T cells, macrophages, NK cells, etc.) are closely related to patient prognosis and treatment response.

[0084] First, ssGSEA analysis was used to analyze breast cancer-related transcriptome data from the TCGA database (as described in Example 1). Expression levels log2(TMP+1) greater than or equal to 1.680052 (median) were defined as high expression, and vice versa. It was found that breast cancer samples with high PDE6G gene expression had higher enrichment scores for tumor-infiltrating immune cells and immune pathways compared to those with low expression. Figure 5 A), and the expression level of the PDE6G gene showed a significant positive correlation with the tumor-infiltrating immune cell enrichment score and the immune pathway enrichment score. Figure 5 B). To further verify the correlation between PDE6G gene expression level and tumor immunity, this embodiment analyzed the correlation between tumor-infiltrating lymphocytes and APCs scores and PDE6G expression levels based on multiple databases such as TIMER, CIBERSORTx, and XCELL. The results showed that ( Figure 5C) The higher the expression level of the PDE6G gene, the higher the enrichment score of infiltrating lymphocytes and APCs, indicating a significant positive correlation. Among them, the expression level of the PDE6G gene showed the strongest positive correlation with the number of infiltrating, activated lymphocytes and APCs in the HER2 subtype. This result is consistent with the results in Example 1 (i.e., the expression level of PDE6G protein is higher in HER2-positive and triple-negative breast cancer), suggesting that the effect of using PDE6G antigen to treat HER2-type breast cancer may be optimal. Furthermore, the DESeq2 algorithm was used to identify differentially expressed genes with significantly upregulated PDE6G expression levels (Fold Change > 2) in breast cancer samples with low PDE6G expression compared to those with high PDE6G expression. GO pathway enrichment analysis was then performed on these genes using the GSEA algorithm. The results showed that these differentially expressed genes were mainly enriched in immune-related pathways, notably including two pathways: "peptide antigen binding" (Figure E) and "MHC protein binding" (Figure F). This suggests that PDE6G protein expressed in breast cancer tissue may bind to MHC molecules in breast cancer tumor cells as an antigenic peptide, thereby being presented to the tumor cell surface and facilitating T cell recognition. In conclusion, it is preliminarily believed that using PDE6G protein as an antigen for immunization can induce a strong immune response, thereby achieving better prevention and treatment of breast cancer.

[0085] Example 3: Synthesis of PDE6G mRNA-LNP vaccine and testing of vaccine performance.

[0086] Based on the tumor-retinal antigen PDE6G, this embodiment designed and synthesized an mRNA (SEQ ID NO:7) encoding the PDE6G antigen, and encapsulated it in an LNP delivery system to construct a PDE6G mRNA-LNP vaccine. The flowchart for vaccine design and preparation is shown below. Figure 6 A. The specific experimental method is as follows:

[0087] 3.1 Design of mRNA

[0088] It is important to understand that the mechanism of action of mRNA vaccines is to introduce mRNA encoding specific antigens of pathogens (such as viruses) into human cells, utilize the host cell's translation system to synthesize antigen proteins, and then stimulate an immune response (including humoral immunity and cellular immunity). Therefore, in order to ensure the safety, efficacy and stability of mRNA vaccines, it is necessary to optimize the core structure of mRNA (such as the 5' cap structure, the 5' and 3' untranslated regions, the antigen coding region and the poly A tail), modification methods, etc. This invention focuses on the optimization of the antigen coding region. The sequence shown in SEQ ID NO:7 is the full-length mRNA sequence, which includes the 5' untranslated region of human β-globin (Humanβ-globin 5'-UTR, SEQ ID NO:8), the antigen coding sequence (SEQ ID NO:9), the 3' untranslated region of human β-globin (Repeat Humanβ-globin 3'-UTR, SEQ ID NO:10), and the poly A sequence (SEQ ID NO:11). The main functions of the 5' and 3' untranslated regions of the β-globin are to enhance the stability, translation efficiency, and intracellular expression level of the mRNA. The antigen coding sequence (SEQ ID NO:9) encodes the antigen amino acid residue sequence shown in SEQ ID NO:12 (i.e., the antigen coding region mentioned above). Furthermore, the antigen amino acid residue sequence (SEQ ID NO:12) comprises a signal peptide (SEQ ID NO:13), a linker arm (SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20), a murine PDE6G protein sequence (SEQ ID NO:15), and tetanus toxoid CD4. + Epitope P2 (tetanus toxoid CD4) + epitopes P2, SEQ ID NO:17), tetanus toxoid CD4 + Epitope P16 (tetanus toxoid CD4) + The functions of the epitopes P16 (SEQ ID NO:19) and the MITD sequence (MHC-ITargeting Domain, SEQ ID NO:21) which increases MHC presentation efficiency are shown in Table 1. It should be noted that in this embodiment, the antigen amino acid residue sequence (SEQ ID NO:12) was designed first, and then the antigen coding sequence (SEQ ID NO:9) was obtained through codon optimization; based on the antigen coding sequence, elements stabilizing the mRNA were introduced to obtain the full-length mRNA sequence (SEQ ID NO:7).

[0089] For mRNA design, please refer to the article Sahin, U., Oehm, P., Derhovanessian, E. et al. An RNAvaccine drives immunity in checkpoint-inhibitor-treated melanoma. Nature 585, 107-112 (2020). (https: / / doi.org / 10.1038 / s41586-020-2537-9).

[0090] Table 1. Components and functions of the antigen amino acid residue sequence

[0091]

[0092] 3.2 mRNA preparation

[0093] mRNA synthesis was performed using in vitro transcription, and the specific experimental steps are as follows:

[0094] The DNA sequence corresponding to the above mRNA (SEQ ID NO:7) was constructed into the pET28a vector (GenScript Biotechnology Co., Ltd.). The constructed vector was digested and purified using BspQ-I enzyme (NEB, R0712L) to obtain the DNA template. The mRNA sequence (SEQ ID NO:7) was obtained using T7 RNA polymerase and an in vitro transcription kit (Vazyme, Cat#TR101-01). During the reaction, UTP was replaced by N1-methylpseudouracil (m1Ψ, Synthgene), which enhanced the stability of the mRNA, reduced immunogenicity, and improved translation efficiency. Capping was performed using commercial reagents, i.e., the m7G5'ppp5'G2'-O-Met-capped (cap1) IVT mRNA was synthesized from vaccinia virus guananylytransferase and 2'-O-methyltransferase (Novoprotein). RNA was purified using RNA cleaning beads; the purified mRNA was then subjected to agarose gel electrophoresis and validated using an Agilent 5400 bioanalyzer to ensure a purity of 95%, and the mRNA concentration was detected using a NanoDrop instrument. The purified IVT mRNA was stored at -20°C.

[0095] For the preparation method of mRNA, please refer to the relevant content in the patent "An engineered mRNA that improves the safety of IL-12 treatment and its preparation method and application" (application number: 202410467277.9).

[0096] 3.3 Preparation of mRNA-LNP

[0097] For the preparation method of mRNA-LNP, please refer to the relevant contents of the patent "Method for generating multifunctional chimeric antigen receptor macrophages in situ and its application in immunotherapy" (application number: 202410159815.8) and the patent "mRNA targeted delivery system based on nucleic acid aptamers for spleen and its subcells" (international application number: PCT / CN2023 / 132420).

[0098] Specifically, ionizable lipids SM102, distearate phosphatidylcholine (DSPC), cholesterol, and polyethylene glycol lipids were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to prepare a lipid organic phase. This lipid mixture was initially mixed with 20 mM sodium citrate-hydrochloric acid buffer (pH=4) containing mRNA (N / P=6) at a volume ratio of 1 (organic phase):2 (aqueous phase). Then, the mRNA aqueous phase and lipid organic phase were rapidly mixed at a flow rate of 2:1 using microfluidic preparation methods to obtain a crude mRNA-LNP solution. The mRNA-LNP solution was concentrated using an Amicon Ultra centrifuge filter (EMD Millipore), and the concentrate was then filtered through a 0.22 μm membrane. The filtrate was the prepared PDE6G. The mRNA-LNP vaccine was used for subsequent experiments; particle size and RNA encapsulation efficiency were evaluated. All formulations had particle sizes between 80 and 100 nm, with an average particle size of 85.06 nm, encapsulation efficiencies exceeding 90%, and a surface zeta potential of +6.47 mV. These parameters indicate that the PDE6G mRNA-LNP particles are uniform in size and have good dispersibility. After aliquoting, the vaccine was stored at 4°C.

[0099] To assess the efficiency of intracellular translation and endocytosis of the PDE6G mRNA-LNP vaccine by APCs (Antigen-Presenting Cells), this embodiment co-incubated a prepared vaccine containing mRNA at concentration gradients of 0-4.0 μg / mL (prepared as described above) with immortalized mouse dendritic cells (DC2.4 cells) derived from C57BL / 6 mouse bone marrow for 24 hours. This allowed the DC2.4 cells to express PDE6G protein after endocytosis of mRNA-LNP particles. Subsequently, total protein was extracted from the DC2.4 cells and analyzed using Western spectroscopy. The expression level of PDE6G protein was detected by blot experiment as follows: DC2.4 cells were lysed on ice using lysis buffer (Beyotime, p0013) and a protease inhibitor mixture (Roche, 5056489001) was added, and lysis was performed for 10 minutes; protein was quantified using a BSA protein quantification kit (Beyotime, p0010); 10-20 μg of crude protein solution was loaded onto a 10%-12% gradient SDS-PAGE gel, and the protein was transferred from the gel to methanol-activated PDE6G at 80V. On a VDF membrane, the transfer time was 2 hours; the PVDF membrane was blocked with 5% skim milk for 1 hour at room temperature; the primary antibodies used for incubation were anti-PDE6G antibody (Abmart, MG163736S; dilution 1:1,000) and anti-GAPDH antibody (CST, 5174S; dilution 1:1,000), incubated overnight at 4°C; the secondary antibodies were anti-mouse IgG (CST, 7076P2; dilution 1:5,000) and anti-rabbit IgG (CST, 7074P2; dilution 1:5,000) for 1 hour at room temperature, and finally imaged using an IMAGE QUANT800 (Cytiva, USA). A negative control was also included, in which the mRNA encoding luciferase (SEQ ID NO:22) was used instead of the mRNA encoding PDE6G (SEQ ID NO:7), with all other components and experimental procedures remaining unchanged. The results showed that ( Figure 6 B) As the concentration of mRNA in the vaccine increased, the expression level of PDE6G protein in DC2.4 cells was upregulated, while the negative control Luc mRNA-LNP treatment group failed to express PDE6G protein. This indicates that the PDE6G mRNA-LNP vaccine can efficiently induce APC cells to produce PDE6G protein, laying the foundation for the vaccine to play its role. The effect is better when the mRNA concentration is greater than 1.0 μg / ml.

[0100] To verify whether mRNA-LNP technology can enable mRNA translation in host cells and the biolocalization of exogenous proteins in vivo, luciferase mRNA was used as a tool to track the in vivo biodistribution of exogenous proteins (luciferase) at different time points. Specifically, BALB / c mice aged 6-8 weeks and weighing 16-20g were subcutaneously injected with 10μg of Luc mRNA-LNP vaccine on the right side, and photographs were taken at 24h, 48h, 72h, and 120h. Luciferase activity was measured using the IVIS Spectrum CT (Caliper Life Sciences, Inc.) in vivo imaging system according to the manufacturer's instructions. Before imaging, d-luciferin potassium salt (Maklin, D812647) was injected intraperitoneally into the mice at a dose of 150mg / kg. In addition, mice were euthanized 24 hours after inoculation with luc mRNA-LNPs, and luciferase activity in the heart, liver, spleen, lungs, kidneys, and right inguinal lymph nodes was measured using a luciferase imaging system; mean radiance of the region of interest (ROI) was measured using M3 vision software.

[0101] The results showed that the expression level of the exogenous protein reached its peak 24 hours after injection, and then decreased over time, with fluorescent expression still detectable within 72 hours. Figure 6 C).

[0102] Based on this, different concentrations (5, 10, and 20 μg / mouse) of Luc mRNA-LNP vaccine were injected subcutaneously into the right side of 6-8 week old BALB / c mice weighing 16-20g. The observation time was 24 hours after injection. It was found that mice treated with doses below 5 μg had relatively lower levels of exogenous protein, while the exogenous protein in the 10 μg Luc mRNA-LNP and 20 μg Luc mRNA-LNP treatment groups was mainly concentrated in the inguinal lymph node region. Figure 6 D) This is because vaccine particles carry a negative charge, allowing them to be recognized and internalized by tissue-resident APC cells, then flowing back into secondary lymphoid structures. This also means that the target protein after mRNA-LNP translation can be effectively presented into secondary lymphoid structures. Furthermore, mice were dissected, and various organs (including inguinal lymph nodes, spleen, heart, liver, lungs, and kidneys) were isolated and their luminescence observed under IVIS SpectrumCT. Figure 6E shows that the fluorescence intensity of the inguinal lymph nodes in the 10 μg Luc mRNA-LNP treatment group was comparable to that in the 20 μg Luc mRNA-LNP treatment group, and in some individuals, the former's fluorescence intensity was even significantly stronger than the latter's. However, only at an injection dose of 20 μg could significant fluorescence be observed in the spleen of mice. This may be because the inguinal lymph nodes are closer to the vaccine injection site, and APC cells first flow back to the inguinal lymph nodes. The spleen is farther away, requiring a larger dose to allow more antigen-engulfing APC cells to flow through the spleen. This also indicates that this dose can stimulate more immune organs and produce a stronger immune response, which can also be achieved through multiple injections. On the other hand, regardless of the injection dose, fluorescence was almost undetectable in other important organs besides the liver. This is partly because LNP has liver affinity, and partly because the vaccine has little effect on most organs in the body. Figure 6 F); combining the results of whole-body and organ luminescence in mice after Luc mRNA-LNP treatment ( Figure 6 G) The overall luminescence intensity of 10μg or 20μg of vaccine is higher than that of 5μg. The effect of 10μg is equivalent to that of 20μg. However, excessively high doses may cause side effects. Therefore, the preferred injection dose is 10μg.

[0103] The above results demonstrate that the mRNA-LNP vaccine has the ability to continuously translate target proteins in vitro and in vivo, and the translated target proteins are significantly enriched in secondary lymphoid organs. These characteristics lay the foundation for the efficacy of the vaccine.

[0104] Example 4: PDE6G mRNA-LNP vaccine can induce PDE6G-specific immune response

[0105] Based on the experimental results of the above embodiments, it is speculated that the mRNA vaccine designed based on the PDE6G antigen has high immunogenicity and may be able to efficiently induce a specific cellular immune response against the PDE6G antigen. Therefore, this embodiment explores this possibility. First, 10 μg of the PDE6G mRNA-LNP vaccine prepared according to the steps described in Example 3 was injected into the BALB / c mice at days 0, 5, and 10, respectively. Figure 7 A) To detect the number and activation status of dendritic cells (DCs), and to perform flow cytometry analysis on the spleens of tumor-bearing mice 18 days after immunization, the spleens of vaccinated mice were collected, spleen cells were isolated, and erythrocytes were lysed. The cells were then analyzed at 4°C using 5 × 10⁻⁶ flow cytometry cells. 5Each splenocyte was co-incubated for 30 minutes with antibodies against mouse CD45 (BV510, BD pharmagen, 563891, dilution 1:50), anti-mouse CD11c (BV421, BD pharmagen, 562782, dilution 1:50), anti-mouse CD80 (PE, BD pharmagen, 561955, dilution 1:50), and anti-mouse CD86 (APC, BD pharmagen, 561964, dilution 1:50). Simultaneously, to detect CD8... + The number of T cells, at 4°C, was 5 × 10⁻⁶. 5 Each splenocyte was co-incubated for 30 minutes with anti-mouse CD45 (BV510, BD pharmagen, 563891, dilution 1:50), anti-mouse CD3 (BV421, BD pharmagen, 562600, dilution 1:50), and anti-mouse CD8a (PerCP-Cy5.5, BD pharmagen, 551162, dilution 1:20) antibodies, and then analyzed on a flow cytometer (CytoFLEX LX, Beckman Coulter). Data were analyzed using FlowJo V10 (v.10.0.7r2). Figure 7 B). This experiment included a PBS blank control group and a Luc mRNA-LNP negative control group, i.e., PBS or Luc mRNA-LNP was used to replace PDE6G mRNA-LNP. The results showed that ( Figure 7 B) Compared with the blank control and negative control, the PDE6G mRNA-LNP vaccine significantly upregulated dendritic cells (specifically CD11c) in the spleen. + The proportion of (cells) and the promotion of DC cells (specifically CD80) + CD86 + The activation of cells also induces CD8 to some extent. + CD3 + The production of T cells demonstrates that PDE6G mRNA-LNP can strongly elicit multiple immune responses.

[0106] To further confirm whether the vaccine-induced immune response is PDE6G specific, this example used ELISpot (Enzyme-Linked Immunospot Assay) technology to detect IFN-γ factor secreted by mouse spleen cells. The specific experimental steps are as follows: On day 7 after the last immunization of mice (i.e. Figure 7 As shown in A, D18), the spleen was ground and red blood cells were lysed, and 5×10 5 Each spleen cell was treated with 5, 10, or 20 μg / ml.-1 The PDE6G peptide pool was co-incubated in vitro for 20 hours; according to the manufacturer's instructions, cells secreting IFN-γ were detected using a mouse IFN-γ pre-coated ELISPOT kit (Kedavi, China, 2210005), and the IFN-γ-secreting factor was measured using an ELISpot reader (Mabtech IRIS). TM The FluoroSpot / ELISpot reader (Mabtech, Inc., USA) counts the developed spots; it also includes blank wells, negative control wells, positive control wells, PBS control group, and Luc mRNA-LNP control group.

[0107] ELISpot analysis results show that ( Figure 7 C) After restimulating mouse spleen cells in vitro with the PDE6G peptide library, compared with the two control groups, the PDE6G mRNA-LNP vaccine group showed more IFN-γ secretion spots, and the PDE6G peptide library concentration of 10 μg / mL was the most effective, which is consistent with the above results. In summary, this example fully demonstrates that the described PDE6G mRNA-LNP vaccine can strongly induce a specific immune response against the PDE6G antigen in vivo.

[0108] Example 5: PDE6G mRNA-LNP vaccine has good anti-tumor effect.

[0109] Example 4 confirms that the PDE6G mRNA-LNP vaccine can induce multiple immune responses in the body, including activation of CD11c. + Cells, CD80 + CD86 + Cells and CD8 + CD3 + T cells, and promote the secretion of IFN-γ, but it is unclear whether the vaccine can be used to treat breast cancer. Therefore, in this embodiment, a suitable mouse tumor-bearing model was constructed, namely, BABL / c mice were anesthetized with isoflurane, the fourth pair of mammary fat pads were grasped with forceps, and a syringe containing 4×100 Matrigel was injected. 5 A tumor cell suspension of 1 cell per 50 μL was slowly injected into the fat pad to establish a tumor-bearing mouse model. Five days after tumor cell inoculation, the tumor size was approximately 50 mm. 3 The model was considered complete at this point; and 10 μg of the PDE6G mRNA-LNP vaccine prepared according to the steps described in Example 3 was injected into the mice on days 5, 10, and 15, respectively, and the tumor growth was observed. See the flowchart for details. Figure 8A. To construct a suitable tumor-bearing animal immune model, this embodiment used Western blot to detect the expression level of PDE6G protein in different mouse breast cancer cell lines (EMT-6 and 4T1 cell lines), both of which were derived from BALB / c mice. Compared with the EMT-6 cell line, a distinct band of PDE6G protein size could be detected in the 4T1 cell line. Figure 8 B) indicates that 4T1 cells can express PDE6G protein, while EMT-6 cells cannot. Therefore, on day 0, 4 × 10⁴ cells were inoculated into the MFP (mammary fat pad) of female BALB / c mice (n=6). 5 4T1 mouse breast cancer cells were used to construct an orthotopic tumor-bearing animal model. On days 5, 10, and 15 after 4T1 cell injection, PBS, luc mRNA-LNP, or PDE6G mRNA-LNP (mRNA concentration 10 μg / mouse) were subcutaneously injected into the right thigh of the mice. Tumor volume was measured every 3 days until day 23. Figure 8 C); On the one hand, after 23 days, the mice were euthanized, the tumors were dissected and isolated, and the tumors were weighed and photographed for recording. Figure 8 D and Figure 8 E); On the other hand, the survival time of tumor-bearing mice after different treatments was statistically analyzed, with 6 replicates for each experimental group. Under normal circumstances, tumor volume grows exponentially, i.e., in the PBS group and the Luc mRNA-LNP group ( Figure 8 C); Conversely, after injection of the PDE6G mRNA-LNP vaccine, the growth of tumor volume and weight in mice was significantly inhibited (C). Figure 8 C~ Figure 8 From 23 dpi (days post-inoculation), the average tumor volume in the PDE6G mRNA-LNP group was only about 25% of that in the control groups (PBS group and Luc mRNA-LNP group), while the average tumor weight was reduced by 50% to 67% compared to the latter. Furthermore, mice treated with the PDE6G mRNA-LNP vaccine achieved a longer survival time. Figure 8 F), these experimental results all show that the PDE6G mRNA-LNP vaccine has a good therapeutic effect on breast cancer.

[0110] To further analyze the mechanism of action of the PDE6G mRNA-LNP vaccine, this embodiment performed a fluorescence immunoblotting experiment. The experimental steps are as follows: Tumor tissue was collected from tumor-bearing mice on day 7 after the last dose of vaccine and fixed with 4% paraformaldehyde (Biosharp, BL539A) for 24 hours at room temperature; the mouse tumors were dewaxed and rehydrated; subsequently, antigen retrieval was performed by incubating slides with citrate buffer (pH=6.0) in an autoclave for 3 minutes; for immunofluorescence, the slides were stained with anti-CD3 (Glyc... oTech, GB15014, dilution 1:400), anti-CD4 (Abcam, ab183685, dilution 1:200) and anti-CD8 (CST, 98941S, dilution 1:200) were stained overnight, then incubated at 37°C with HRP-conjugated secondary antibody, goat anti-rabbit IgG (Abcam, ab205718, 1:1000) and goat anti-mouse IgG (Abcam, ab6789) for 1 hour; subsequently, tyramine solution (Biolite; Cy3) was added. Tyramide (dilution ratio 1:400, 1:1065); XFD488 tyramide (dilution ratio 1:100, 1:1070); Cy5 tyramide (dilution ratio 1:200, 1:1066) were applied to each sample and incubated at 37°C for 30 minutes. Cell nuclei were counterstained using DAPI (Solarbio, C0065). Images were captured using a Pannoracic 250FLASH III digital scanner (3DHISTECH, Hungary). Stained cells were quantified in at least five fields of view for each slice, and the calculations were verified by two independent researchers. A PBS group and a Luc mRNA-LNP group were included as controls. Multiplex immunofluorescence assays showed that, compared to the two control groups, the PDE6G mRNA-LNP vaccine group had significantly higher levels of CD3 infiltrating CD3+ in tumor tissue. + CD8 + T cells were significantly more abundant, but no significant CD4 count was observed. + CD3 + T cell infiltration, ( Figure 8 G~ Figure 8 I) Explanation: PDE6GmRNA-LNP vaccine mainly works by inhibiting CD3... + CD8 + T cells are recruited to tumor tissue to play a role, while CD3 + CD8 +T cells (cytotoxic T cells, CTLs) are the core effector cells of adaptive immunity. Their main function is to recognize and kill virus-infected cells, tumor cells, and other abnormal cells. This further explains why the PDE6G mRNA-LNP vaccine can mobilize the body's own immune function to kill breast tumor cells, thereby achieving a good therapeutic effect.

[0111] To further investigate whether the anti-tumor immune response induced by the PDE6G mRNA-LNP vaccine is specific, this embodiment also constructed a mouse tumor-bearing model using EMT6 cells (which do not express PDE6G protein), using the same construction and immunization methods as described above. Although the tumors generated by EMT6 cells were 30% smaller than those generated by 4T1 cells, no inhibition of tumor growth was observed after three immunizations with the PDE6G mRNA-LNP vaccine. Figure 8 K) indicates that the vaccine cannot immunize tumor cells that do not express the PDE6G antigen.

[0112] To further verify the specificity of the anti-tumor immune response of the PDE6G mRNA-LNP vaccine, this embodiment also constructed an EMT6 cell line overexpressing the PDE6G protein using lentivirus. Specifically, Beijing Qingke Biotechnology Co., Ltd. was commissioned to insert the 264bp nucleotide sequence (SEQ ID NO:2) encoding the PDE6G protein into the pLV4ltr-puro-CMV plasmid to obtain the recombinant plasmid pLV4ltr-PDE6G, and the successful construction of the recombinant plasmid was confirmed by DNA sequencing. Using the lipo2000 reagent (Thermo Scientific), the above plasmid and the viral packaging plasmid (including pMD2.G and PSPAX2) were simultaneously transfected into HEK293T cells in DMEM high-glucose medium containing 10% fetal bovine serum. After 48 hours, the virus-containing medium was collected, filtered to remove cell debris, and 1×10 5 EMT6 cells were seeded into six-well plates. After 24 hours, the EMT6 cell line was transduced with virus-containing medium supplemented with 10 μg / mL Polybrene (Beyotime, CO351-1 ml). After two viral infections, the cells were selected with 1-2 μg / ml (preferably 1 μg / ml) puromycin (Beyotime, ST551-10 mg) to establish an EMT6 cell line that stably expresses PDE6G protein. Western blot analysis (the experimental procedure is the same as described in Example 3) then confirmed the successful construction of the cell line. Figure 8 J), that is, protein bands of the expected size and with clear edges appeared; subsequently, a tumor-bearing model was constructed using the modified EMT6 cell line, and the steps for model construction and immunization of mice were the same as described above. The results showed that (Figure 8 After three immunizations with the PDE6G mRNA-LNP vaccine, the growth rate of tumors generated by the EMT6 cell line that overexpresses PDE6G protein was effectively controlled, which further confirms the anti-tumor specificity of the PDE6G mRNA-LNP vaccine.

[0113] Based on the experimental results of Examples 4 and 5, the following conclusions can be drawn: the PDE6G mRNA-LNP vaccine can efficiently stimulate the body to produce a multi-level immune response targeting only the PDE6G antigen, thereby achieving an effective treatment effect for breast cancer.

[0114] Example 6: The PDE6G mRNA-LNP vaccine exhibits good biosafety.

[0115] Example 3 mentions that exogenous proteins (antigens) produced by the Luc mRNA-LNP vaccine accumulate in the liver, and endogenous PDE6G protein is also produced within the body (referring to the retina). It is unclear whether the PDE6G mRNA-LNP vaccine will cause damage to organs such as the liver or autoimmunity. Therefore, this example analyzes the biosafety of the PDE6G mRNA-LNP vaccine. The specific steps are as follows:

[0116] 6.1 Using the BALB / c mouse model (as described in Example 4), PBS, luc mRNA-LNP, or PDE6G mRNA-LNP (mRNA concentration 10 μg / mouse) were subcutaneously injected into the right thigh of BALB / c mice on days 0, 5, and 10, respectively. Mouse body weight was measured every 5 days until day 20. Figure 9 A).

[0117] 6.2 To assess the toxicity of the vaccine to various organs, on day 18 after vaccination (i.e., 8 days after the last dose of vaccine), mouse organs (including heart, liver, spleen, lung, and kidney) were collected for HE staining. Specifically, mouse heart, liver, spleen, lung, and kidney were fixed with 4% paraformaldehyde (Biosharp, BL539A) at room temperature for 24 hours. The fixed tissues were dehydrated, cleared, embedded in paraffin, sectioned, dewaxed, and hydrated. Hematoxylin staining was performed for 5-15 minutes (preferably 10 minutes in this embodiment), followed by rinsing with running water. Differentiation with 1% hydrochloric acid alcohol for a few seconds removed excess staining, followed by blueing with running water for 10-30 minutes (preferably 20 minutes in this embodiment). Counterstaining with 0.5% eosin for 1-3 minutes, followed by graded alcohol dehydration, clearing with xylene, and mounting with neutral resin were performed. Finally, the tissue structure was observed under a microscope. Figure 9 B).

[0118] 6.3 To assess the toxicity of the vaccine to the retina, ophthalmological observation was performed using a Micron IV system (Phoenix, USA) 8 days after the last dose of vaccine. Before image capture, mice were anesthetized, and their pupils were dilated with 1% tropicamide. The restrained mice were moved to the experimental table, and the cornea was directly aligned with the microscope lens to ensure sufficient light beam entry into the eye. The position and focus of the ophthalmoscope were adjusted to ensure clear display and capture of the retinal image for ophthalmoscopy analysis. HE staining was performed on the mouse retina, following the same procedure as described above. Figure 9 C).

[0119] 6.4 To further evaluate the toxicity of the vaccine to the hematopoietic system and liver and kidneys, 0.2 mL of venous blood was collected from mice 8 days after the last dose of vaccine for complete blood count and blood biochemistry tests. The complete blood count included white blood cell count (CBC). Figure 9 D) Red blood cells ( Figure 9 E), platelets ( Figure 9 The detection of F) levels, the blood biochemistry test including serum alanine aminotransferase (ALT) (F) Figure 9 G), Aspartate aminotransferase (AST) Figure 9 H), urea (Urea) Figure 9 I) Creatinine (CR) Figure 9 J) level.

[0120] All of the above experiments included a PBS group and a Luc mRNA-LNP group as controls, with 6 replicates in each group.

[0121] After three immunizations with the PDE6G mRNA-LNP vaccine, no significant change in body weight was observed in mice compared to the control group. Figure 9 A); The cells in the HE-stained sections of the three groups were of uniform size and structure, with even chromatin distribution, intact nuclei, and close cell arrangement, indicating that PDE6G mRNA-LNP vaccine immunization did not cause morphological changes in important organs. Figure 9 B); meanwhile, fundus examination showed no obvious inflammatory exudative changes in the mouse fundus, and HE staining of the retina did not reveal immune cell infiltration, indicating that the retina was not damaged. Figure 9 C); Further routine blood tests and serum biochemical analyses of mice showed that, compared to the control group, the white blood cell count in the PDE6GmRNA-LNP vaccine group was significantly higher. Figure 9 D), this may be because the vaccine triggered an immune response in the body, which is a normal phenomenon, and the other indicators did not show any abnormalities. Figure 9 E~ PDE6G antigen coding regionJ) indicates that the PDE6G mRNA-LNP vaccine has no significant blood, liver, or kidney toxicity. The experimental results of this embodiment confirm that the PDE6G mRNA-LNP vaccine possesses good biocompatibility. Combined with the efficacy demonstrated in Examples 3-5, it can be concluded that the PDE6G mRNA-LNP vaccine shows great promise in the clinical treatment of breast cancer.

[0122] Example 7: Optimization of elements in the PDE6G antigen coding region

[0123] Examples 3-6 demonstrated the efficacy of the PDE6G mRNA-LNP vaccine. To further optimize the overall performance of the vaccine, Examples 7 and 8 optimized the PDE6G antigen coding region and mRNA components, respectively, by combining different components to explore the combination that best inhibits breast cancer tumor growth. First, different mRNA vaccines were prepared according to the steps described in Example 3, except that the PDE6G antigen coding region sequence was different. Then, a mouse tumor-bearing model was constructed using the 4T1 cell line, and the tumor-bearing mice were immunized three times with the PDE6G mRNA-LNP vaccine. On day 7 after the third immunization, the tumor size and tumor inhibition rate were counted. The specific steps were the same as those described in Example 5. The specific results are shown in Table 2. Among them, "HIV-1gp160 signal peptide-PDE6G antigen-P2-P16-MITD" corresponds to the PDE6G antigen coding region (SEQ ID NO:12) described in Example 3. The tumor inhibition rate = (average tumor volume of experimental group - average tumor volume of Luc mRNA-LNP control group) / average tumor volume of Luc mRNA-LNP control group × 100%.

[0124] Table 2. Effects of the PDE6G antigen coding region on the tumor-inhibiting effect of PDE6G mRNA-LNP vaccine.

[0125] Tumor inhibition rate HIV-1 gp160 signal peptide-PDE6G antigen-P2-P16-MITD Immunoglobulin kappa light chain signal peptide-PDE6G antigen-P2-P16-MITD 75％ Melittin signal peptide-PDE6G antigen-P2-P16-MITD 50％ HIV-1 gp160 signal peptide-PDE6G antigen-P2-P30-MITD 55％ HIV-1 gp160 signal peptide-PDE6G antigen-P2-DT8-MITD 65％ HIV-1 gp160 signal peptide-PDE6G antigen-P2-P16-P30-MITD 60％ HIV-1 gp160 signal peptide-PDE6G antigen-P2-P16 70％ PDE6G antigen 60％ mRNA structure 45％

[0126] Note: The PDE6G antigen sequence is shown in SEQ ID NO:15; the HIV-1 gp160 signal peptide is MRVTAPRTLILLLSGALALTETWAGS (SEQ ID NO:13); the immunoglobulin κ light chain signal peptide sequence is METDTLLLWVLLLWVPGSTG; the bee venom peptide signal peptide sequence is MKLFVLSLVFMVAFVYLYW; P2 represents tetanus toxoid CD4. + Epitope P2 (SEQ ID NO:17); P16 represents tetanus toxoid CD4. + Epitope P16 (SEQ ID NO:19); P30 represents tetanus toxoid CD4. +Epitope P30 (FNNFTVSFWLRVPKVSASHLE); DT-8 represents diphtheria toxoid epitope DT-8 (YVDEYEYLQKFEKLTN); MITD sequence is shown in SEQ ID NO:21.

[0127] As shown in Table 2, without optimization of the PDE6G antigen coding region (i.e., the PDE6G antigen coding region contains only the PDE6G antigen), the corresponding mRNA vaccine's tumor inhibition rate was only 45%. This result indicates, on the one hand, the role of the PDE6G antigen itself in treating breast cancer; on the other hand, it also demonstrates that introducing a signal peptide and immune-enhancing elements into the PDE6G antigen coding region can improve the vaccine's tumor-inhibiting effect to varying degrees (5%–30%). Among them, the HIV-1gp160 signal peptide-PDE6G antigen-P2-P16-MITD combination showed the best effect. This may be due to the structure of the PDE6G antigen itself, that is, the fusion protein formed by the PDE6G antigen, the signal peptide, and the immune-enhancing elements has a more stable conformation, binds to MHC molecules more efficiently, and is presented to the cell membrane, thereby being recognized by more immune cells, causing a stronger immune response, and ultimately achieving a better effect in inhibiting breast cancer tumors. It is worth noting that the effect of including all three toxoid epitopes (i.e., HIV-1 gp160 signal peptide-PDE6G antigen-P2-P30-MITD) is not as good as that of including only P12 and P16. This may be because the more toxoid epitopes there are, the larger the synthesized protein becomes, and the lower the translation efficiency. Alternatively, the increase in toxoid epitopes may cover the PDE6G protein, thereby inducing an immune response against toxoids rather than PDE6G, thus failing to improve the tumor-suppressive effect. In summary, the preferred PDE6G antigen coding region is composed of HIV-1 gp160 signal peptide, P2, P16, MITD, and PDE6G antigen.

[0128] Example 8: Optimization of mRNA elements

[0129] As shown in Example 3, the mRNA in the PDE6G mRNA-LNP vaccine of the present invention contains multiple stable mRNA elements and has undergone 5' end capping and m1Ψ modification. Based on the results of Examples 3 and 7, this example optimizes the mRNA elements to further improve the performance of the vaccine. The vaccine preparation steps are the same as those in Example 3, except that the specific mRNA sequence is different. The experimental steps for testing the antitumor effect of the vaccine are the same as those in Examples 5 and 7. The specific results are shown in Table 3. Among them, the β-globulin 5'UTR-antigen coding sequence-β-globulin 3'UTR corresponds to SEQ ID NO:7 described in Example 3.

[0130] Table 3. Effects of mRNA elements on the tumor-suppressing efficacy of PDE6G mRNA-LNP vaccine.

[0131] Tumor inhibition rate Beta globulin 5' UTR-antigen coding sequence-beta globulin 3' UTR Human hydroxysteroid dehydrogenase 5' UTR-antigen coding sequence-beta globulin 3' UTR 75％ TMV omega sequence 5' UTR-antigen coding sequence-beta globulin 3' UTR 60％ Beta globulin 5' UTR-antigen coding sequence-alpha globulin 3' UTR 65％ Antigen coding sequence 70％ ​ 50％

[0132] Note: The antigen coding sequence is shown in SEQ ID NO:9; the β-globulin 5'UTR is shown in SEQ ID NO:8; the human hydroxysteroid dehydrogenase 5'UTR sequence is CGGGGCUCCGCCGCCACC; the TMVΩ sequence 5'UTR is GCCACCAUUGACUACUUAAUGGAGACGGCCAAUUCGA; the β-globulin 3'UTR is shown in SEQ ID NO:10. α The 3'UTR of globulin is UGCAUAUUUGCUUCACCAUAA.

[0133] As shown in Table 3, the tumor inhibition rate of vaccines without the introduction of mRNA stabilizing elements was only 50%. However, adding different UTR sequences to the 5' and 3' ends of the antigen coding sequence improved the tumor inhibition effect of the vaccine to varying degrees (10%–25%). Among them, the combination of β-globulin 5'UTR-antigen coding sequence-β-globulin 3'UTR showed the best effect. This may be because, for the sequence encoding the PDE6G antigen (SEQ ID NO:9), the β-globulin 5'UTR sequence can best improve the translation efficiency of mRNA, while the β-globulin 3'UTR sequence can best prolong the half-life of mRNA. Therefore, the mRNA can be translated to generate the most antigen within a certain period of time, thereby causing the strongest immune response and ultimately achieving the best effect in inhibiting breast cancer tumors. Therefore, it is preferred that the β-globulin 5'UTR sequence, the β-globulin 3'UTR sequence, and the antigen coding sequence are used to form the mRNA.

[0134] In summary, in addition to the PDE6G antigen itself, the therapeutic effect of the PDE6G mRNA-LNP vaccine of the present invention is also affected by the signal peptide that matches the antigen, the elements that enhance the immune response, and the elements that stabilize the mRNA.

[0135] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description. Therefore, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. An mRNA vaccine, characterized in that, The vaccine comprises RNA sequences capable of encoding one or more of PDE6G protein, PDE6D protein and recoverin protein.

2. The vaccine as described in claim 1, characterized in that, The RNA comprises a nucleotide sequence capable of encoding PDE6G protein.

3. The vaccine as described in claim 1, characterized in that, The RNA further comprises any one or more of untranslated region and poly A sequences.

4. The vaccine of claim 1, wherein the antigen is a protein or a polypeptide. The RNA sequence has 60% or more homology with the sequence shown in SEQ ID NO: 7 and / or SEQ ID NO:

9.

5. The vaccine of claim 1, wherein the antigen is a protein or a polypeptide. The total amount of the RNA is 5 μg to 100 mg.

6. The vaccine as described in claim 1, characterized in that, The vaccine further comprises liposomes comprising one or more of ionizable lipids, neutral phospholipids, cholesterol and polyethylene glycol lipids.

7. The vaccine as described in claim 6, characterized in that, The molar ratio of the ionizable lipids, neutral phospholipids, cholesterol and polyethylene glycol lipids is (25-50):(1-10):(20-38.5):(0.1-1.5).

8. Use of RNA having 60% or more homology with the sequence shown in SEQ ID NO: 7 and / or SEQ ID NO: 9 for the preparation of a medicament for the treatment of breast cancer.

9. Use of RNA having 60% or more homology with the sequence shown in SEQ ID NO: 7 and / or SEQ ID NO: 9 for the preparation of a medicament for stimulating an immune response in a subject.

10. The use according to claim 9, characterized in that, The immune response comprises activation of immune cells and production of immune factors.

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