A dual-target mRNA vaccine, preparation method and application thereof

Abstract

This invention provides a dual-target mRNA vaccine, its preparation method, and its applications, belonging to the field of medical preparation technology. The mRNA is transcribed from a target gene, which includes TROP2 scFv and NKG2D scFv. The nucleotide sequence of TROP2 scFv is shown in SEQ ID NO.2 of the sequence listing, and the nucleotide sequence of NKG2D scFv is shown in SEQ ID NO.4 of the sequence listing. The vaccine of this invention, by targeting the two antigens TROP2 and NKG2DL, enhances antigen targeting and can improve the therapeutic effect on triple-negative breast cancer. This invention improves the therapeutic effect of the LNP / mRNA-TROP2-NKG2DL-CAR vaccine on triple-negative breast cancer by optimizing the artificial nucleic acid sequences of TROP2 scFv and NKG2D scFv.

Description

Technical Field

[0001] This invention relates to a dual-target mRNA vaccine, its preparation method, and its application, belonging to the field of medical preparation technology. Background Technology

[0002] Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by a lack of significant expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR), accounting for approximately 15-20% of invasive breast cancers. In the absence of well-defined targets, patients with triple-negative breast cancer cannot benefit from endocrine therapy or other available targeted therapies (monoclonal antibodies). To date, standard treatment still relies on surgery, adjuvant chemotherapy, and radiotherapy. Therefore, there is an urgent need to develop effective treatment strategies for patients with triple-negative breast cancer.

[0003] Bioinformatics analysis shows that trophoblast cell surface antigen 2 (TROP2) is a calcium-conducting transmembrane protein with high expression levels in many malignant tumors, especially triple-negative breast cancer; and TROP2 expression is associated with poor prognosis, thus becoming a potential therapeutic target for triple-negative breast cancer.

[0004] Members of the natural killer cell group 2D ligand (NKG2DL) family are expressed on cancer cells but are not typically found in healthy tissues; therefore, they are promising tumor antigens for novel immunotherapies. Studies have found that NKG2DL is highly expressed in triple-negative breast cancer tissues, making it an ideal candidate for targeted therapy against TNBC.

[0005] CN117903317A discloses a TROP2-targeted CAR molecule, CAR-T cells, and their applications. This patent uses the high expression of the TROP2 antigen cluster in triple-negative breast cancer tumor cells as a new treatment strategy to construct CAR-T cells that can specifically bind to the TROP2 anti-epitope of tumor cells, thereby playing a targeted killing role. This provides a newer, more effective, and feasible cell therapy option for triple-negative breast cancer patients, while reducing damage to the patient's own tissues and organs.

[0006] The aforementioned patent mainly targets TROP2-based single-target CAR-T cells. However, single-target CAR-T cell therapy is not as effective as dual-target CAR-T cell therapy. The killing rate of this patent against triple-negative breast cancer cells is only about 60%, and the treatment effect is not ideal.

[0007] CN120960402B discloses the application of fibrinogen α chain in CAR-T therapeutic drugs. This patent provides an application of fibrinogen α chain in the preparation of CAR-T therapeutic drugs. The fibrinogen α chain, by activating the TLR4-NF-κB signaling pathway, significantly increases the expression level and proportion of TROP2 in triple-negative breast cancer, demonstrating enhanced sensitivity to TROP2-targeted CAR-T therapy.

[0008] The aforementioned patent also belongs to single-target CAR-T cell therapy. Although it activates the TLR4-NF-κB signaling pathway through fibrinogen α chain to increase the killing rate of TROP2-CAR-T against TNBC cells, its killing effect is significantly lower than 80%.

[0009] Traditional single-target CAR-T therapy can only kill cells that express that target, while tumor cells that do not express it will be "missed," leading to reduced efficacy or even drug resistance.

[0010] The use of lipid nanoparticles that target TROP2 and NKG2DL dual-target mRNA for the treatment of triple-negative breast cancer is not yet publicly available in the current technology. Summary of the Invention

[0011] To address the shortcomings of existing technologies, this invention provides a dual-target mRNA vaccine, its preparation method, and its application, which improves the therapeutic effect on triple-negative breast cancer by targeting two antigens, TROP2 and NKG2DL.

[0012] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A dual-target mRNA vaccine, wherein the mRNA is transcribed from a target gene, the target gene including TROP2scFv and NKG2DscFv, the nucleotide sequence of TROP2scFv is shown in SEQ ID NO.2 of the sequence listing, and the nucleotide sequence of NKG2DscFv is shown in SEQ ID NO.4 of the sequence listing.

[0013] The target gene is obtained by sequentially connecting the following modules: CD8 signal peptide, TROP2 scFv, Linker, NKG2D scFv, CD8 Hinge region, CD8α transmembrane region, 4-1BB co-stimulatory region, CD3ε signal transduction region, T2A, IL-15.

[0014] The nucleotide sequence of the CD8 signal peptide is shown in SEQ ID NO.1 of the sequence listing; the nucleotide sequence of the linker is shown in SEQ ID NO.3 of the sequence listing; the nucleotide sequence of the CD8 Hinge region is shown in SEQ ID NO.5 of the sequence listing; the nucleotide sequence of the CD8α transmembrane region is shown in SEQ ID NO.6 of the sequence listing; the nucleotide sequence of the 4-1BB co-stimulatory region is shown in SEQ ID NO.7 of the sequence listing; the nucleotide sequence of the CD3ε signal transduction region is shown in SEQ ID NO.8 of the sequence listing; the nucleotide sequence of T2A is shown in SEQ ID NO.9 of the sequence listing; and the nucleotide sequence of IL-15 is shown in SEQ ID NO.10 of the sequence listing.

[0015] The mRNA vaccine is packaged using LNP.

[0016] The LNP comprises DLin-MC3-DMA, DSPC, cholesterol, and PEG200-DMG in a mass ratio of (10.2-10.6):(2.6-2.8):(4.7-5.0):(2.0-2.4), preferably 10.4:2.7:4.9:2.2.

[0017] The method for preparing the dual-target mRNA vaccine involves constructing a recombinant plasmid of the target gene, linearizing and digesting the DNA fragment with enzymes to obtain mRNA, and then using LNP to encapsulate the mRNA to prepare the dual-target mRNA vaccine.

[0018] The application of the dual-target mRNA vaccine in the preparation of drugs for treating triple-negative breast cancer.

[0019] Compared with the prior art, the present invention achieves the following beneficial effects: (1) The vaccine of the present invention enhances antigen targeting by targeting two antigens, TROP2 and NKG2DL, and can improve the therapeutic effect on triple-negative breast cancer compared with vaccines prepared by single targets.

[0020] (2) This invention optimizes the artificial nucleic acid sequences of TROP2 scFv and NKG2D scFv, thereby improving the killing rate of LNP / mRNA-TROP2-NKG2DL-CAR vaccine against triple-negative breast cancer cells, making it more effective against tumors and improving the survival rate of triple-negative breast cancer mice. Attached Figure Description

[0021] Figure 1 A schematic diagram showing the connections of each module in the TROP2 and NKG2DL dual-target CAR; Figure 2Bar chart showing cell viability after co-incubation of LNP / mRNA-CAR and T cells for different times; In this chart, A represents the cell viability histogram of LNP / mRNA-TROP2-NKG2DL-CAR and T cells after co-incubation for different time periods; B represents the cell viability histogram of LNP / mRNA-TROP2-NKG2DL-CAR and T cells after co-incubation for different time periods; C represents the cell viability histogram of LNP / mRNA-TROP2-CAR and T cells after co-incubation for different time periods; and D represents the cell viability histogram of LNP / mRNA-NKG2DL-CAR and T cells after co-incubation for different time periods. Figure 3 Flow cytometry plot of transfection efficiency for LNP / mRNA-CAR-T cells; In the diagram, A is a flow cytometry plot showing the transfection efficiency of LNP / mRNA-TROP2-NKG2DL-CAR-T cells; B is a flow cytometry plot showing the transfection efficiency of LNP / mRNA-TROP2-NKG2DL-CAR2-T cells; C is a flow cytometry plot showing the transfection efficiency of LNP / mRNA-TROP2-CAR-T cells; and D is a flow cytometry plot showing the transfection efficiency of LNP / mRNA-NKG2DL-CAR-T cells. Figure 4 A bar chart showing the killing rate of LNP / mRNA-CAR transfected T cells against triple-negative breast cancer cells; Figure 5 This is a statistical chart showing the survival rate of mice in each group within 50 days in Example 6. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. However, the uses and purposes of these exemplary embodiments are only for illustrating the present invention and do not constitute any limitation on the actual scope of protection of the present invention, nor are they intended to limit the scope of protection of the present invention to these embodiments.

[0023] Example 1: Construction of a dual-target CAR expression vector for TROP2 and NKG2DL The connection diagram of each module of the TROP2 and NKG2DL dual-target CAR is shown below. Figure 1 As shown, the modules and nucleotide sequences of the TROP2 and NKG2DL dual-target CAR expression vector are as follows: (1) CD8 signal peptide (SEQ ID NO.1) (2) Optimized TROP2 scFv (SEQ ID NO.2) (3)Linker (SEQ ID NO.3) (4) Optimized NKG2D scFv (SEQ ID NO.4) (5) CD8 Hinge region (SEQ ID NO.5) (6) CD8α transmembrane region (SEQ ID NO.6) (7) 4-1BB co-stimulatory region (SEQ ID NO.7) (8) CD3ε signal transduction region (SEQ ID NO.8) (9) T2A (SEQ ID NO.9) (10) IL-15 (SEQ ID NO.10) The above sequences were sequentially linked, and the entire expression cassette was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and inserted into the standard vector pUC57 to obtain pUC-TROP2-NKG2DL-CAR-IL-15. This was then transformed into E. coli (DH5α), and after successful sequencing, the recombinant plasmid was extracted from the positive clone and named TROP2-NKG2DL-CAR.

[0024] The optimized TROP2 scFv (SEQ ID NO. 2) and optimized NKG2D scFv (SEQ ID NO. 4) were replaced by unoptimized TROP2 scFv (SEQ ID NO. 11) and unoptimized NKG2D scFv (SEQ ID NO. 12), respectively. The nucleotide sequences of SEQ ID NO. 1, SEQ ID NO. 11, SEQ ID NO. 3, SEQ ID NO. 12, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, and SEQ ID NO. 10 were sequentially ligated to construct a recombinant plasmid named TROP2-NKG2DL-CAR2.

[0025] Omit SEQ ID NO.3 and SEQ ID NO.4, and sequentially connect the nucleotide sequences of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 to construct a recombinant plasmid named TROP2-CAR according to the above method.

[0026] Omit SEQ ID NO.2 and SEQ ID NO.3, and sequentially connect the nucleotide sequences of SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 to construct a recombinant plasmid named NKG2DL-CAR according to the above method.

[0027] The concentration of the above recombinant plasmids was 1.0 μg / μL.

[0028] Example 2: In vitro transcription into mRNA (1) Linearization of recombinant plasmids, enzyme digestion and DNA recovery The enzyme digestion system (total 50 μL) is as follows: 10×Buffer: 5 μL; NotI enzyme: 3 μL; recombinant plasmid: 2 μL (1 μg / μL); water: 40 μL.

[0029] The four recombinant plasmids (TROP2-NKG2DL-CAR, TROP2-NKG2DL-CAR2, TROP2-CAR, and NKG2DL-CAR) prepared in Example 1 were subjected to single-enzyme digestion reactions. Specifically, the digestion was performed in a 37°C water bath for 1 hour, followed by rapid transfer to a 65°C water bath for 5 minutes to terminate the digestion. After the digestion, the DNA was identified by 1% agarose gel electrophoresis, and the size of the obtained DNA fragments was as expected.

[0030] The target DNA fragment was excised and recovered using an agarose gel DNA recovery kit. The concentration of the recovered DNA fragment was measured using a spectrophotometer. The concentration of the DNA fragment was adjusted to 1 μg / μL and stored at -20℃ for later use.

[0031] (2) Transcription into mRNA in vitro The DNA fragments obtained after linearization and enzyme digestion of the above four recombinant plasmids were used to transcribe mRNA in vitro. Using the capped mMESSAGE mMACHINE T7 kit (Ambion), 5′ capped mRNA was synthesized in vitro. At the same time, a poly(A) tail was added to the 3′ end of the mRNA to obtain mRNA with 5′ capped and 3′ tailed structures. The mRNA was then purified to obtain purified mRNA, which was named mRNA-TROP2-NKG2DL-CAR, mRNA-TROP2-NKG2DL-CAR2, mRNA-TROP2-CAR, and mRNA-NKG2DL-CAR, respectively. The concentration of the purified mRNA was adjusted to 2 μg / μL and stored at -80℃ for later use.

[0032] The purified mRNA was placed in a 70°C water bath for 10 min, then on ice for 3 min, and then electrophoresed in a urea-acrylamide gel at a constant voltage of 100V for 30 min. The gel was photographed to verify the size and integrity of the mRNA. The electrophoresis results showed that the mRNA fragments were consistent with the expectations.

[0033] Example 3: Preparation of LNPs loaded with mRNA-CAR (1) Preparation of ethanol solution of liposome mixture Weigh 10.4 mg DLin-MC3-DMA, 2.7 mg DSPC, 4.9 mg cholesterol and 2.2 mg PEG200-DMG, add 1.25 mL ethanol, stir thoroughly to dissolve, and obtain an ethanol solution of the liposome mixture.

[0034] (2) Preparation of mRNA aqueous solution Dissolve 1 μL of the purified mRNA (2 μg / μL) prepared in Example 2 in 1.25 mL of 10 mM citrate buffer (pH=4) and mix by pipetting 50 times. Add 2.5 mL of PBS and mix by pipetting 50 times to obtain an aqueous solution of mRNA.

[0035] (3) Preparation of LNP particles loaded with mRNA-CAR Using the Ignite nanoparticle synthesis system, ethanol solutions of liposome mixtures and aqueous mRNA solutions were mixed at a 1:3 ratio (volume ratio) to synthesize LNP particles encapsulating mRNA-CAR. The obtained LNP particles were dialyzed in PBS solution (pH 7.4) for 24 h, concentrated to a volume of 1 mL using an Amicon ultracentrifuge filter, and filtered twice through a 0.22 μm filter membrane to obtain an LNP / mRNA-CAR solution, which was stored at -20 °C for later use.

[0036] In Example 2, mRNA-TROP2-NKG2DL-CAR, mRNA-TROP2-NKG2DL-CAR2, mRNA-TROP2-CAR, and mRNA-NKG2DL-CAR were prepared according to the above methods to obtain LNP / mRNA-TROP2-NKG2DL-CAR solution, LNP / mRNA-TROP2-NKG2DL-CAR2 solution, LNP / mRNA-TROP2-CAR solution, and LNP / mRNA-NKG2DL-CAR solution, respectively.

[0037] Blank control: The preparation method is the same as above, except that in step (2), an equal amount of DEPC water is used to replace the purified mRNA, and the blank control is represented by LNP / blank.

[0038] (4) Characterization analysis of LNP particles The average diameter and dispersion index (PDI) of the prepared LNP / mRNA-CAR were analyzed using dynamic light scattering (DLS) method, and the encapsulation efficiency of LNP particles was measured using the Quant-iT™ RiboGreen® RNA Reagent and Kit (purchased from Invitorgen).

[0039] The results are shown in Table 1. The encapsulation rate of LNP particles in the LNP / mRNA-CAR solution was over 90%, and almost all of the mRNA was encapsulated.

[0040] Table 1. Characterization of nanoparticles

[0041] Example 4: Cytotoxicity and in vitro transfection experiments of LNP / mRNA-CAR in T cells (1) Preparation of T cells Peripheral blood (PMBCs) were obtained by separating 50 mL of peripheral blood using TBD sample density separation medium (purchased from Tianjin Haoyang Huake Biotechnology). After 24 h of induction culture in DMEM medium (purchased from Corning Pharmaceutical) containing 1000 IU / mL recombinant interferon α2a (purchased from Shenyang Sansheng Pharmaceutical), 1000 IU / mL (final concentration) of recombinant IL-2 (purchased from Shenyang Sansheng Pharmaceutical), 50 ng / mL (final concentration) of OKT-3, and 5 vol% (final concentration) of patient autologous plasma were added, and the culture was continued for another 24 h. Medium was added serially every two days until day 14. Flow cytometry was used to detect the positive expression rates of CD3+ and CD56+ in T cells (CD3-FITC and CD16 / CD56-PE antibodies were purchased from Beckman Coulter, A07735). Results showing a CD3+ positivity rate >90% and a CD3+CD56+ double positivity rate >20% were considered successful T cell induction, resulting in activated T cells.

[0042] (2) Cytotoxicity study of LNP / mRNA-CAR Activated T cells were seeded into 96-well plates (5 × 10⁶ cells / well). 3 Cells / well were cultured overnight, and then LNP / mRNA-TROP2-NKG2DL-CAR, LNP / mRNA-TROP2-NKG2DL-CAR2, LNP / mRNA-TROP2-CAR, and LNP / mRNA-NKG2DL-CAR (final concentration of 2 μg / mL based on mRNA) were added and incubated together for 0 h, 6 h, 12 h, 18 h, and 24 h. T cell viability was then measured. The results are as follows: Figure 2 As shown.

[0043] Depend on Figure 2 It can be seen that none of the four LNP / mRNA-CARs showed significant cytotoxicity to T cells, indicating good safety.

[0044] (3) In vitro transfection experiment Activated T cells were seeded into 24-well plates (6 × 10⁶ cells / well). 4 Cells were divided into four groups (50 ng / well, based on mRNA) and incubated for 24 hours. After incubation, LNP / mRNA-TROP2-NKG2DL-CAR, LNP / mRNA-TROP2-NKG2DL-CAR2, LNP / mRNA-TROP2-CAR, and LNP / mRNA-NKG2DL-CAR (50 ng / well, based on mRNA) were added to each well. Cells were then collected after 48 hours of incubation. Transfection efficiency was determined by detecting GFP expression rate using flow cytometry. Results are shown below. Figure 3 As shown in Table 2.

[0045] Depend on Figure 3 As shown in Table 2, the transfection efficiencies of LNP / mRNA-TROP2-NKG2DL-CAR, LNP / mRNA-TROP2-NKG2DL-CAR2, LNP / mRNA-TROP2-CAR and LNP / mRNA-NKG2DL-CAR vectors on the surface of T cells were 40.7%, 38.6%, 37.2% and 38.1%, respectively.

[0046] Table 2 Transfection efficiency of LNP / mRNA-CAR-T cells

[0047] Example 5: Evaluation of the in vitro tumor-killing ability of the LNP / mRNA-CAR vaccine Activated T cells were seeded into 24-well plates (6 × 10⁶ cells / well). 4 After 24 hours, the cells were divided into five groups and incubated with PBS, LNP / mRNA-TROP2-NKG2DL-CAR, LNP / mRNA-TROP2-NKG2DL-CAR2, LNP / mRNA-TROP2-CAR, and LNP / mRNA-NKG2DL-CAR (50 ng / well, based on mRNA), respectively, for a total of 12 hours. Then, luciferase-labeled triple-negative breast cancer cell line MDA-MB-468 (6 × 10⁻⁶ cells / well) was added. 4 Cells were cultured for 4 hours (1 cell / well). Cells were collected and the tumor cell killing effect in each group was measured using an ELISA reader.

[0048] The specific groups are as follows: Experimental group A: LNP / mRNA-TROP2-NKG2DL-CAR-T cells and MDA-MB-468 cells were co-cultured; Experimental group B: LNP / mRNA-TROP2-NKG2DL-CAR2-T cells and MDA-MB-468 cells were co-cultured; Experimental group C: LNP / mRNA-TROP2-CAR-T cells and MDA-MB-468 cells were co-cultured; Experimental group D: LNP / mRNA-NKG2DL-CAR-T cells and MDA-MB-468 cells were co-cultured; Control group: Activated T cells and MDA-MB-468 cells were co-cultured; The results are as follows Figure 4 As shown in Table 3, the dual-target LNP / mRNA-TROP2-NKG2DL-CAR-T cells (experimental group A) and dual-target LNP / mRNA-TROP2-NKG2DL-CAR2-T cells (experimental group B) of this invention showed significantly higher killing efficiency against triple-negative breast cancer cells than single-target LNP / mRNA-TROP2-CAR-T cells (experimental group C) and LNP / mRNA-NKG2DL-CAR-T cells (experimental group D), and the differences were statistically significant. Furthermore, compared to dual-target LNP / mRNA-TROP2-NKG2DL-CAR2-T cells (experimental group B), the dual-target LNP / mRNA-TROP2-NKG2DL-CAR-T cells (experimental group A) of this invention showed a higher killing rate against triple-negative breast cancer cells, indicating that the optimization of the nucleic acid sequences of TROP2 and NKG2DL can significantly improve the in vitro killing rate of CAR-T cells against triple-negative breast cancer cells, giving them higher anti-tumor activity.

[0049] Table 3. Kill rate of T cells transfected with different LNP / mRNA-CAR against triple-negative breast cancer cells.

[0050] Example 6: In vivo pharmacodynamic evaluation of LNP / mRNA-CAR vaccine A triple-negative breast cancer mouse model was established using female mice. Log-phase human triple-negative breast cancer MDA-MB-468 cells were collected, washed with sterile PBS, and resuspended to a final cell concentration of 1 × 10⁻⁶. 7 Cells / mL were collected to obtain a cell suspension. Mice were anesthetized with isoflurane inhalation, and 100 μL of the cell suspension was slowly injected into the fat pad of the left fourth mammary gland. Mice were fed under routine aseptic conditions, and tumor volume and mouse weight were monitored regularly. When the tumor volume reached 80–120 mm³, the mice were randomly divided into 5 groups (n = 10). Mice were administered PBS (150 μL), LNP / mRNA-TROP2-NKG2DL-CAR, LNP / mRNA-TROP2-NKG2DL-CAR2, LNP / mRNA-TROP2-CAR, and LNP / mRNA-NKG2DL-CAR (0.3 mg / kg, based on mRNA) via tail vein injection, once a week for a total of two administrations (one week apart). Mice were monitored daily for 50 days following the injection, and survival curves were plotted. The results are shown below. Figure 5 As shown in Table 4.

[0051] Depend on Figure 5 It can be seen that, under the same mRNA, the survival rate of mice injected with dual-target LNP / mRNA-CAR vaccine (experimental group A, experimental group B) is higher than that of mice injected with single-target LNP / mRNA-CAR vaccine (experimental group C, experimental group D), indicating that dual-target LNP / mRNA-CAR vaccine has a higher killing effect on tumors and can prolong the survival rate of tumor mice.

[0052] Furthermore, compared to the dual-target LNP / mRNA-TROP2-NKG2DL-CAR2 vaccine (experimental group B), the dual-target LNP / mRNA-TROP2-NKG2DL-CAR vaccine (experimental group A) provided by this invention showed the best survival improvement effect, with a survival rate of up to 80% within 50 days. This indicates that the optimization of the nucleic acid sequences of TROP2 and NKG2DL can significantly improve the in vivo efficacy of the final vaccine.

[0053] Table 4. Survival rate of mice within 50 days

[0054] In summary, the dual-target LNP / mRNA-TROP2-NKG2DL-CAR vaccine constructed in this application can induce a strong immune response in cells and has a significant effect on killing tumor cells.

Claims

1. A dual-target mRNA vaccine, characterized in that: The mRNA is transcribed from the target gene, which includes TROP2 scFv and NKG2D scFv. The nucleotide sequence of TROP2 scFv is shown in SEQ ID NO.2 in the sequence listing, and the nucleotide sequence of NKG2D scFv is shown in SEQ ID NO.4 in the sequence listing.

2. The dual-target mRNA vaccine according to claim 1, characterized in that: The target gene is obtained by sequentially connecting the following modules: CD8 signal peptide, TROP2 scFv, Linker, NKG2D scFv, CD8 Hinge region, CD8α transmembrane region, 4-1BB co-stimulatory region, CD3ε signal transduction region, T2A, IL-15.

3. The dual-target mRNA vaccine according to claim 2, characterized in that: The nucleotide sequence of the CD8 signal peptide is shown in SEQ ID NO.1 of the sequence listing; the nucleotide sequence of the linker is shown in SEQ ID NO.3 of the sequence listing; the nucleotide sequence of the CD8 Hinge region is shown in SEQ ID NO.5 of the sequence listing; the nucleotide sequence of the CD8α transmembrane region is shown in SEQ ID NO.6 of the sequence listing; the nucleotide sequence of the 4-1BB co-stimulatory region is shown in SEQ ID NO.7 of the sequence listing; the nucleotide sequence of the CD3ε signal transduction region is shown in SEQ ID NO.8 of the sequence listing; the nucleotide sequence of T2A is shown in SEQ ID NO.9 of the sequence listing; and the nucleotide sequence of IL-15 is shown in SEQ ID NO.10 of the sequence listing.

4. The dual-target mRNA vaccine according to claim 1, characterized in that: The mRNA vaccine is packaged using LNP.

5. The method for preparing the dual-target mRNA vaccine according to any one of claims 1-4, characterized in that: The method for preparing the dual-target mRNA vaccine involves constructing a recombinant plasmid of the target gene, linearizing and digesting the DNA fragment with enzymes to obtain mRNA, and then using LNP to encapsulate the mRNA to prepare the dual-target mRNA vaccine.

6. The use of the dual-target mRNA vaccine according to any one of claims 1-4 in the preparation of a medicament for treating triple-negative breast cancer.

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