An mRNA vaccine for treating HER2 low expression type breast cancer and a preparation method and use thereof

Abstract

The application discloses an mRNA vaccine for treating HER2 low-expression breast cancer and a preparation method and application thereof, and belongs to the technical field of tumor immunotherapy. The mRNA vaccine comprises mRNA molecules encapsulated by folate-targeted lipid nanoparticles (LNP), wherein the mRNA molecules encode a tPA signal peptide, a HER2 polyepitope string, a transmembrane domain and an intracellular domain fragment connected in sequence, the HER2 polyepitope string comprises six neoantigen peptides (SEQ ID NO. 1-SEQ ID NO. 6), the mRNA is modified by 1-methyl-pseudouridine and optimized by codon, the 5'-end is a Cap1 structure, and the 3'-end contains 120 nt Poly(A). The mRNA vaccine can be used for treating HER2 low-expression (IHC 1+ or 2+ / FISH negative) breast cancer patients, and can be used alone or in combination with an immune checkpoint inhibitor and a small molecule HER2 inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy technology, specifically relating to an mRNA vaccine for treating HER2-low expression breast cancer. More specifically, it relates to an mRNA vaccine encoding key epitopes (containing neoantigen peptides) of the extracellular and transmembrane domains of human HER2 protein, its lipid nanoparticle (LNP) delivery system, preparation process, and the use of this mRNA vaccine in postoperative adjuvant therapy and postoperative maintenance therapy for HER2-low expression (IHC 1+ or 2+ / FISH negative) breast cancer patients. It can be used alone or in combination with immune checkpoint inhibitors and small molecule HER2 inhibitors. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide, and HER2 (human epidermal growth factor receptor 2) expression status is one of the core indicators for clinical treatment selection. Currently, for HER2-positive breast cancer (IHC 3+ or 2+ / FISH positive), there are various targeted therapies such as trastuzumab, pertuzumab, T-DM1, and DS-8201, which have significantly improved patient prognosis. However, approximately 45-55% of all breast cancer patients have low HER2 expression (immunohistochemical IHC 1+ or 2+ / FISH negative). This group has traditionally been classified as "HER2 negative" and lacks targeted therapies. Clinical treatment is still mainly chemotherapy, which has limited efficacy and significant adverse reactions, a high risk of postoperative recurrence, and a poor prognosis, indicating a huge unmet clinical need for treatment.

[0003] As an important branch of tumor immunotherapy, tumor vaccines aim to induce a specific anti-tumor immune response in the body to eliminate tumor cells or inhibit tumor growth, and have the advantages of high specificity and few adverse reactions. Traditional protein vaccines or peptide vaccines have drawbacks such as weak immunogenicity, the need for adjuvants to assist immunization, easy degradation by the body, and difficulty in inducing a strong cellular immune response, which limit their clinical application.

[0004] mRNA vaccines have become a research hotspot in the field of tumor immunotherapy in recent years due to their unique advantages: mRNA vaccines can endogenously express target antigens in the body's cells, and can simultaneously induce the body to produce specific CD8+ cytotoxic T cell responses (to eliminate tumor cells) and humoral immune responses (to produce anti-HER2 antibodies and block tumor cell proliferation); at the same time, by modifying mRNA nucleosides, optimizing codons, and using lipid nanoparticle (LNP) delivery systems, the stability of mRNA can be significantly improved, its immunogenicity reduced (avoiding unnecessary IFN-α storms), and the safety and efficacy of vaccines enhanced, thus solving many of the drawbacks of traditional vaccines.

[0005] Currently, some mRNA vaccines targeting HER2-positive tumors have entered clinical trials. However, there are no reports of mRNA vaccines specifically targeting HER2-low expression breast cancer. Furthermore, most existing HER2-related mRNA vaccines only cover epitopes of a single HLA subtype. HLA-A02:01 and HLA-A24:02 are high-frequency HLA subtypes in my country and other Asian populations (with a combined coverage rate of ≥80%), and existing vaccines cannot effectively cover this mainstream population. In addition, existing vaccines do not integrate sequences related to immune microenvironment regulation, making it difficult to reverse the immunosuppressive state of the tumor microenvironment in HER2-low expression breast cancer patients. This results in limited immune response induction efficiency, failing to effectively eliminate residual microlesions after surgery and thus failing to meet clinical treatment needs.

[0006] Based on the shortcomings of the existing technologies, developing an mRNA vaccine specifically targeting HER2-low expression breast cancer, covering mainstream HLA subtypes, capable of regulating the tumor immune microenvironment, and possessing high safety and strong immunogenicity, along with its preparation method, has significant clinical significance and application value. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, such as the lack of specific vaccines for breast cancer with low HER2 expression, limited coverage of existing vaccines, insufficient immunogenicity, and inability to regulate the tumor immune microenvironment, this invention provides an mRNA vaccine that can induce a potent, durable, and specific anti-HER2 immune response in the general population (HLA-A02:01 and HLA-A24:02 genotyping coverage ≥80%), as well as its preparation method and uses.

[0008] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide an mRNA molecule whose nucleotide sequence from the 5' end to the 3' end comprises: (a) Nucleotide sequence encoding human tPA signal peptide; (b) A nucleotide sequence encoding a HER2 multiepitope string, wherein the HER2 multiepitope string is composed of neoantigen peptides with amino acid sequences as shown in SEQ ID NO.1-SEQ ID NO.6, connected in series by flexible linkers; (c) The nucleotide sequence encoding the HER2 transmembrane domain; and (d) Nucleotide sequence encoding the intracellular domain segment of HER2; The uridine in the mRNA molecule is replaced by 1-methyl-pseudouridine, the 5' end has a Cap1 structure, and the 3' end has a polyadenylate tail of 100-150 nucleotides in length.

[0009] Furthermore, the amino acid sequence of the human tPA signal peptide is shown in SEQ ID NO.7; the amino acid sequence of the HER2 transmembrane domain is shown in SEQ ID NO.11; and the amino acid sequence of the HER2 intracellular domain fragment is shown in SEQ ID NO.12.

[0010] Furthermore, the coding sequence of the mRNA molecule has been optimized with human codons, and the complete amino acid sequence is shown in SEQ ID NO.8.

[0011] Furthermore, the backbone structure of the mRNA molecule is: 5'-Cap1-Kozak sequence-5'-UTR-coding sequence-3'-UTR-Poly(A)120-restriction spacer sequence, wherein the Kozak sequence is GCCACC, the 5'-UTR sequence is shown in SEQ ID NO.9, and the 3'-UTR sequence is shown in SEQ ID NO.10.

[0012] A second objective of this invention is to provide an mRNA vaccine comprising the mRNA molecule and lipid nanoparticles encapsulating the mRNA molecule; the lipid nanoparticles comprise ionizable cationic lipids, neutral cofactor lipids, cholesterol and polyethylene glycol-modified lipids, and the lipid nanoparticles further comprise folic acid-modified polyethylene glycol-modified lipids.

[0013] Furthermore, in the lipid nanoparticles, the molar ratio of ionizable cationic lipids, neutral cofactor lipids, cholesterol, PEGylated lipids, and folic acid-modified PEGylated lipids is (45-55):(8-12):(35-40):(0.5-1.5):(0.4-0.6); the nitrogen-to-phosphorus ratio of the lipid nanoparticles to mRNA is 4-8; the particle size of the lipid nanoparticles is 75-85 nm, PDI<0.1, and the mRNA encapsulation efficiency is ≥95%.

[0014] The third objective of this invention is to provide a method for preparing the mRNA vaccine, comprising the following steps: (1) Provide the mRNA molecule; (2) Dissolve the mRNA molecule in an aqueous buffer to obtain an aqueous phase; dissolve the lipid component in an organic solvent to obtain an organic phase, wherein the lipid component includes ionizable cationic lipids, neutral auxiliary lipids, cholesterol, polyethylene glycol-modified lipids and folic acid-modified polyethylene glycol-modified lipids; (3) The aqueous phase and the organic phase are mixed by microfluidics to encapsulate the mRNA molecules with lipid nanoparticles; (4) The obtained lipid nanoparticles were purified and concentrated to obtain an mRNA vaccine.

[0015] Further, in step (1), mRNA molecules are obtained by in vitro transcription and the Cap1 structure is introduced by co-transcription capping; in step (2), the aqueous buffer is citrate buffer with pH 3.5-4.5, the mRNA concentration is 0.1-0.3 mg / mL, and the organic solvent is anhydrous ethanol; in step (3), the volume ratio of the aqueous phase to the organic phase is (2-4):1, and the microfluidic mixing flow rate is 8-15 mL / min; in step (4), ethanol is removed by ultrafiltration or dialysis, and buffer replacement is performed with phosphate buffer, and then the mixture is filtered through a 0.22 μm filter membrane for sterilization.

[0016] The fourth objective of this invention is to provide the application of the mRNA vaccine in the preparation of a medicament for treating HER2-positive or HER2-low expression breast cancer.

[0017] Furthermore, the HER2-low expression breast cancer is IHC 1+ and / or 2+ / FISH negative breast cancer; the drug is used for adjuvant therapy and / or maintenance therapy after breast cancer surgery; the drug can be used in combination with immune checkpoint inhibitors or HER2 small molecule inhibitors.

[0018] Compared with the prior art, the present invention has the following significant advantages: (1) Highly targeted, filling a clinical gap: This invention is specifically designed for breast cancer patients with low HER2 expression, who account for 45-55% of breast cancer patients. Traditional targeted therapies are lacking. This vaccine can effectively fill the clinical gap in this field and provide new treatment options for these patients.

[0019] (2) Wide population coverage and strong immunogenicity: The six novel antigenic peptides screened in this invention can cover the two major high-frequency HLA subtypes, HLA-A02:01 and HLA-A24:02, with a population coverage rate of ≥80%. Simultaneously, through multi-epitope string design, signal peptide fusion, codon optimization, and nucleoside modification, the immunogenicity of the vaccine is significantly enhanced, inducing potent specific CD8+ T cell responses and humoral immune responses. In vitro DC loading experiments showed that the proportion of IFN-γ+ CD8+ T cells induced by this vaccine reached 28.7%, significantly higher than the unmodified mRNA group (11.2%, p<0.01); ELISPOT detection results ( Figure 3 The results showed that the number of HER2-specific spots induced by this vaccine was significantly higher than that in the control group, indicating its excellent immunogenicity.

[0020] (3) High delivery efficiency and good targeting: The present invention uses folic acid modified LNP as a delivery system. On the one hand, it can effectively protect mRNA from degradation by nucleases in the body, improve the stability of mRNA and cell uptake efficiency; on the other hand, folic acid can specifically bind to the folic acid receptor-α highly expressed in breast cancer tissue, realize active targeted delivery, increase the drug concentration in the target tissue, reduce damage to normal tissue, and reduce adverse reactions.

[0021] (4) Significant anti-tumor effect, which can delay postoperative recurrence: In a mouse model of HER2-low breast cancer after surgery (4T1 / HER2-low), the vaccine was administered on the 7th day after surgery. The results showed that the number of lung metastatic nodules in mice decreased from 42±6 in the control group to 8±3 (p<0.001), and the median survival time increased from 38 days in the control group to 67 days (p<0.001). Figure 4 This indicates that the vaccine can effectively eliminate residual lesions after surgery, inhibit tumor metastasis, and prolong survival.

[0022] (5) Good safety and high tolerability: This invention effectively reduces the immunogenicity of mRNA through mRNA nucleoside modification (U→1-methyl-ψ), avoiding the occurrence of IFN-α storm; at the same time, the components and ratios of the LNP delivery system have been optimized, resulting in low cytotoxicity. Repeated-dose toxicity tests in cynomolgus monkeys (dose of 0, 1, and 3 mg / kg, administered once every 2 weeks for a total of 5 times) showed no systemic toxicity reactions of Grade ≥2, and the peak values ​​of serum IL-6 and TNF-α were not statistically different from those in the blank control group, indicating that the vaccine has good safety, high tolerability, and is suitable for clinical application.

[0023] (6) The preparation process is stable and suitable for industrial production: The present invention uses microfluidic technology to prepare LNP-mRNA nanoparticles. The process parameters are controllable, and the prepared vaccine has uniform particle size and high encapsulation rate. The entire preparation process is simple and efficient, and can realize large-scale industrial production and reduce production costs.

[0024] (7) Excellent combined drug effect and broad application prospects: The mRNA vaccine of the present invention can be used in combination with PD-1 inhibitors and small molecule HER2 inhibitors, which has a significant synergistic anti-tumor effect, can further improve the treatment effect, expand the applicable population, and has broad application prospects. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the mRNA structure in this invention, in which the positions of 5'-cap (Cap1 structure), Kozak sequence, 5'-UTR, antigen coding sequence (tPA-PES-TM-intracellular domain fragment), 3'-UTR, Poly(A)120 and restriction enzyme spacer sequence are marked.

[0026] Figure 2 This is a histogram of the particle size distribution of lipid nanoparticles (LNPs) in this invention; the particle size distribution curve shows that the LNP particle size is concentrated in 75-85 nm, and the PDI < 0.1.

[0027] Figure 3 The figure shows the ELISPOT assay results of HER2-specific T cell responses induced by the mRNA vaccine of this invention. The horizontal axis represents different treatment groups (blank control group, unmodified mRNA group, and vaccine group of this invention), and the vertical axis represents the number of specific IFN-γ spots / 10^5 cells. The results show that the number of spots in the vaccine group of this invention is significantly higher than that in the other two groups, indicating that it can induce a strong specific T cell response.

[0028] Figure 4 The graph shows the survival curve of the mRNA vaccine of the present invention in a mouse model (4T1 / HER2-low) of HER2-low breast cancer after surgery; where the horizontal axis represents survival time (days) and the vertical axis represents survival rate (%). The results show that the median survival time of the mice in the vaccine group of the present invention is significantly longer than that in the control group, indicating that it can effectively prolong the survival time of patients after surgery. Detailed Implementation

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0030] 1. Purpose of the invention The purpose of this invention is to overcome the shortcomings of existing technologies, such as the lack of specific vaccines for HER2-low expression breast cancer, the limited coverage of existing vaccines, insufficient immunogenicity, and inability to regulate the tumor immune microenvironment. The invention provides an mRNA vaccine that can induce a potent, durable, and specific anti-HER2 immune response in the general population (HLA-A02:01 and HLA-A24:02 genotyping coverage ≥80%). This vaccine can effectively clear residual microlesions after surgery in patients with HER2-low expression breast cancer, delay tumor recurrence, and prolong patient survival.

[0031] Another objective of this invention is to provide a method for preparing the above-mentioned mRNA vaccine, which is stable and controllable, and produces a vaccine with high purity, good stability, and high encapsulation rate, making it suitable for large-scale industrial production.

[0032] Another object of the present invention is to provide the use of the above-mentioned mRNA vaccine, specifically its use in the preparation of adjuvant therapy drugs and maintenance therapy drugs for breast cancer with low HER2 expression. The vaccine can be used alone or in combination with PD-1 inhibitors or small molecule HER2 inhibitors to further improve the therapeutic effect.

[0033] 2. Technical Solution 2.1 Antigen Sequence Design The antigen sequence encoded by the mRNA vaccine of this invention has undergone rigorous epitope screening and sequence optimization, and is specifically designed as follows: (1) The extracellular domain II region (amino acid sites aa195-420) and IV region (amino acid sites aa563-652) of the human HER2 gene (NM_004448.4) encoded protein were selected as epitope screening regions. Using NetMHCpan-4.1 software, 9-10 mer neoantigen peptides with IC50 < 50 nM (high binding affinity) and immunogenicity score > 0.75 (strong immunogenicity) were screened out, totaling 6 peptides. Their amino acid sequences are shown in SEQ ID NO.1-SEQ ID NO.6, respectively. These 6 neoantigen peptides can be presented by HLA-A02:01 or HLA-A24:02 molecules, respectively, to achieve coverage of the mainstream population.

[0034] (2) The above 6 new antigen peptides are connected in series through GGPPG flexible linker to form a poly-epitopestring (PES). The flexible linker can avoid the spatial steric hindrance between epitopes, ensure that each epitope can be effectively presented, and enhance the solubility of the antigen.

[0035] (3) A human tPA signal peptide (amino acid sequence as shown in SEQ ID NO.7) is fused upstream of the multi-epitope string (PES). This signal peptide can guide the antigen protein to achieve secretory expression, promote the effective capture and cross-presentation of the antigen by dendritic cells (DCs), and enhance the efficiency of immune response induction.

[0036] (4) The transmembrane domain (amino acid sites aa 655-678, sequence SEQ ID NO.11: ISAVVGILLVVVLGVVFGILIKRR) and intracellular domain fragment (amino acid sites aa 679-690, containing T-helper epitopes, sequence SEQ ID NO.12: QQKIRKYTMRRL) of HER2 protein are fused downstream of the multi-epitope string (PES). The transmembrane domain can promote the anchoring of antigen proteins on the cell surface and prolong the antigen presentation time. The T-helper epitope can activate CD4+ helper T cells, provide auxiliary signals for the activation of CD8+ cytotoxic T cells, and enhance the persistence of the immune response.

[0037] (5) The complete antigen coding sequence (tPA signal peptide-multi-epitope string-transmembrane domain-intracellular domain fragment) was optimized with human codons. The optimized codon fitness index (CAI) was 0.92 (close to 1, indicating that the sequence is highly expressed in human cells), and the GC content was 58% (to avoid the formation of secondary structures and ensure the stability and translation efficiency of mRNA). At the same time, three U→1-methyl-ψ (1-methylpseuuridine) modifications were introduced into the sequence. This modification can effectively eliminate the activation of TLR7 / 8 receptors by mRNA, reduce the risk of IFN-α storm in the body, and improve the safety of the vaccine. The optimized complete antigen coding sequence is shown in SEQ ID NO.8.

[0038] 2.2 mRNA backbone design To further improve the stability, translation efficiency, and safety of mRNA, this invention optimizes the mRNA backbone structure, as follows: (1) 5'-UTR (5' untranslated region): The 5'-UTR of the human β-globin gene (as shown in SEQ ID NO.9) was used, and the Kozak optimized sequence (GCCACC) was added downstream of it. The Kozak sequence can enhance the binding ability of ribosomes to mRNA and significantly improve the translation efficiency of mRNA.

[0039] (2) 3'-UTR (3' untranslated region): AES-mtRNR1 double fragment (sequence shown in SEQ ID NO.10) is used. This double fragment can effectively prolong the half-life of mRNA in the cell, reduce mRNA degradation, and ensure that the antigen protein can be continuously and stably expressed.

[0040] (3) Poly(A) tail: 120 nt in length. A 10-nt restriction site spacer sequence is introduced at the end of the Poly(A) tail. This spacer sequence facilitates the recovery of linearized plasmids after in vitro transcription and improves the efficiency and purity of mRNA preparation.

[0041] (4) 5'-cap (5' cap structure): The capping method is adopted by co-transcription and the capping is performed using CleanCap® Reagent AG reagent to ensure the capping efficiency. The final proportion of Cap1 structure obtained is ≥95%. The Cap1 structure can protect mRNA from degradation by 5'-exonuclease, while promoting nuclear export of mRNA and translation initiation, further improving the stability and translation efficiency of mRNA.

[0042] The complete mRNA structure of this invention is: 5'-cap-5'-UTR-Kozak sequence-antigen coding sequence (tPA-PES-transmembrane domain-intracellular domain)-3'-UTR-Poly(A)120-restriction spacer sequence, as shown in the schematic diagram. Figure 1 As shown.

[0043] 2.3 Composition of Lipid Nanoparticles (LNPs) To achieve efficient mRNA delivery, improve cellular uptake efficiency, and reduce in vivo degradation, this invention employs lipid nanoparticles (LNPs) as an mRNA delivery system. These LNPs are specifically modified for targeted delivery and have the following composition: (1) Lipid components and molar ratio: The molar ratio of ionized cationic lipid DLin-MC3-DMA, neutral lipid DSPC, cholesterol, and PEG-modified lipid PEG2000-DMG is 50:10:38.5:1.5. Among them, DLin-MC3-DMA, as a cationic lipid, can bind to negatively charged mRNA through electrostatic interaction to achieve mRNA encapsulation; DSPC can enhance the structural stability of LNP; cholesterol can regulate the fluidity of the lipid bilayer and improve the in vivo stability of LNP; PEG2000-DMG can reduce the clearance of LNP by the reticuloendothelial system (RES) in vivo and prolong its circulation time in vivo.

[0044] (2) Key parameters: The N / P ratio (nitrogen-phosphorus ratio) of LNP to mRNA is 6. This ratio can ensure that mRNA is efficiently encapsulated while avoiding cytotoxicity caused by excessive cationic lipids. The prepared LNP-mRNA nanoparticles have a particle size of 75-85 nm, a polydispersity index (PDI) of <0.1 (indicating that the particle size is uniform and the dispersibility is good), and an mRNA encapsulation rate of ≥95% (ensuring that the mRNA is not degraded by nucleases in vivo and is effectively delivered into target cells).

[0045] (3) Targeted modification: 0.5 mol% folic acid-PEG2000-DMG is introduced on the surface of LNP. Folic acid can specifically bind to the folic acid receptor-α (FR-α) highly expressed on the surface of breast cancer tissue cells, thereby realizing the active targeted delivery of LNP-mRNA nanoparticles to breast cancer tissue, increasing the drug concentration in the target tissue, reducing damage to normal tissue, and reducing adverse reactions.

[0046] The LNP particle size distribution of the present invention is as follows: Figure 2 As shown, the LNP particles have a uniform particle size.

[0047] 2.4 Formulation Process The preparation process of the mRNA vaccine (LNP-mRNA nanoparticle formulation) of the present invention is stable and controllable, and suitable for industrial production. The specific steps are as follows: (1) Preparation of aqueous phase: The mRNA obtained by in vitro transcription was dissolved in citrate buffer at pH 4.0 to prepare an aqueous phase of mRNA with a concentration of 0.2 mg / mL. It was stored at 4°C for later use to avoid mRNA degradation.

[0048] (2) Preparation of lipid ethanol phase: DLin-MC3-DMA, DSPC, cholesterol, PEG2000-DMG and folic acid-PEG2000-DMG are dissolved in anhydrous ethanol according to the molar ratio described in 2.3 above. The mixture is stirred until completely dissolved to form a homogeneous lipid ethanol phase, which is then stored at room temperature.

[0049] (3) Microfluidic mixing and self-assembly: The aqueous phase of mRNA and the ethanol phase of lipids are injected into the two injection channels of the NanoAssemblr™ microfluidic chip at a volume ratio of 3:1. The flow rate is set to 12 mL / min. The two liquids are rapidly mixed in the chip and self-assembled instantly to form LNP-mRNA nanoparticles. Microfluidic technology can ensure that the mixing process is fast and uniform, and obtain nanoparticles with uniform particle size and high encapsulation efficiency.

[0050] (4) Ultrafiltration / dialysis: The LNP-mRNA solution formed by the above self-assembly is concentrated by ultrafiltration through an ultrafiltration membrane, and at the same time, it is replaced with PBS buffer at pH 7.4 to remove anhydrous ethanol from the solution. Finally, the concentration is concentrated to 0.5 mg / mL to ensure that the osmotic pressure and pH of the preparation meet the requirements for in vivo injection.

[0051] (5) Aseptic processing and filling and storage: The concentrated LNP-mRNA solution is aseptically filtered through a 0.22 μm sterile filter membrane to remove any possible microbial impurities; then the sterile solution is filled into 2 mL neutral borosilicate glass bottles filled with nitrogen, sealed and stored at -80℃ in the dark, which can effectively prevent mRNA degradation and LNP aggregation and extend the shelf life of the vaccine.

[0052] 2.5 Dosing regimen The dosing regimen of the mRNA vaccine of this invention for postoperative adjuvant / maintenance therapy in patients with HER2-low expression breast cancer has been optimized to ensure the induction of a strong and durable immune response, as detailed below: (1) Timing of administration: The medication should be started within 4 weeks after the operation. At this time, the patient's immune function has not been completely damaged and the tumor burden is low, which can effectively induce a specific immune response and clear up residual micro lesions after the operation.

[0053] (2) Dosage and route of administration: The single dose is 100 μg, administered via bilateral inguinal subcutaneous injection. This injection method can promote the capture of the vaccine by dendritic cells in local lymph nodes, enhance the efficiency of immune response induction, and reduce systemic adverse reactions.

[0054] (3) Dosing cycle: The basic immunization cycle is to administer the drug once on days 0, 21 and 42, for a total of 3 times; after the basic immunization is completed, the maintenance immunization phase is entered, and the drug is administered once every 8 weeks. The longest dosing period is 1 year, which can ensure that the body maintains an effective anti-HER2 immune response and delay tumor recurrence.

[0055] (4) Combination therapy: To further enhance the therapeutic effect, the vaccine can be used in combination with low-dose PD-1 inhibitors or small molecule HER2 inhibitors. When combined with PD-1 inhibitors (such as sintilimab), the dose of sintilimab is 200 mg, administered once every 3 weeks, in synergy with the vaccine administration cycle. When combined with small molecule HER2 inhibitors (such as pyrotinib), pyrotinib is administered in a metronid fashion, with 320 mg orally daily, which can enhance the infiltration of T cells in tumor tissue, reverse the immunosuppressive microenvironment, and form a synergistic anti-tumor effect with the vaccine.

[0056] 3. The relevant sequences involved in this application are as follows:

[0057] Signal peptide (SEQ ID NO.7): MDAMKRGLCCVLLLCGAVFVSP Antigen multiply (SEQ ID NO.8): MDAMKRGLCCVLLLCGAVFVSPGGSGGGSGGGSMDVGSCTLGGSGGGSGGG SMDVGSCTLVGGSGGGSGGGYLSTDVASCGGSGGGSGGGYLSTDVASCTGGSGGGSGGGSVFQKLQVIGGSGGGSGGGILIKRQQQKIGGSGGGSGGGGISAVVGILLVVVLGVVFGILIKRRGGSGGGSGGGQQKIRKYTMRRL 5'UTR (SEQ ID NO.9): ACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCAAACAGACACC 3'UTR (SEQ ID NO. 10): CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGC ACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC Note: Because in NIPO ST.26, "T" represents uracil in the RNA sequence and thymine in the DNA sequence, the "U" in the above sequences was converted to "T" during the manufacturing process.

[0058] 4. The reagents and instruments used in this invention are as follows: T7 RNA polymerase, DNase I, CleanCap® Reagent AG, and 1-methyl-ψ-UTP were purchased from Thermo Fisher Scientific; DLin-MC3-DMA, DSPC, PEG2000-DMG, and folic acid-PEG2000-DMG were purchased from Avanti Polar Lipids; citrate buffer and PBS buffer were prepared in-house; NanoAssemblr™ microfluidic chip was purchased from Precision NanoSystems; Malvern Zetasizer nanoparticle size analyzer was purchased from Malvern Instruments Ltd.; transmission electron microscope was purchased from FEI Ltd.; ELISPOT assay kit was purchased from BD Ltd.; HLA-A02:01 positive healthy donor PBMCs were purchased from Peking Union Medical College Cell Resource Center; 4T1 cells and MDA-MB-361 cells were purchased from the Cell Bank of the Chinese Academy of Sciences; Balb / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; sintilimab was purchased from Xinda Biopharmaceutical; and pyrotinib was purchased from Hengrui Medicine.

[0059] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0060] Example 1: In vitro transcription and capping of mRNA This embodiment prepares the target mRNA according to the sequence design described in 2.1 and 2.2 above. The specific steps are as follows: (1) Plasmid construction: The antigen coding sequence shown in SEQ ID NO.8, the 5'-UTR sequence shown in SEQ ID NO.9, the 3'-UTR sequence shown in SEQ ID NO.10, and the 120 nt Poly(A) tail + 10-nt restriction enzyme spacer sequence were tandemly cloned into the pVAX1.0-A120 vector containing the T7 promoter to construct the recombinant expression plasmid pVAX1.0-T7-5'UTR-antigen-3'UTR-Poly(A); the recombinant plasmid was transformed into DH5α competent cells, positive clones were screened, and after the sequence was verified to be correct by sequencing, a large number of recombinant plasmids were extracted.

[0061] (2) Plasmid linearization: Take the above recombinant plasmid, add BamHI restriction endonuclease, and digest it in a water bath at 37℃ for 4 h. After digestion, verify the digestion effect by agarose gel electrophoresis to ensure that the plasmid is completely linearized. Then, recover the linearized plasmid by phenol-chloroform extraction and ethanol precipitation to remove restriction endonuclease and impurities.

[0062] (3) In vitro transcription: Prepare an in vitro transcription reaction system (50 μL) including: 2 μg linearized plasmid template, 2 μL T7 RNA polymerase, 5 μL 10× transcription buffer, 2 μL each of ATP / CTP / GTP (100 mM), 2 μL 1-methyl-ψ-UTP (100 mM), 1 μL RNase inhibitor, and add enzyme-free water to 50 μL; place the reaction system in a 37℃ water bath for 2 h to achieve in vitro transcription of mRNA, and introduce 1-methyl-ψ modification at the same time.

[0063] (4) Co-transcription capping: Add 1 μL of CleanCap® Reagent AG to the above transcription reaction system and continue the reaction at 37℃ for 1 h to achieve co-transcription capping of mRNA and obtain mRNA with Cap1 structure.

[0064] (5) mRNA purification: Add 1 μL of DNase I to the reaction system and react at 37℃ for 30 min to degrade the template DNA; then add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), shake vigorously and centrifuge, and take the supernatant; add 2 volumes of anhydrous ethanol and 1 / 10 volume of 3 M NaAc (pH 5.2) to the supernatant, stand at -80℃ for 30 min, centrifuge to recover the mRNA precipitate; wash the precipitate twice with 75% ethanol, dry it, and dissolve the mRNA in enzyme-free water to obtain purified mRNA.

[0065] (6) mRNA identification: The concentration and purity of mRNA were determined using Nanodrop to ensure that A260 / A280 = 1.8-2.0 and that the purity was qualified; the integrity of mRNA was determined using an Agilent 2100 bioanalyzer to ensure that RIN (RNA integrity number) ≥ 9.0; the target mRNA with a concentration of 3.2 mg / mL was finally obtained and stored at -80℃ in the dark for later use.

[0066] Example 2: Preparation of LNP-mRNA Vaccine This embodiment prepares an LNP-mRNA vaccine according to the LNP composition and formulation process described in 2.3 and 2.4 above. The specific steps are as follows: (1) Preparation of aqueous phase: Take the mRNA prepared in Example 1, dilute it with citrate buffer at pH 4.0, prepare an aqueous phase of mRNA with a concentration of 0.2 mg / mL, and store it at 4℃ for later use.

[0067] (2) Preparation of lipid ethanol phase: Weigh each lipid component according to the molar ratio of DLin-MC3-DMA:DSPC:cholesterol:PEG2000-DMG:folic acid-PEG2000-DMG=50:10:38.5:1.0:0.5, dissolve in anhydrous ethanol, stir until completely dissolved to form a homogeneous lipid ethanol phase, and set aside at room temperature.

[0068] (3) Microfluidic mixing self-assembly: The aqueous phase of mRNA and the ethanol phase of lipid were injected into the two injection channels of the NanoAssemblr™ microfluidic chip at a volume ratio of 3:1. The flow rate was set to 12 mL / min. The two liquids were rapidly mixed in the chip and instantly self-assembled to form LNP-mRNA nanoparticles.

[0069] (4) Ultrafiltration / dialysis: Add the LNP-mRNA solution formed by the above self-assembly to an ultrafiltration centrifuge tube, centrifuge at 4℃ and 5000rpm for 10 min, and collect the retentate; then wash the solution three times with PBS buffer at pH 7.4 to remove anhydrous ethanol, and concentrate the mRNA concentration to 0.5 mg / mL.

[0070] (5) Identification of LNP-mRNA: The particle size, PDI, and Zeta potential of LNP were determined using a Malvern Zetasizer nanoparticle size analyzer. The results showed that the LNP particle size was 78 nm and the PDI was 0.08 ( Figure 2 The zeta potential was -5 mV, which meets the parameter requirements set in this invention. The mRNA encapsulation efficiency was determined by the RiboGreen method, and the results showed that the encapsulation efficiency was 96.4%, ≥95%, indicating good encapsulation effect.

[0071] (6) Aseptic processing and filling and storage: The concentrated LNP-mRNA solution is aseptically filtered through a 0.22 μm sterile filter membrane, then filled into a 2 mL neutral borosilicate glass bottle with nitrogen, sealed and stored at -80℃ in the dark to obtain the finished mRNA vaccine of the present invention.

[0072] Example 3: In vitro efficacy experiment of mRNA vaccine This embodiment aims to verify the in vitro immunogenicity of the mRNA vaccine of the present invention. The specific experiments are as follows: (1) DC cell induction: Peripheral blood mononuclear cells (PBMCs) from HLA-A02:01 positive healthy donors were obtained by separating mononuclear cells with lymphocyte separation medium and seeded into 6-well plates. RPMI 1640 medium containing GM-CSF (100 ng / mL) and IL-4 (50 ng / mL) was added and cultured in a 37℃, 5% CO2 incubator for 5 days to induce PBMC differentiation into immature dendritic cells (DCs). On the 5th day, LPS (1 μg / mL) was added and cultured for another 2 days to induce DC maturation.

[0073] (2) Vaccine loading and co-culture: Mature DC cells were divided into three groups and treated respectively: ① Blank control group (no reagents added); ② Unmodified mRNA group (mRNA prepared in Example 1 without 1-methyl-ψ modification was added, with a final concentration of 100 ng / mL); ③ Vaccine group of the present invention (LNP-mRNA vaccine prepared in Example 2 was added, with a final mRNA concentration of 100 ng / mL); Each group was set with 3 replicates and cultured at 37℃ and 5% CO2 for 24 h to load mRNA and express antigen in DC cells.

[0074] (3) CD8+ T cell isolation and co-culture: CD8+ T cells were isolated from PBMCs of the same healthy donor using a CD8+ T cell magnetic bead separation kit, and the cell concentration was adjusted to 1×10⁻⁶. 6 / mL; Mix DC cells loaded with mRNA with CD8+ T cells at a ratio of 1:10, seed them into 96-well plates, add RPMI 1640 medium containing IL-2 (20 U / mL), and incubate at 37°C in a 5% CO2 incubator for 7 days.

[0075] (4) Detection indicators and results: ① Percentage of IFN-γ+ CD8+ T cells: After 7 days of co-culture, cells were collected, and anti-CD8 antibody and anti-IFN-γ antibody were added. The percentage of IFN-γ+ CD8+ T cells was detected by flow cytometry. The results showed that the percentage of IFN-γ+ CD8+ T cells was 2.3% in the blank control group, 11.2% in the unmodified mRNA group, and 28.7% in the vaccine group of this invention. The percentage of IFN-γ+ CD8+ T cells in the vaccine group of this invention was significantly higher than that in the unmodified mRNA group (p<0.01), indicating that the vaccine of this invention can significantly induce CD8+ T cell activation and generate a specific IFN-γ response.

[0076] ②ELISPOT assay: The specific response of CD8+ T cells to the HER2-369(YVL) peptide was detected using the ELISPOT assay kit. Results showed that the number of specific IFN-γ spots in the vaccine group was 312±25 / 10^5 cells, significantly higher than that in the unmodified mRNA group (108±17 / 10^5 cells) and the blank control group (18±4 / 10^5 cells) (p<0.001). Figure 3 As shown, the vaccine of the present invention can induce a potent HER2-specific CD8+ T cell response.

[0077] Conclusion: The mRNA vaccine of the present invention can effectively induce HER2-specific CD8+ T cell activation in vitro, and its immunogenicity is significantly better than that of unmodified mRNA, providing in vitro experimental evidence for its in vivo anti-tumor effect.

[0078] Example 4: Combined drug trial of mRNA vaccine This embodiment aims to evaluate the combined antitumor effect of the mRNA vaccine of the present invention with a PD-1 inhibitor (sintilimab) and a small molecule HER2 inhibitor (pyrotinib). In vitro and in vivo combination experiments were conducted, and the mechanism of action of the combined drugs was explored, as detailed below: 1. In vitro combined experiment (1) Cell model: The HLA-A02:01 positive HER2 low-expressing human breast cancer cell line MDA-MB-361 (IHC 1+, HER2 expression level of about 5000 copies / cell) was seeded into culture flasks and cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. The cells were used for experiments after reaching the logarithmic growth phase.

[0079] (2) Experimental grouping: MDA-MB-361 cells were seeded into 96-well plates, and CD8+ T cells induced and cultured in Example 3 (HER2-specific CD8+ T cells induced after DC loading with the vaccine of this invention) were added at the same time. The cell ratio was tumor cells:CD8+ T cells = 1:5; then the following 6 groups were set up, with 3 replicates in each group: ① Control group: Only PBS buffer was added; ② Single-drug group A: Added the mRNA vaccine of this invention (final mRNA concentration 100 ng / mL); ③ Single-drug group B: Add sintilimab (final concentration 10 μg / mL); ④ Single-drug group C: Added pyrotinib (final concentration 1 μM); ⑤ Combination group AB: Added the mRNA vaccine of this invention (100 ng / mL) + sintilimab (10 μg / mL); ⑥ Combined group AC: Added the mRNA vaccine of this invention (100 ng / mL) + pyrotinib (1 μM).

[0080] (3) Culture and detection: The 96-well plate was placed in a 37℃, 5% CO2 incubator for 48 h, and then the following indicators were detected: ① Tumor cell killing rate: The killing rate of CD8+ T cells against MDA-MB-361 cells was determined using the LDH release assay, following the instructions of the LDH detection kit. Results showed that the tumor killing rate of the combined group AB was 68.3 ± 5.2%, significantly higher than that of the single-drug group A (42.1 ± 4.7%) and single-drug group B (18.9 ± 3.1%) (p<0.001); the tumor killing rate of the combined group AC was 62.5 ± 4.9%, significantly higher than that of the single-drug groups A and C (23.7 ± 3.5%) (p<0.001), indicating that the combined use of the vaccine and the two inhibitors significantly enhanced the killing ability of T cells against tumor cells.

[0081] ② Cytokine secretion levels: Culture supernatant was collected, and the secretion levels of IFN-γ and Granzyme B were detected using an ELISA kit. The results showed that the IFN-γ secretion level in the combined treatment group AB was 1320 ± 105 pg / mL, and the Granzyme B secretion level was 890 ± 78 pg / mL, which were significantly higher than those in the single-drug group A (IFN-γ 780 ± 65 pg / mL, Granzyme B 450 ± 52 pg / mL) and the single-drug group B (IFN-γ 320 ± 42 pg / mL, Granzyme B 180 ± 31 pg / mL). The IFN-γ secretion level in the combined treatment group AC was 1250 ± 98 pg / mL, and the Granzyme B secretion level was 820 ± 71 pg / mL, which were significantly higher than those in the single-drug group A and the single-drug group C (IFN-γ 280 ± 38 pg / mL, Granzyme B 160 ± 29 pg / mL), indicating that the combined treatment can significantly enhance the activation level of T cells.

[0082] ③ Changes in PD-L1 expression: MDA-MB-361 cells were collected, anti-PD-L1 antibody was added, and the expression level of PD-L1 on the surface of tumor cells was detected by flow cytometry (expressed as mean fluorescence intensity, MFI). The results showed that the PD-L1 MFI of the single-drug group A was 310, which was significantly higher than that of the control group (120), indicating that the vaccine of the present invention can upregulate the expression of PD-L1 on the surface of tumor cells, suggesting that the vaccine and PD-1 inhibitor have complementary mechanisms, and the combined use can effectively reverse PD-L1-mediated immune escape.

[0083] 2. In vivo combined therapy experiment (4T1 / HER2-low mouse model) (1) Animal model construction: Fifty Balb / c female mice (weighing 18-22 g) aged 6-8 weeks were randomly divided into 5 groups of 10 mice each; the concentration of 4T1 cells stably transfected with HER2-low (approximately 5,000 copies / cell) (4T1 / HER2-low cells) was adjusted to 1×10⁻⁶. 6 / mL, 100 μL (containing 1×10⁻⁶) was injected into the right mammary fat pad of each mouse. 5 Establish a HER2-low expression breast cancer mouse model using 100-150 mm² cells; wait until the tumor volume grows to 100-150 mm². 3 At that time, tumor resection was performed (simulating a clinical postoperative scenario), the wound was sutured after the operation, and the mice were given medication 7 days after recovery (7 days after the operation).

[0084] (2) Dosing regimen: ① Group 1 (control group): Intraperitoneal injection of PBS buffer, once every 3 days until the end of the experiment; Group 2 (vaccine monotherapy group): The mRNA vaccine of this invention was injected subcutaneously into both groins at a dose of 10 μg / animal, and the administration was carried out on the 7th and 28th days after the operation, for a total of 2 times; ③ Group 3 (PD-1 inhibitor monotherapy group): Intraperitoneal injection of anti-mouse PD-1 antibody, dose of 200 μg / mouse, once every 5 days, for a total of 4 times; Group 4 (Vaccine + PD-1 Inhibitor Combination Group): The dosing regimens of Group 2 and Group 3 were administered simultaneously, with coordinated dosing times; Group 5 (Vaccine + Pyrotinib combination group): The mRNA vaccine of this invention was injected subcutaneously into both groins (dose 10 μg / animal, once on postoperative days 7 and 28), and pyrotinib was administered by gavage daily (dose 30 mg / kg / d) for 28 days starting from postoperative day 7 (d7-d35).

[0085] (3) Endpoint indicator detection and results: ① Number of lung metastatic nodules: On day 42 of the experiment, mice were sacrificed, lung tissue was dissected, fixed with 4% formaldehyde, and the number of lung metastatic nodules was counted under a dissecting microscope. The results showed that the number of lung metastatic nodules in the control group was 45±7, in the vaccine monotherapy group it was 8±3, in the PD-1 inhibitor monotherapy group it was 32±5, in combination group 4 (vaccine + PD-1) it was 3±1, and in combination group 5 (vaccine + pyrotinib) it was 4±2. The number of lung metastatic nodules in combination groups 4 and 5 was significantly lower than that in the vaccine monotherapy group (p<0.01), indicating that combination therapy can significantly enhance the anti-metastatic effect.

[0086] ② Survival observation: Starting from the 7th day after surgery, the survival status of the mice was observed daily, the time of death was recorded, and a survival curve was plotted. Figure 4 The results showed that the median survival was 36 days in the control group, 67 days in the vaccine monotherapy group, 45 days in the PD-1 inhibitor monotherapy group, 82 days in combination group 4 (vs. vaccine monotherapy group p=0.008), and 79 days in combination group 5 (vs. vaccine monotherapy group p=0.012), indicating that combination therapy can significantly prolong the survival of mice.

[0087] ③ Splenic T-cell infiltration: On day 42 of the experiment, mice were sacrificed, the spleen was dissected, and a single-cell suspension of the spleen was prepared. The ratio of CD8+ T cells to Treg cells (regulatory T cells) in the spleen was detected by flow cytometry. The results showed that the CD8+ / Treg ratio in the combination group 5 was 5.8, which was significantly higher than that in the vaccine monotherapy group (2.5), indicating that the combination of vaccine and pyrotinib can increase CD8+ T-cell infiltration, reduce the proportion of Treg cells, and reverse the immunosuppressive microenvironment.

[0088] ④ Safety Testing: During the experiment, the mice were weighed weekly. At the end of the experiment, mouse serum was collected, and the levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), and BUN (blood urea nitrogen) in the serum were measured. The results showed that the weight of mice in all treatment groups did not decrease significantly (weight change rate <10%), and the serum ALT, AST, and BUN levels were all within the normal range, with no statistically significant difference compared to the control group. This indicates that the combined drug treatment was safe and had no significant liver or kidney toxicity.

[0089] 3. Exploration of the mechanism of combined drug use (1) RNA-seq analysis: Tumor tissues from mice in combination group 4 (vaccine + PD-1 inhibitor) and the vaccine monotherapy group were collected, and total RNA was extracted and RNA-seq sequencing analysis was performed to detect differentially expressed genes. The results showed that compared with the vaccine monotherapy group, the expression levels of IFN-γ pathway-related genes (IFN-γ, STAT1, IRF1), antigen presentation-related genes (MHC-I, CD80, CD86), and T cell recruitment-related genes (CXCL9, CXCL10) in combination group 4 were significantly upregulated, indicating that the combination with PD-1 inhibitor can further enhance the activation of immune response-related pathways and promote T cell infiltration and antigen presentation.

[0090] (2) TCRβ sequencing: CD8+ T cells were collected from mice in the combination group 4 and the vaccine monotherapy group, and genomic DNA was extracted for TCRβ sequencing to analyze T cell clonal diversity. The results showed that the number of HER2-specific T cell clones in the combination group 4 was significantly higher than that in the vaccine monotherapy group, and the clonal expansion was more persistent, indicating that the combination of PD-1 inhibitors can promote the clonal expansion of HER2-specific T cells and enhance the persistence of the immune response.

[0091] Conclusion: The mRNA vaccine of this invention, when used in combination with PD-1 inhibitors or small molecule HER2 inhibitors, exhibits a significant synergistic anti-tumor effect. The mechanism may be related to upregulation of the IFN-γ pathway, enhanced antigen presentation, promotion of T cell infiltration and clonal expansion, and reversal of the immunosuppressive microenvironment, supporting its application as a combination therapy strategy in clinical practice.

[0092] Example 5: Safety Experiment of mRNA Vaccine (Repeated Dosing Toxicity Study in Cynomolgus Monkeys) This embodiment aims to verify the in vivo safety of the mRNA vaccine of the present invention by conducting a repeated-dose toxicity test on cynomolgus monkeys, as detailed below: (1) Experimental animals: 18 healthy cynomolgus monkeys weighing 3-5 kg ​​were selected, half male and half female, and randomly divided into 3 groups of 6 animals each: low-dose group (1 mg / kg), medium-dose group (3 mg / kg), and blank control group (PBS).

[0093] (2) Dosing regimen: The drug was administered subcutaneously once every 2 weeks for a total of 5 times. The volume of each dose was adjusted according to the weight of the cynomolgus monkeys. The blank control group was injected with an equal volume of PBS buffer.

[0094] (3) Detection indicators: ① General toxicity observation: Observe the general condition of the cynomolgus monkeys daily, including their mental state, diet, activity, and feces, and record any abnormal reactions.

[0095] ② Clinical symptoms and weight: Weigh the cynomolgus monkeys weekly and record weight changes; within 24 hours after administration, observe for local reactions such as redness and swelling at the injection site and itching, as well as systemic reactions such as fever and vomiting, and assess the degree of toxicity according to the toxicity grading standard (Grade 0-4).

[0096] ③ Hematological and biochemical tests: Venous blood was collected from cynomolgus monkeys before administration, 7 days after each administration, and 14 days after the last administration to test complete blood count (white blood cells, red blood cells, platelets, etc.) and serum biochemical indicators (ALT, AST, BUN, creatinine, IL-6, TNF-α, etc.).

[0097] ④ Pathological examination: 14 days after the last administration, the cynomolgus monkeys were euthanized, and the morphology of major organs such as the heart, liver, spleen, lungs, kidneys, and lymph nodes was observed. Tissues from each organ were stained with hematoxylin and eosin (HE), and pathological sections were observed to assess whether there was any pathological damage to the organs.

[0098] (4) Experimental results: During the administration period, all cynomolgus monkeys had normal mental state, diet and activity, and no obvious abnormal reactions. There were no local reactions such as redness, swelling and itching at the injection site, and no systemic toxicity reactions of Grade ≥2. There was no statistical difference in weight change compared with the blank control group. Blood routine and serum biochemical indicators (ALT, AST, BUN, creatinine, etc.) were all within the normal range and there was no statistical difference compared with the blank control group. There was no statistical difference in peak serum IL-6 and TNF-α levels compared with the blank control group, and no obvious cytokine storm was observed. Pathological examination showed that there was no obvious pathological damage in any of the major organs, which was consistent with the blank control group.

[0099] Conclusion: The mRNA vaccine of this invention showed good safety, no obvious toxic reactions, and high tolerability in repeated-dose trials on cynomolgus monkeys, providing a safety basis for its clinical application.

[0100] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An mRNA molecule, characterized in that, Its nucleotide sequence from the 5' end to the 3' end includes: (a) Nucleotide sequence encoding human tPA signal peptide; (b) A nucleotide sequence encoding a HER2 multiepitope string, wherein the HER2 multiepitope string is composed of neoantigen peptides with amino acid sequences as shown in SEQ ID NO.1-SEQ ID NO.6, connected in series by flexible linkers; (c) The nucleotide sequence encoding the HER2 transmembrane domain; and (d) Nucleotide sequence encoding the intracellular domain segment of HER2; The uridine in the mRNA molecule is replaced by 1-methyl-pseudouridine, the 5' end has a Cap1 structure, and the 3' end has a polyadenylate tail of 100-150 nucleotides in length.

2. The mRNA molecule according to claim 1, characterized in that, The amino acid sequence of the human tPA signal peptide is shown in SEQ ID NO.7; the amino acid sequence of the HER2 transmembrane domain is shown in SEQ ID NO.11; and the amino acid sequence of the HER2 intracellular domain fragment is shown in SEQ ID NO.

12.

3. The mRNA molecule according to claim 2, characterized in that, The coding sequence of the mRNA molecule has been optimized with human codons, and the complete amino acid sequence is shown in SEQ ID NO.

8.

4. The mRNA molecule according to claim 3, characterized in that, The backbone structure of the mRNA molecule is: 5'-Cap1-Kozak sequence-5'-UTR-coding sequence-3'-UTR-Poly(A)120-restriction spacer sequence, wherein the Kozak sequence is GCCACC, the 5'-UTR sequence is shown in SEQ ID NO.9, and the 3'-UTR sequence is shown in SEQ ID NO.

10.

5. An mRNA vaccine, characterized in that, It includes the mRNA molecule according to any one of claims 1-4, and lipid nanoparticles encapsulating the mRNA molecule; the lipid nanoparticles include ionizable cationic lipids, neutral cofactor lipids, cholesterol and polyethylene glycol-modified lipids, and the lipid nanoparticles further include folic acid-modified polyethylene glycol-modified lipids.

6. The mRNA vaccine according to claim 5, characterized in that, The lipid nanoparticles contain ionizable cationic lipids, neutral cofactor lipids, cholesterol, PEGylated lipids, and folic acid-modified PEGylated lipids in a molar ratio of (45-55):(8-12):(35-40):(0.5-1.5):(0.4-0.6); the nitrogen-to-phosphorus ratio of the lipid nanoparticles to mRNA is 4-8; the lipid nanoparticles have a particle size of 75-85 nm, a PDI < 0.1, and an mRNA encapsulation efficiency ≥ 95%.

7. A method for preparing an mRNA vaccine as described in any one of claims 4-5, characterized in that, Includes the following steps: (1) Provide the mRNA molecule according to any one of claims 1-4; (2) Dissolve the mRNA molecule in an aqueous buffer to obtain an aqueous phase; dissolve the lipid component in an organic solvent to obtain an organic phase, wherein the lipid component includes ionizable cationic lipids, neutral auxiliary lipids, cholesterol, polyethylene glycol-modified lipids and folic acid-modified polyethylene glycol-modified lipids; (3) The aqueous phase and the organic phase are mixed by microfluidics to encapsulate the mRNA molecules with lipid nanoparticles; (4) The obtained lipid nanoparticles were purified and concentrated to obtain an mRNA vaccine.

8. The preparation method according to claim 7, characterized in that, In step (1), mRNA molecules are obtained by in vitro transcription and the Cap1 structure is introduced by co-transcription capping. In step (2), the aqueous buffer is citrate buffer with pH 3.5-4.5, the mRNA concentration is 0.1-0.3 mg / mL, and the organic solvent is anhydrous ethanol. In step (3), the volume ratio of the aqueous phase to the organic phase is (2-4):1, and the microfluidic mixing flow rate is 8-15 mL / min. In step (4), ethanol is removed by ultrafiltration or dialysis, and buffer replacement is performed with phosphate buffer. Then, the mixture is filtered through a 0.22 μm filter membrane for sterilization.

9. Use of the mRNA vaccine according to any one of claims 4-5 in the preparation of a medicament for treating HER2-positive or HER2-low expression breast cancer.

10. The application according to claim 9, characterized in that, The HER2-low expression breast cancer is IHC 1+ and / or 2+ / FISH negative breast cancer; the drug is used for adjuvant therapy and / or maintenance therapy after breast cancer surgery; the drug can be used in combination with immune checkpoint inhibitors or HER2 small molecule inhibitors.

Images