An mRNA drug for enhancing t cell efficacy and application

Abstract

The application discloses an mRNA drug for enhancing T cell efficacy and application, and belongs to the technical field of biological medicine. The mRNA drug encodes a protein, which is a BH3 domain of pro-apoptotic protein Puma or Bim fused with an aptamer scaffold; and the administration mode is an intratumoral administration dosage form. The application provides an mRNA anti-tumor drug, which is locally administered intratumorally in the form of the mRNA drug, induces local cell apoptosis of a lesion, and can synergistically enhance T cell-mediated killing effect. Finally, the effect of synergistically inhibiting growth of a solid tumor in vivo is achieved. The novel mRNA drug provided by the application exhibits a strong and effective tumor inhibition effect in a solid tumor model.

Description

An mRNA drug that enhances T-cell therapy and its application Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an mRNA drug and its application that enhances the therapeutic effect of T cells. Background Technology

[0002] While TCR-T cell therapy has emerged as a promising treatment option for solid tumors, its development still faces numerous obstacles, including high drug resistance due to the suppressive tumor immune microenvironment and insufficient T cell function (Klebanoff et al., 2016; Sadelain et al., 2017). Furthermore, the in vitro engineered manufacturing of T cells faces challenges such as substantial investment and low yields with high failure rates. Current research focuses on combining other drugs to enhance the efficacy of engineered T cells, such as combining immune checkpoint inhibitors and neoantigen tumor vaccines (Li et al., 2022; Srivastava et al., 2021; Wiede et al., 2022; Puig-Saus et al., 2023; Reinhard et al., 2020), and combining oncolytic viruses to improve T cell homing (Chalise et al., 2022; Evgin et al., 2022; Rezaei et al., 2022; Wang et al., 2023), thereby further enhancing the potential of T cell therapy. However, even with combinations of other drugs, the activation of the immune cycle often fails, and the final tumor treatment effect remains unsatisfactory. Therefore, there is an urgent need to develop new and effective anti-tumor drugs to synergistically enhance the efficacy of T cell therapy.

[0003] Although T-cell-based immunotherapies have multiple mechanisms of action, their ultimate goal is to induce apoptosis in cancer cells. Apoptosis is a classic form of programmed cell death. As the smallest unit for inducing apoptosis, the pro-apoptotic BH3 domain has been considered an effective target for cancer immunotherapy (Hossini and Eberle, 2008; Kelekar and Thompson, 1998). Subfamilies containing only the BH3 domain, such as BIM, BID, BAD, NOXA, and PUMA, utilize the binding of their BH3 domain to the hydrophobic pockets of pro-survival protein families to induce activation of the apoptosis pathway cascade (Adams and Cory, 2007; Warren et al., 2019). Enhancing the apoptosis sensitivity of cancer cells has been considered a key strategy for improving anti-cancer activity. Previous studies have shown that modulating the initiation state of mitochondrial apoptosis in cancer cells with pro-apoptotic drugs can drive cancer cells to cross the apoptosis threshold (Pan et al., 2022). This can also increase the number of effector memory T cells infiltrating tumors (Kohlhapp et al., 2021).

[0004] mRNA, as a novel drug form that can replace DNA and recombinant proteins, offers controllable expression and high safety, attracting increasing attention in the field of cancer immunotherapy. mRNA drugs encoding the pro-apoptotic BH3 domain can initiate apoptosis in cancer cells at the lesion site, while simultaneously synergistically enhancing T cell-mediated killing effects. In summary, given the limited efficacy of existing T cell therapies in tumor treatment, developing an anti-tumor mRNA drug to enhance T cell efficacy is highly significant. This could substantially reduce the number of T cells required for T cell therapy and lower economic costs, providing a safe, efficient, and cost-effective mRNA drug for clinical use. Summary of the Invention

[0005] The purpose of this invention is to address the limitations of current T-cell therapy alone in terms of therapeutic efficacy, the large number of T cells required, and high immunogenicity. This invention provides an mRNA drug and its application that enhances the therapeutic effect of T-cell therapy, thereby improving the anti-tumor activity of single-T-cell therapy.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides an mRNA drug that enhances the therapeutic effect of T cells. The mRNA drug comprises a pharmaceutical carrier and an active ingredient. The active ingredient comprises an mRNA molecule encoding a 5' to 3' end coding sequence as shown in SEQ ID NO.4 or SEQ ID NO.5, for encoding the BH3 domain of the pro-apoptotic protein Puma or Bim fused to the aptamer scaffold. The mRNA drug is a local intratumoral dosage form.

[0008] The mRNA molecule comprises two parts: a Puma or Bim BH3 domain sequence from the pro-apoptotic protein family, encoded as shown in SEQ ID NO.1 and SEQ ID NO.2; and a Stefin A quadruple aptamer scaffold sequence, encoded as shown in SEQ ID NO.3.

[0009] Preferably, the aptor scaffold is a Stefin A quadruple aptor scaffold, with the coding sequence shown in SEQ ID NO.3.

[0010] Preferably, the mRNA molecule has a capping modification. The 5' end of the mRNA molecule has an m7G-PPPNm structure.

[0011] The mRNA drug described in this invention is an intratumoral local drug delivery formulation. Through intratumoral local administration, the mRNA molecule is taken up and expressed by tumor cells, achieving local expression. Studies have shown that this delivery method is biosafety-free. The mRNA molecule provided by this invention does not easily enter systemic circulation and has low toxicity.

[0012] Preferably, the pharmaceutical carrier is a lipid nanoparticle (LNP), and the mRNA molecule is encapsulated within the lipid nanoparticle. Studies have shown that using lipid nanoparticles as an mRNA delivery carrier helps improve the expression efficiency of mRNA molecules in tumor cells.

[0013] Preferably, the anti-tumor mRNA vaccine provided by the present invention should be administered at multiple intervals. Preferably, the dosing interval is 2 days, and the number of doses is 3.

[0014] Secondly, the present invention also provides a method for preparing the mRNA drug that enhances T-cell therapeutic efficacy, comprising the following steps:

[0015] DNA fragments encoding the Puma or Bim BH3 domains of the pro-apoptotic protein family and the Stefin A quadruple aptamer scaffold were amplified using polymerase chain reaction (PCR). Using these fragments as templates, homologous arms were added via PCR to extend the DNA and obtain a fusion gene. The fusion gene was then ligated into an in vitro transcription vector to obtain a recombinant plasmid. After enzyme digestion, an in vitro transcription linearization template was obtained. The mRNA molecule was then obtained through in vitro transcription, modification, and purification. Finally, the mRNA molecule was loaded onto a pharmaceutical vector to prepare an mRNA drug.

[0016] All mRNA drugs are individually packaged.

[0017] Preferably, lipid nanoparticles (LNPs) are used to encapsulate mRNA molecules. The preparation process includes: diluting mRNA molecules in a citrate-sodium citrate buffer solution at pH 4.5, adding a lipid ethanol solution and mixing well, repeatedly blowing and dialysis to obtain lipid nanoparticles loaded with mRNA; the lipid ethanol solution contains SM102, DSPC, CHO-HP and PEG2000 in a molar ratio of 50:10:38.5:1.5.

[0018] Thirdly, the present invention also provides the application of the mRNA drug in the preparation of tumor therapeutic drugs and in synergistically enhancing the efficacy of T cell therapy.

[0019] The tumor is a solid tumor, including at least: melanoma, colon cancer, and breast cancer.

[0020] Animal experiments of this invention show that the application of this mRNA-LNP local therapy has a significant inhibitory effect on tumor growth in a melanoma solid tumor model, and can synergistically enhance the anti-tumor effect of T-cell therapy.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention provides an mRNA antitumor drug for enhancing T-cell therapy. Utilizing the form of an mRNA drug for local intratumoral administration, it induces apoptosis in local cells at the lesion site, while simultaneously synergistically enhancing the T-cell-mediated killing effect, ultimately achieving a synergistic inhibitory effect on solid tumor growth in vivo. The novel mRNA drug provided by this invention exhibits strong and effective tumor-suppressive activity in solid tumor models. It is highly versatile and has a wide range of applications, significantly reducing the number of T cells required and the cost of T-cell therapy, thus providing a safe, efficient, and economical antitumor drug model. Attached Figure Description

[0023] Figure 1 shows a map of the in vitro transcription template plasmid T7-St-Puma-BH3.

[0024] Figure 2 shows the map of the in vitro transcription template plasmid T7-St-Bim-BH3.

[0025] Figure 3 shows the cytotoxicity assay of different concentrations of mRNA-LNP on tumor cell lines and normal cell lines.

[0026] Figure 4 shows the curves illustrating the synergistic effect of mRNA-LNP in enhancing the killing efficacy of T cells against melanoma tumor cell lines with different effector-to-target ratios in vitro. Figure 5 shows the drug administration timeline and tumor growth inhibition curves of locally applied mRNA therapy in a melanoma model, where (A) is the drug administration timeline of locally applied mRNA therapy in the melanoma model, (B) is the average tumor growth curve, and (C) are the individual tumor growth curves of the control group, LNP group, Puma-BH3 domain mRNA, and Bim-BH3 domain mRNA drugs, respectively.

[0027] Figure 6 shows the flow cytometry results of the antitumor immune response level of locally applied mRNA drugs in a melanoma model; where (A) is the proportion of T cells infiltrating the tumor, (B) is the proportion of CD8 T cells infiltrating the tumor to the total number of T cells, (C) is the proportion of CD4 T cells infiltrating the tumor to the total number of T cells, (D) is the proportion of macrophages infiltrating the tumor, (E) is the proportion of M1 macrophages infiltrating the tumor to the total number of macrophages, and (F) is the proportion of mature dendritic cells in the draining lymph nodes.

[0028] Figure 7 shows the tumor growth inhibition curves of combined mRNA drug and T cell therapy in a melanoma model. (A) is the administration time axis of combined mRNA drug and T cell therapy in the melanoma model, (B) is the average tumor growth curve, and (C) are the individual tumor growth curves of the control group, the T cell therapy group alone, the T cell therapy group combined with Puma-BH3 domain mRNA therapy group, and the T cell therapy group combined with Bim-BH3 domain mRNA therapy group, respectively.

[0029] Figure 8 shows the survival curves of mice in a melanoma model that received a combination of mRNA drugs and T-cell therapy.

[0030] Figure 9 shows the flow cytometry results of the anti-tumor immune response level of infiltrating CD8 T cells in a melanoma model after combined application of mRNA drugs and T cell therapy; where (A) is the proportion of CD8 T cells that highly express granzyme B and perforin in the tumor, and (B) is the proportion of CD8 T cells that highly express granzyme B and perforin in the spleen.

[0031] Figure 10 shows the biosafety assessment of LNP and LNP-mRNA administration in a melanoma model, where (A) is tissue section of each major organ and (B) is serum ALT and AST levels. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and 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.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0034] This invention provides an mRNA anti-tumor drug that enhances the anti-tumor activity of single T-cell therapy, can be used to enhance T-cell efficacy, has high biosafety, and provides a new strategy and approach for cancer treatment. Simultaneously, it can significantly reduce the number of T cells required for in vitro expansion in T-cell therapy, reducing treatment time and economic costs for cancer patients. Furthermore, this mRNA drug induces apoptosis of tumor cells at the lesion site locally, promoting enhanced tumor antigen presentation, thereby inducing a stronger anti-tumor specific immune response. When used in synergy with T-cell therapy, it can enhance the T-cell-mediated killing effect, ultimately achieving the goal of inhibiting or even eliminating solid tumor tissue.

[0035] Example 1: Construction of linear in vitro transcription vectors for T7-St-Puma-BH3 mRNA and T7-St-Bim-BH3 mRNA

[0036] The mRNA drug encodes the BH3 domain of the pro-apoptotic protein Puma or Bim, which is fused to the aptamer scaffold. The recombinant plasmid was obtained using conventional molecular biology techniques through the following steps.

[0037] (1) Acquisition of target antigen fragments

[0038] The nucleotide sequences of Puma-BH3 and Bim-BH3 were obtained through NCBI and then synthesized by Beijing Qingke Biotechnology Co., Ltd. The length of each sequence is 78 bp, and the nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0039] (2) Acquisition of fusion genes

[0040] A Stefin A quadruple aptamer scaffold was fused before and after the Puma-BH3 or Bim-BH3 fragment to stabilize the BH3 domain conformation for physiological function. The nucleotide length was 294 bp, and the sequence is shown in SEQ ID NO.3. The fusion genes were obtained and named St-Puma-BH3 and St-Bim-BH3, respectively. The length of each fusion gene was 372 bp. They were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the nucleotide sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0041] (3) Construction of recombinant plasmids

[0042] The fusion genes St-Puma-BH3 and St-Bim-BH3 fragments obtained by fusing Puma-BH3 or Bim-BH3 fragments with aptamers were ligated to the linearized template vector Cloning Kit for mRNA Template (Takara, Cat:6143) via homologous recombination and transformed into DH5α competent cells. Correct single-clone colonies were selected to obtain the in vitro transcription template plasmids T7-St-Puma-BH3 and T7-St-Bim-BH3. The plasmid maps are shown in Figures 1 and 2, respectively, and the sequencing sequences were correct.

[0043] (4) Obtaining the linearization template

[0044] The obtained recombinant plasmid was digested overnight at 37°C with the restriction endonuclease HindIII. Subsequently, the linear fragment was separated by 1.5% gel electrophoresis, and the digestion products were recovered using a DNA recovery kit. Finally, the DNA concentration was determined by Nanodrop, yielding the linearized template for subsequent in vitro mRNA transcription.

[0045] Example 2: In vitro transcription, modification and purification of mRNA

[0046] Using the EasyCap T7 Co-transcription Kit with CAG Trimer (Vazyme, Cat:DD4203-01), in vitro transcribed Puma-BH3 mRNA and Bim-BH3 mRNA were generated respectively through the following steps.

[0047] (1) On ice, add the following reagents to 200 μL microcentrifuge tubes according to Table 1.

[0048] Table 1

[0049] Component Dosage and Final Concentration: T7 RNA polymerase Mix 2 μL, 10x Co-Reaction Buffer 2 μL, 1× ATP solution (100 mM) 1 μL, 5 mM MTP solution (100 mM) 1 μL, 5 mM CTP solution (100 mM) 1 μL, 5 mM MUTTP solution (100 mM) 1 μL, 3.75 mM CAG Trimer 0.8 μL, Inerted template 1 μL (1 μg) 50 ng / μL, RNAase-Free ddH2O y μL, Total 20 μL surface

[0050] After thorough mixing and centrifugation, the reaction tubes were incubated at 37°C for 4 hours. Subsequently, to remove template DNA, 1.5 μL of DNase I and 2 μL of DNase buffer (10×) (Vazyme, Cat:DD4104) were added to the reaction tubes, and the mixture was incubated at 37°C for 30 minutes.

[0051] (2) Use The RNAPurification Kit (TransGen Biotech, Cat:ER701-01) was used to purify in vitro transcribed Puma-BH3 mRNA and Bim-BH3 mRNA products using the following steps.

[0052] Take the in vitro transcription product, add nuclease-free water to a final volume of 100 μL, and transfer to a 1.5 mL centrifuge tube. Add 350 μL of BB12 (containing 1% β-mercaptoethanol) and vortex to mix. Add 900 μL of anhydrous ethanol and vortex again. Add the mixture to a centrifuge column in two portions, centrifuge at 12000×g for 1 minute, and discard the eluent. Add 500 μL of WB12 and centrifuge at 12000×g for 1 minute, discarding the eluent. Repeat the above steps. Centrifuge at 12000×g for 2 minutes to completely remove residual ethanol. Transfer the centrifuge column to a new 1.5 mL nuclease-free centrifuge tube, add 30 μL of nuclease-free water to the column, incubate at room temperature for 2 minutes, and centrifuge at 12000×g for 1 minute. Determine the concentration and purity of the purified in vitro transcribed mRNA product using Nanodrop, and assess its quality by 1.5% agarose gel electrophoresis.

[0053] Example 3: Preparation of the corresponding mRNA lipid nanoparticle complex

[0054] (1) Preparation of lipid ethanol solution. SM102 (Cat:O02010), distearate phosphatidylcholine DSPC (Cat:S01005), high-purity cholesterol CHO-HP (Cat:57-88-5), and DMG-PEG2000 (Cat:O02005) purchased from Aivit (Shanghai) Pharmaceutical Technology Co., Ltd. were dissolved in anhydrous ethanol and prepared into lipid ethanol solution according to the molar percentages in Table 2 for later use.

[0055] Table 2

[0056] Component molar percentage: SM10 250% DSPC 10% CHO-HP 38.5% PEG2000 1.5% surface

[0057] (2) Dilute the purified mRNA with citrate-sodium citrate buffer (pH=4.5) and set aside. Mix the above lipid ethanol solution with the mRNA dilution solution thoroughly and repeatedly pipette.

[0058] (3) Place 120 μL of the above mixture in Slide-A-Lyzer TM In a mini dialysis cup (10 kWh / mL, 0.1 mL), a centrifuge tube was filled with PBS solution and incubated overnight on a shaker at 4°C. This yielded the mRNA-lipid nanoparticle complex.

[0059] Example 4: Cytotoxicity assessment of normal and tumor cell lines transfected with mRNA-LNP

[0060] (1) Lay 1.5×10⁻⁶ m³ of the substrate in a 96-well plate overnight in advance. 4 mRNA-LNPs encoding the Puma or Bim BH3 domains, prepared using the above method, were transfected into different cell lines at different concentrations using T293, B16F10, and CT-26. Cytotoxicity was assessed using a CCK8 assay 24 hours after transfection. The results showed that mRNA-LNPs encoding the BH3 domain selectively killed tumor cell lines without significantly affecting the proliferation of normal cell lines (Figure 3).

[0061] Example 5: Evaluation of the in vitro synergistic enhancement of T cell-mediated killing effects with different effector-to-target ratios by mRNA-LNP

[0062] (1) Extraction and in vitro activation of CD8 T cells from OT-1 mice

[0063] OT-1 mouse spleens were sterilized in a biosafety cabinet, filtered through a 0.45 μm filter, and centrifuged at 1000 rpm. After lysis with erythrocyte lysis buffer for 3 minutes, the spleen cells were resuspended in DPBS. CD8a was used... + T-cell isolation kit (Beaver Biosciences, Inc.) to obtain purified CD8a + T cell suspension. 1×10 6 CD8 T cells were resuspended in 24-well plates (RPMI 1640 medium supplemented with 5 mM Hepes, 2 mM Glutamax, 50 μg / ml Pen / Strep, 5 mM NEAA, 5 mM sodium pyruvate, 10% heat-inactivated FBS, 1% human a / B serum, 50 μM β-mercaptoethanol, and 30 IU / ml IL-2). 25 μL of mouse T cell activator CD3 / CD28 Dynabeads (ThermoFisher Scientific) was added to each well, and the plates were incubated at 37°C for 72 hours.

[0064] (2) Evaluation of the synergistic effect of mRNA drugs on T cell killing efficacy with different target ratios

[0065] 7×10 mm thick sheets were laid in a 24-well plate overnight. 4 B16-OVA melanoma cells stably overexpressing chicken ovalbumin (OVA) were injected with different numbers of activated T cells at effector-to-target ratios of 1:4, 1:2, 2:1, 4:1, and 10:1, along with a certain dose of mRNA drug for synergistic killing. Apoptosis levels were detected by flow cytometry using an Annexin V FITC / PE kit 24 hours after transfection. The results showed that mRNA-LNP encoding the BH3 domain synergistically enhanced the killing efficacy of T cells at different effector-to-target ratios against tumor cell lines, and this synergistic effect was more pronounced at low effector-to-target ratios (Figure 4).

[0066] Example 6: Evaluation of the antitumor effect of locally injected mRNA-LNP drugs encoding the BH3 domain in a solid tumor model.

[0067] (1) Experimental animals and solid tumor transplantation models:

[0068] Six-week-old female C57 mice, purchased from Hangzhou Pasio Biotechnology Co., Ltd., were used to construct a melanoma model. They were purchased one week prior to the experiment and had free access to water and food. Mouse melanoma cells (B16F10) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibiotics. Culture conditions were 37°C and 5% CO2. Cells were grown at a rate of 5 × 10⁶ cells per mouse. 5 One B16F10 cell was used to subcutaneously inoculate cancer cells into the right posterior dorsal side of C57 mice.

[0069] (2) mRNA-LNP administration:

[0070] 8-10 days after tumor transplantation, when the tumor volume in mice grows to 50-100 mm 3 Mice were randomly divided into four groups of 7-8 mice each. Group 1 was the control group, receiving intratumoral administration of saline. Group 2 was the LNP control group, receiving intratumoral administration of empty lipid nanoparticles. Groups 3 and 4 were mRNA-LNP administration groups, receiving intratumoral administration of mRNA-LNPs encoding the Puma-BH3 and Bim-BH3 domains, respectively. The dosage was 10 μg mRNA per mouse, administered every two days for a total of three administrations. Body weight and tumor volume were recorded every two days, calculated using the formula: tumor volume = 0.5 × length × width. 2 Nineteen days after tumor implantation, mice were euthanized by cervical dislocation. Histological sections of the major organs (heart, liver, spleen, lung, and kidney) from each group were collected, and H&E staining was used to analyze the pathological condition of each major organ. Serum samples from each group were collected for ALT and AST analysis.

[0071] (3) Detection of anti-tumor immune response level:

[0072] After euthanizing the mice, tumors from each experimental group were collected, minced with scissors, digested with tissue digestion solution, and cleaved to obtain tumor single-cell suspensions. Cells were labeled with mouse APC-anti-CD45, PE-anti-CD3, FITC-anti-CD4, and PerCP-anti-CD8a antibodies, and tumor invasive CD3 was measured. + / CD8 + The proportion of cytotoxic T cells and CD3 + / CD4 + Helper T cell ratio. Cells were also labeled with PE-anti-CD11b, APC-anti-F4 / 80, FITC-anti-CD80, and PerCP-Cy5.5-anti-CD206 antibodies to determine the ratio of tumor-infiltrating M1 and M2 macrophages.

[0073] Inguinal lymph nodes from each group of mice were collected, digested with tissue digestion solution, and centrifuged to obtain single-cell suspensions. Surface markers were applied using mouse PE-anti-CD11c, FITC-anti-CD80, and APC-anti-CD86 antibodies. Mature dendritic cells (CD11c) were analyzed by flow cytometry. + / CD80 + CD11c + / CD86 + )Proportion.

[0074] (4) Results Analysis

[0075] Analysis of the tumor growth curves of mice in each group showed that local administration of mRNA drugs encoding the BH3 domain significantly inhibited the growth of solid tumors in the melanoma model (Figure 5).

[0076] Flow cytometry data showed that intratumoral administration of both mRNA drugs encoding the BH3 domain significantly increased intratumoral CD3 levels. + / CD8 + The proportion of T cells had no significant effect on the polarization level of macrophages within the tumor (Figure 6). Meanwhile, the mRNA-LNP encoding the Bim-BH3 domain effectively induced the maturation of dendritic cells in the lymph nodes (Figure 6).

[0077] HE pathological sections of major organs in mice and serum ALT / AST levels were used to assess the biosafety of this strategy (Figure 10).

[0078] Example 7: Evaluation of the antitumor effect of combined mRNA-LNP drugs and T-cell therapy in a solid tumor model

[0079] (1) Experimental animals and solid tumor transplantation models:

[0080] Six-week-old female C57 mice, purchased from Hangzhou Pasio Biotechnology Co., Ltd., were used to construct a B16-OVA melanoma model. The mice were purchased one week prior to the experiment and had free access to water and food. B16-OVA mouse melanoma cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibiotics. Culture conditions were 37°C and 5% CO2. Cells were grown at a rate of 5 × 10⁶ cells per mouse. 5 One B16-OVA cell was used to subcutaneously inoculate cancer cells into the right posterior dorsal side of C57 mice.

[0081] (2) Combined administration of mRNA-LNP to T cells:

[0082] 8-10 days after tumor transplantation, when the tumor volume in mice grows to 50-100 mm 3 Mice were randomly divided into four groups, with 7-8 mice in each group. Group 1 was the control group, receiving intratumoral administration of saline. Group 2 was the T-cell therapy group alone. Groups 3 and 4 were BH3 domain mRNA groups, receiving intratumoral administration of mRNA-LNPs encoding the Puma-BH3 and Bim-BH3 domains, respectively, at a dose of 10 μg mRNA per mouse. The day after administration, 2 × 10⁻⁶ in vitro activated OT-1CD8 T cells were intravenously infused. 6 Each mouse was counted individually. Body weight and tumor volume were recorded for each group of mice every two days. The calculation formula was: Tumor volume = 0.5 × length × width. 2 The mice were euthanized when the tumors grew to 15 mm.

[0083] (3) Detection of anti-tumor immune response level:

[0084] After euthanizing the mice, tumor and spleen tissues were collected from each group of mice. The tissues were minced with scissors, digested with tissue digestion solution, and cleaved to obtain tumor single-cell suspensions. Cytotoxic T cells were labeled with mouse Pacific Blue anti-CD45, Brilliant Violet 510 anti-CD3, and PerCP anti-CD8a antibodies, and the effector function of cytotoxic T cells was measured using FITC anti-Granzyme B and PE anti-Perforin.

[0085] (4) Results Analysis

[0086] Analysis of tumor growth curves in each group of mice showed that the combined use of mRNA drugs encoding the BH3 domain significantly enhanced the tumor-suppressive effect of T-cell therapy in a melanoma model (Figure 7), and also significantly improved the survival rate of the tumor model mice (Figure 8). Flow cytometry data indicated that the combined use of mRNA drugs encoding the BH3 domain enhanced the effector function of cytotoxic T cells within the tumor and spleen (Figure 9).

[0087] Therefore, this local administration strategy of mRNA encoding the BH3 domain can synergistically enhance the killing effect of T cells on tumor tissue, thereby enhancing the therapeutic effect of T cells.

[0088] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The application of an mRNA drug in the preparation of a drug that enhances the efficacy of adoptive T cells against melanoma, characterized in that, The mRNA drug comprises a pharmaceutical carrier and an active ingredient, wherein the active ingredient comprises an mRNA molecule encoding a 5' to 3' end coding sequence as shown in SEQ ID NO. 4 or SEQ ID NO. 5, for encoding the BH3 domain of the pro-apoptotic protein Puma or Bim fused to the aptamer scaffold, and the mRNA drug is a local intratumoral dosage form.

2. The application according to claim 1, characterized in that, The adopted T cells mentioned are TCR-T cells.

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