MRNA (messenger ribonucleic acid) medicine for enhancing curative effect of T cells and application

Abstract

The invention discloses an mRNA (messenger Ribonucleic Acid) medicine for enhancing the curative effect of T cells and application, and belongs to the technical field of biological medicines. The mRNA drug coding protein is a BH3 structural domain of pro-apoptotic protein Puma or Bim fused into a ligand scaffold; the administration mode is an intratumor administration dosage form. The invention provides an mRNA (messenger Ribonucleic Acid) anti-tumor drug, which is locally administered in a tumor in the form of an mRNA drug, induces local cell apoptosis of a focus, and can synergistically enhance a T cell mediated killing effect at the same time. Finally, the effect of synergistically inhibiting the growth of the solid tumor in vivo is achieved. The novel mRNA drug provided by the invention shows a strong and effective tumor inhibition effect in a solid tumor model.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an mRNA drug for enhancing the therapeutic effect of T cells and its application. Background Art

[0002] As TCR-T cell therapy has developed into a promising treatment for solid tumors, its development still faces many obstacles, including high drug resistance caused by the suppressive tumor immune microenvironment and insufficient T cell functionality (Klebanoff et al., 2016; Sadelain et al., 2017). In addition, the in vitro engineering manufacturing of T cells faces the problems of huge investment, low yield and high failure rate. Current research is committed to combining other drugs to improve 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), combining oncolytic viruses to improve T cell homing (Chalise et al., 2022; Evgin et al., 2022; Rezaei et al., 2022; Wang et al., 2023) to further enhance the therapeutic potential of T cells. However, even in combination with other drugs, the activation of the immune cycle often fails, and the final tumor treatment effect is still unsatisfactory. Therefore, there is an urgent need to develop new and effective anti-tumor drugs to synergistically enhance the efficacy of T cells.

[0003] Although T cell-based immunotherapy has multiple mechanisms of action, its ultimate goal is to achieve cancer cell apoptosis. Apoptosis is a classic type of programmed cell death. As the smallest unit for inducing apoptosis, the pro-apoptotic BH3 domain is considered an effective target for cancer immunotherapy (Hossini and Eberle, 2008; Kelekar and Thompson, 1998). Subfamilies containing only BH3 domains, such as BIM, BID, BAD, NOXA, and PUMA, use their BH3 domains to bind to the hydrophobic pocket of the pro-survival protein family to induce activation of the apoptotic pathway cascade (Adams and Cory, 2007; Warren et al., 2019). Improving the apoptotic sensitivity of cancer cells has been considered a key strategy to improve anti-cancer activity. Previous studies have shown that regulating the initiation state of cancer cell mitochondrial apoptosis by pro-apoptotic drugs can push cancer cells across the apoptotic threshold (Pan et al., 2022). It can also increase tumor-infiltrating effector memory T cells (Kohlhapp et al., 2021).

[0004] As a new drug form that can replace DNA and recombinant proteins, mRNA has controllable expression and high safety, and has received increasing attention in the field of cancer immunotherapy. mRNA drugs encoding pro-apoptotic BH3 domains can be used to initiate apoptosis of local cancer cells in lesions, while synergistically enhancing the killing effect mediated by T cells. In summary, due to the limited effectiveness of existing T cell therapies in tumor treatment, it is very meaningful to develop an anti-tumor mRNA drug for enhancing the efficacy of T cells, which can greatly reduce the number of T cells required for T cell therapy and the economic cost, and provide a safe, efficient and economical mRNA drug for clinical use. Summary of the invention

[0005] The purpose of the present invention is to solve the problems of limited therapeutic ability of T cell therapy alone, large number of T cells required and high immunogenicity, and to provide an mRNA drug and application that enhances the efficacy of T cells, which has the effect of improving the anti-tumor activity of single T cell therapy.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides an mRNA drug for enhancing the therapeutic effect of T cells, the mRNA drug comprising a pharmaceutical carrier and an active ingredient, the active ingredient comprising an mRNA molecule having a 5'-end to 3'-end coding sequence such as that shown in SEQ ID NO.4 or SEQ ID NO.5, which is used to encode the BH3 domain of the pro-apoptotic protein Puma or Bim fused with an adaptor scaffold, and the mRNA drug is a dosage form for local intratumoral administration.

[0008] The mRNA molecule includes two parts, namely, the pro-apoptotic protein family Puma or Bim BH3 domain sequence, the coding sequence is shown in SEQ ID NO.1 and SEQ ID NO.2, and the Stefin A quadruple aptamer scaffold sequence, the coding sequence is shown in SEQ ID NO.3.

[0009] Preferably, the aptamer scaffold is a Stefin A quadruple aptamer scaffold, and the coding sequence is 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 the present invention is a local intratumoral administration dosage form. Through the local intratumoral administration, the mRNA molecule is taken up by tumor cells and expressed, thereby achieving local expression. Studies have shown that this administration method has biosafety. The mRNA molecule provided by the present invention is not easy to enter the systemic circulation and has low toxicity.

[0012] Preferably, the pharmaceutical carrier is a lipid nanoparticle (LNP), and the mRNA molecule is encapsulated in the lipid nanoparticle. Studies have shown that using lipid nanoparticles as mRNA molecule delivery carriers helps to 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 multiple times at intervals. Preferably, the administration interval is 2 days and the number of administrations is 3.

[0014] In a second aspect, the present invention further provides a method for preparing the mRNA drug for enhancing T cell efficacy, comprising the following steps:

[0015] DNA fragments encoding the pro-apoptotic protein family Puma or Bim BH3 domain and the Stefin A quadruple aptamer scaffold are respectively obtained by polymerase chain reaction amplification, and the fragments are used as templates to add homologous arms for chain extension reaction to obtain fusion genes; the fusion genes are then connected to an mRNA in vitro transcription vector to obtain a recombinant plasmid, and an in vitro transcription linearization template is obtained after enzyme digestion; then the mRNA molecules are obtained through in vitro transcription, modification and purification; finally, the mRNA molecules are loaded on a pharmaceutical carrier to obtain an mRNA drug.

[0016] The mRNA drugs are all packaged independently.

[0017] Preferably, lipid nanoparticles LNP are used to encapsulate mRNA molecules, and the preparation process includes: diluting the mRNA molecules in a citric acid-sodium citrate buffer with a pH of 4.5, adding a lipid ethanol solution to mix, repeatedly blowing and dialyzing 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] In a third aspect, the present invention also provides the use of the mRNA drug in preparing tumor treatment drugs and synergistically improving the efficacy of T cells.

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

[0020] The animal test results of the present invention show that the application of the mRNA-LNP local therapy has a significant inhibitory effect on the tumor growth of melanoma solid tumor models, and can synergistically improve the anti-tumor effect of T cell therapy.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides an mRNA anti-tumor drug for enhancing the efficacy of T cells. The mRNA drug is used for local administration into the tumor to induce apoptosis of local cells in the lesion, while synergistically enhancing the killing effect mediated by T cells, and finally achieving the effect of synergistically inhibiting the growth of solid tumors in vivo. The new mRNA drug provided by the present invention shows a strong and effective tumor inhibition effect in a solid tumor model, has strong universality and a wide range of applications, can greatly reduce the number and cost of T cells required for T cell therapy, and provides a safe, efficient and economical anti-tumor drug model. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0025] Figure 3 The cytotoxicity of different concentrations of mRNA-LNP on tumor cell lines and normal cell lines was determined.

[0026] Figure 4 The curve shows that mRNA-LNP synergistically improves the efficacy of T cells with different effector-target ratios in killing melanoma tumor cell lines in vitro. Figure 5 The figures are the dosing timeline and tumor growth inhibition curve of topically applied mRNA therapy in a melanoma model, wherein (A) is the dosing timeline of topically applied mRNA therapy in a 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 The results of flow cytometry analysis of the anti-tumor immune response of locally applied mRNA drugs in a melanoma model; (A) is the proportion of T cells infiltrating the tumor, (B) is the proportion of CD8 T cells infiltrating the tumor to total T cells, (C) is the proportion of CD4 T cells infiltrating the tumor to total T cells, (D) is the proportion of macrophages infiltrating the tumor, (E) is the proportion of M1 macrophages infiltrating the tumor to total macrophages, and (F) is the proportion of mature dendritic cells in the draining lymph nodes.

[0028] Figure 7Figure 2 is the tumor growth inhibition curve of the combined use of mRNA drugs and T cell therapy in the melanoma model, where (A) is the administration timeline of the combined use of mRNA drugs 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 treatment group alone, the T cell combined with Puma-BH3 domain mRNA treatment group, and the T cell combined with Bim-BH3 domain mRNA treatment group.

[0029] Figure 8 This is the survival curve of mice treated with combined mRNA drug and T cell therapy in a melanoma model.

[0030] Fig. 9 The results of flow cytometry analysis of the anti-tumor immune response of infiltrating CD8 T cells in a melanoma model using a combination of mRNA drugs and T cell therapy; (A) is the proportion of CD8 T cells with high expression of granzyme B and perforin in the tumor, and (B) is the proportion of CD8 T cells with high expression of granzyme B and perforin in the spleen.

[0031] Fig.10 This is the biosafety assessment of LNP and LNP-mRNA administration in a melanoma model, where (A) is the tissue sections of major organs and (B) is the ALT and AST levels in serum. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0034] The present invention provides an mRNA anti-tumor drug, which has the function of improving the anti-tumor activity of single T cell therapy, can be used to enhance the therapeutic effect of T cells, has high biosafety, and provides a new strategy and idea for the treatment of cancer; at the same time, it can greatly reduce the number of T cells required for in vitro expansion in T cell therapy, and reduce the treatment time and economic cost of tumor patients; in addition, the mRNA drug induces apoptosis of tumor cells in the lesion site locally, promotes enhanced tumor antigen presentation, and thus induces a stronger anti-tumor specific immune response. When used in conjunction with T cell therapy, it can enhance the killing effect mediated by T cells, and ultimately achieve the purpose of inhibiting or even clearing solid tumor tumor tissue.

[0035] Example 1. Construction of linear vectors for in vitro transcription of 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 fused with an adaptor scaffold, and the recombinant plasmid thereof is obtained by the following steps using conventional molecular biological techniques.

[0037] (1) Obtaining target antigen fragments

[0038] The nucleotide sequences of Puma-BH3 and Bim-BH3 were obtained through NCBI and submitted to Beijing Qingke Biotechnology Co., Ltd. for gene synthesis. The length of both sequences was 78 bp, and the nucleotide sequences were shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0039] (2) Acquisition of fusion genes

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

[0041] (3) Recombinant plasmid construction

[0042] The fusion gene St-Puma-BH3 and St-Bim-BH3 fragments obtained by fusing the Puma-BH3 or Bim-BH3 fragments to the adapter scaffold were connected to the linearized template vector Cloning Kit for mRNA Template (Takara, Cat: 6143) by homologous recombination and transformed into DH5α competent cells. The correct monoclonal colonies were picked to obtain the in vitro transcription template plasmids T7-St-Puma-BH3 and T7-St-Bim-BH3. The plasmid maps are as follows Figure 1 and Figure 2 As shown, the sequence was correct.

[0043] (4) Linearization template acquisition

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

[0045] Example 2. mRNA in vitro transcription, modification and purification

[0046] Using 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 by the following steps.

[0047] (1) Add the following reagents according to Table 1 to 200uL microcentrifuge tubes on ice.

[0048] Table 1

[0049] Components Dosage Final concentration T7 RNA polymerase Mix 2μL 10x Co-Reaction Buffer 2μL 1× ATP solution (100 mM) 1μL 5mM GTP solution (100 mM) 1μL 5mM CTP solution (100mM) 1μL 5mM UTP solution (100mM) 1μL 3.75mM CAG Trimer 0.8μL Linearized template xμL(1μg) 50ng / μL <![CDATA[RNase-Free ddH2O]]> yμL Total 20μL

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

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

[0052] Take the in vitro transcription product, make up to a volume of 100 μL with nuclease-free water, and transfer it 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 to mix again. Add the above mixture to the centrifuge column twice, centrifuge at 12,000 × g for 1 minute, and discard the effluent. Add 500 μL of WB12 and centrifuge at 12,000 × g for 1 minute, and discard the effluent. Repeat the above steps. Centrifuge at 12,000 × g for 2 minutes to completely remove the 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 centrifuge column, let it stand at room temperature for 2 minutes, and centrifuge at 12,000 × g for 1 minute. The concentration and purity of the purified in vitro transcribed mRNA product were determined by Nanodrop, and its quality was tested by 1.5% agarose gel electrophoresis.

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

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

[0055] Table 2

[0056] Components Mole Percent SM102 50％ DSPC 10％ CHO-HP 38.5％ PEG2000 1.5％

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

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

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

[0060] (1) 1.5×10 4The mRNA-LNP encoding the BH3 domain of Puma or Bim prepared by the above method was transfected into different cell lines at different concentrations, and the cytotoxicity was detected by CCK8 kit 24 hours after transfection. The results showed that the mRNA-LNP encoding the BH3 domain could selectively kill tumor cell lines, but had no significant effect on the proliferation of normal cell lines ( Figure 3 ).

[0061] Example 5. Evaluation of mRNA-LNP synergistically enhancing the killing effect mediated by T cells with different effector-target ratios in vitro

[0062] (1) Isolation and in vitro activation of OT-1 mouse CD8 T cells

[0063] The spleen of OT-1 mice was sterilized in a biosafety cabinet, filtered through a 0.45 μm filter, and centrifuged at 1000 rpm. After lysis with red blood cell lysis buffer for 3 minutes, the spleen cells were resuspended in DPBS. + Purified CD8a T cell isolation kit (BeaverBiosciences Co., Ltd.) + T cell suspension. 1×10 6 CD8 T cells were resuspended in a 24-well plate (the RPMI1640 medium contained 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) were added to each well and incubated at 37°C for 72 hours.

[0064] (2) Evaluation of mRNA drug synergistic killing efficacy of T cells with different effector-target ratios

[0065] 7 × 10 cells were plated in a 24-well plate overnight. 4 Different numbers of activated T cells were added to the melanoma cells B16-OVA stably overexpressing chicken ovalbumin OVA at effector-target ratios of 1:4, 1:2, 2:1, 4:1, and 10:1, respectively, and a certain dose of mRNA drugs were added for synergistic killing. The level of cell apoptosis was detected by flow cytometry using the Annexin V FITC / PE kit 24 hours after transfection. The results showed that the use of mRNA-LNP encoding the BH3 domain can synergistically improve the killing efficacy of T cells with different effector-target ratios against tumor cell lines, and this synergistic effect is more obvious at low effector-target ratios ( Figure 4 ).

[0066] Example 6 Evaluation of the tumor-suppressing effect of local injection of mRNA-LNP drugs encoding BH3 domains in solid tumor models

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

[0068] Six-week-old female C57 mice purchased from Hangzhou Paisiao Biotechnology Co., Ltd. were used to construct a melanoma model. They were purchased one week before the experiment and were free to drink water and eat. Mouse melanoma cells B16F10 were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. The culture conditions were 37°C and 5% CO2. 5×10 cells were placed in each mouse. 5 B16F10 cells were subcutaneously inoculated into the right dorsal flank of C57 mice.

[0069] (2) mRNA-LNP administration:

[0070] 8-10 days after tumor implantation, when the tumor volume of mice grows to 50-100mm 3 The mice were randomly divided into 4 groups, with 7-8 mice in each group. The first group was the control group, which was intratumorally administered with normal saline; the second group was the LNP control group, which was intratumorally administered with empty lipid nanoparticles; the third and fourth groups were both mRNA-LNP administration groups, which were intratumorally administered with mRNA-LNP encoding Puma-BH3 and Bim-BH3 domains, respectively, as experimental groups. The dose was 10 μg mRNA / mouse each time, administered once every 2 days, for a total of 3 times. The body weight and tumor volume of each group of mice were recorded every 2 days, and the calculation formula was tumor volume = 0.5 × length × width 2 . After the mice were killed by cervical dislocation 19 days after tumor implantation, the main organs of the mice in each group were taken for tissue sections: heart, liver, spleen, lung, and kidney, and then the pathological conditions of the main organs were analyzed using H&E staining. The serum of the mice in each group was taken for ALT and AST analysis.

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

[0072] After killing the mice, the tumors of the mice in each experimental group were taken, cut into pieces with scissors, digested with tissue digestion solution, and then lysed to obtain a single tumor cell suspension. The cells were labeled with mouse APC-anti-CD45, PE-anti-CD3, FITC-anti-CD4, and PerCP-anti-CD8a antibodies to measure the tumor infiltration CD3 + / CD8 + Cytotoxic T cell ratio and CD3 + / CD4 +The ratio of helper T cells. PE-anti-CD11b, APC-anti-F4 / 80, FITC-anti-CD80, and PerCP-Cy5.5-anti-CD206 antibodies were used to label cells and determine the ratio of tumor-infiltrating M1 and M2 macrophages.

[0073] Inguinal lymph nodes of mice in each group were obtained, digested with tissue digestion solution and centrifuged to obtain single cell suspension. Mouse PE-anti-CD11c, FITC-anti-CD80, and APC-anti-CD86 antibodies were used for surface marker labeling, and flow cytometry was used to analyze the mature dendritic cells (CD11c + / CD80 + , CD11c + / CD86 + )Proportion.

[0074] (4) Results analysis

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

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

[0077] HE pathological sections of the main organs of mice and serum ALT / AST were used to evaluate the biosafety of this strategy. Fig.10 ).

[0078] Example 7. Evaluation of the tumor inhibition effect of combined mRNA-LNP drug and T cell therapy in solid tumor models

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

[0080] Six-week-old female C57 mice purchased from Hangzhou Paisiao Biotechnology Co., Ltd. were used to construct the B16-OVA melanoma model. They were purchased one week before the experiment and were free to drink water and eat. Mouse melanoma cells B16-OVA were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody. The culture conditions were 37°C and 5% CO2. 5×10 per mouse were used. 5 B16-OVA cells were subcutaneously inoculated into the right dorsal flank of C57 mice.

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

[0082] 8-10 days after tumor implantation, when the tumor volume of mice grows to 50-100mm 3 The mice were randomly divided into 4 groups, with 7-8 mice in each group. The first group was the control group, which was intratumorally administered with normal saline; the second group was the T cell treatment group; the third and fourth groups were BH3 domain mRNA groups, which were intratumorally administered with mRNA-LNP encoding Puma-BH3 and Bim-BH3 domains, respectively, at a dose of 10 μg mRNA per mouse each time; the next day after administration, in vitro activated OT-1CD8 T cells were intravenously infused at a dose of 2×10 6 The weight and tumor volume of each group of mice were recorded every 2 days, and the calculation formula was tumor volume = 0.5 × length × width 2 When the tumor grew to 15 mm, the mice were euthanized.

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

[0084] After killing the mice, the tumor and spleen tissues of each group of mice were obtained, cut into pieces with scissors, digested with tissue digestion solution, and then lysed to obtain tumor single cell suspension. Mouse Pacific Blue-anti-CD45, Brilliant Violet 510-anti-CD3, PerCP-anti-CD8a antibodies were used to label cytotoxic T cells, and FITC-anti-Granzyme B and PE-anti-Perforin were used to determine the effector function of cytotoxic T cells.

[0085] (4) Results analysis

[0086] Analysis of the tumor growth curves of each group of mice showed that the combined use of mRNA drugs encoding the BH3 domain could significantly enhance the tumor suppression effect of T cell therapy in melanoma models ( Figure 7 ), and significantly improved the survival rate of tumor model mice ( Figure 8 ). Flow cytometry data showed that the combination of mRNA drugs encoding BH3 domains can enhance the effector function of cytotoxic T cells in tumors and spleens ( Fig. 9 ).

[0087] It can be seen that this local administration strategy of mRNA encoding the BH3 domain can synergistically enhance the killing effect of T cells on tumor tissues, thereby achieving the effect of enhancing the efficacy of T cells.

[0088] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. An mRNA drug for enhancing T cell efficacy, characterized in that: The mRNA drug comprises a pharmaceutical carrier and an active ingredient, wherein the active ingredient comprises an mRNA molecule with a 5' to 3' coding sequence such as SEQ ID NO.4 or SEQ ID NO.5, which is used to encode the BH3 domain of the pro-apoptotic protein Puma or Bim fused with an adaptor scaffold, and the mRNA drug is a dosage form for local intratumoral administration.

2. The mRNA drug for enhancing T cell efficacy according to claim 1, characterized in that: The BH3 domain coding sequence of the pro-apoptotic protein family Puma or Bim is shown in SEQ ID NO.1 or SEQ ID NO.2; the aptamer scaffold sequence is the Stefin A quadruple aptamer scaffold, and the coding sequence is shown in SEQ ID NO.

3.

3. The mRNA drug for enhancing T cell efficacy according to claim 1, characterized in that: The mRNA molecule has a capping modification.

4. The mRNA drug for enhancing T cell efficacy according to claim 1, characterized in that: The pharmaceutical carrier is a lipid nanoparticle, and the mRNA molecule is encapsulated in the lipid nanoparticle.

5. A method for preparing an mRNA drug for enhancing T cell efficacy as claimed in claim 1, characterized in that: The following steps are involved: DNA fragments encoding the pro-apoptotic protein family Puma or Bim BH3 domain and the Stefin A quadruple aptamer scaffold are respectively obtained by polymerase chain reaction amplification, and the fragments are used as templates to add homologous arms for chain extension reaction to obtain fusion genes; the fusion genes are then connected to an mRNA in vitro transcription vector to obtain a recombinant plasmid, and an in vitro transcription linearization template is obtained after enzyme digestion; then the mRNA molecules are obtained through in vitro transcription, modification and purification; finally, the mRNA molecules are loaded on a pharmaceutical carrier to obtain an mRNA drug.

6. The method for preparing the mRNA drug for enhancing T cell efficacy according to claim 5, characterized in that: The method of loading the mRNA molecule onto the pharmaceutical carrier comprises: diluting the mRNA molecule into a citric acid-sodium citrate buffer solution with a pH of 4.5, adding a lipid ethanol solution to mix, repeatedly blowing and beating, and dialyzing to obtain lipid nanoparticles loaded with the mRNA; the lipid ethanol solution contains SM102, DSPC, CHO-HP and PEG2000 in a molar ratio of 50:10:38.5:1.

5.

7. Use of the mRNA drug for enhancing T cell efficacy according to any one of claims 1 to 4, characterized in that: The uses include: (1) for preparing tumor treatment drugs; (2) for combined administration with T cells to enhance the efficacy of T cells.

8. The use according to claim 7, characterized in that: The tumor is a solid tumor, including at least melanoma, colon cancer, and breast cancer.

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