Novel mRNA tumor vaccine for treating mouse liver cancer

Abstract

The invention relates to the technical field of tumor vaccines, and provides a novel mRNA (messenger Ribonucleic Acid) tumor vaccine for treating mouse liver cancer, which is prepared by the following steps: S1, specific neoantigen prediction: carrying out bioinformatics analysis on a next-generation sequencing database of tumor patients in the hospital, and selecting tumor specific neoantigens through the following steps; s11, low-quality readings are removed through a filtering step; s12, detecting single nucleotide variation by using a Mutect method and a VarScan method; s13, annotating missense mutation by using ANNOVAR software; in-vivo and in-vitro experiments prove that the vaccine has a remarkable anti-tumor effect, is designed and synthesized aiming at a newly screened new antigen mutation sequence and has high specificity and pertinence, and experimental results show that the vaccine can remarkably improve immune response of specific CTLs cells, induce T cell response of new antigen specificity, and has a good anti-tumor effect in a tumor-bearing mouse model. The vaccine has a remarkable inhibiting effect on tumor growth, and the problems in the prior art are solved through the technical scheme.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor vaccines, and specifically, to a novel mRNA tumor vaccine for treating mouse liver cancer. Background Art

[0002] In recent years, tumor-specific antigen vaccines have become a research hotspot. In particular, neoantigen peptide vaccines have shown certain clinical treatment effects. However, due to problems such as long time consumption, high cost, large molecular weight, and difficulty in effectively transferring into antigen-presenting cells in neoantigen long peptide vaccines, it is very difficult to be popularized in clinical practice. Therefore, it is urgent to develop more efficient and lower-cost tumor neoantigen vaccines for pre-clinical application research. Tumor neoantigen mRNA vaccines have unique advantages compared with tumor neoantigen peptide vaccines: through precise sequence design, mRNA vaccines can encode one or more tumor-specific antigens (TSA), and after protein translation and antigen processing in cells, they bind to the major histocompatibility antigen complex I (MHC I) in antigen-presenting cells, and finally are presented to T cells to induce a strong tumor-specific T cell response to kill tumor cells. At the same time, different from peptide vaccines, tumor mRNA vaccines can encode full-length tumor antigens, allowing APCs to perform simultaneous presentation or cross-presentation, which may stimulate a more extensive T cell response. In addition, the synthesis and processing process of mRNA vaccines takes shorter time and lower cost, and is easier to be popularized in clinical practice, so it is worthy of in-depth study. However, the efficacy of tumor neoantigen RNA vaccines is restricted by three important factors: First, the design of the RNA sequence. Some studies have shown that the sequence of the neoantigen RNA vaccine needs to compare the exon sequencing results of tumor tissues and normal tissues to find the unique mutations in tumor tissues, and then further verify the immunogenicity to determine the RNA sequence for preparing the vaccine. Second is the loading and delivery of the neoantigen RNA vaccine. Because RNA is easily degraded by enzymes, the neoantigen RNA vaccine needs to be delivered by a specific carrier, hoping to not only protect the RNA from degradation but also enhance the targeted delivery effect. Third is the tumor immune microenvironment suitable for immune cells to exert their efficacy; To address these three issues, our laboratory conducted bioinformatics analysis on the next-generation sequencing database of cancer patients in our hospital, listed the gene high-frequency mutation map, predicted and identified 85 neoantigen epitopes, screened out hot mutations from 5 high-frequency driver mutation genes (TP53, PIK3CA, ERBB2, ARID1A, and CDKN2A genes), designed and synthesized neoantigen long peptides, and carried out anti-tumor immunity research. The results showed that DC cells loaded with neoantigen peptides could significantly increase the secretion of cytokines such as IFN-γ and IL-2 by CTLs cells, promote the increase in the total number of effector T cells, and produce a certain anti-tumor effect. Using these 5 neoantigen peptide segments to design a novel mRNA vaccine, wrapping the mRNA with LNP, a neoantigen mRNA vaccine was successfully prepared, and its anti-tumor effect was verified in a mouse liver cancer model. Summary of the Invention

[0003] The present invention provides a novel mRNA tumor vaccine for treating mouse liver cancer, which solves the problems in the related art.

[0004] The technical solution of the present invention is as follows: A novel mRNA tumor vaccine for treating mouse liver cancer includes S1. Specific neoantigen prediction: We conducted bioinformatics analysis on the next-generation sequencing database of cancer patients in our hospital, and selected tumor-specific neoantigens through the following steps; S11. Remove low-quality reads through a filtering step; S12. Detect single nucleotide variations using the Mutect and VarScan methods; S13. Annotate missense mutations using the ANNOVAR software; S14. Evaluate the MHC I affinity of tumor neoantigens and the corresponding WT sequences using the PSSMHCpan algorithm; S15. Analyze the expression level of neoantigens in the transcriptome; S16. Determine the coding sequence (CDS) of the target gene, list the gene high-frequency mutation map, predict and identify 85 neoantigen epitopes, screen out hot mutations from 5 high-frequency driver mutation genes (TP53, PIK3CA, ERBB2, ARID1A, and CDKN2A genes), design and synthesize neoantigen long peptides, and carry out anti-tumor immunity research.

[0005] As a preferred embodiment of the present invention, S2. mRNA sequence design mainly includes the design of 5 elements: 5' cap (cap structure), 5'UTR, ORF, 3'UTR, and poly A tail (polyadenylate tail); Select the pUC57 plasmid cassette as the vector. First, insert the designed DNA sequence of the RNA vaccine between the MluI and XhoI restriction sites of the pUC57 plasmid, and amplify the plasmid carrying the RNA vaccine sequence. Then, use the in vitro transcription kit (mMESSAGE mMACHINE T7 ULTRA Transcription Kit) to perform in vitro transcription on the linearized plasmid. The transcription product is then processed by a purification kit (MEGAclear Transcription Clean-Up Kit) to obtain the RNA vaccine sequence for subsequent experiments. Detect the concentration, purity, and gel electrophoresis of the synthesized RNA vaccine. The absorbance at A260 of the synthesized RNA product is 23.646, and the absorbance at A280 is 12.38. Calculate the A260 / A280 value to be 1.91, indicating good purity of the synthesized product. Calculate the RNA concentration to be 0.83 mg / ml. At the same time, perform sequencing detection on the synthesized RNA. The sequencing results confirm the correctness of the RNA sequence obtained by IVT and can be used for further RNA vaccine experiments.

[0006] As a preferred embodiment of the present invention, S3. Since mRNA molecules are negatively charged, it is difficult for them to pass through the cell membrane and they are easily degraded by nucleases in the body. Therefore, a delivery technology, the LNP (lipid nanoparticle) system, which is composed of ionizable lipids, helper lipids, cholesterol, and polyethylene glycol-modified lipids, is used to effectively encapsulate and protect mRNA and promote its entry into cells. S3.1. Prepare the lipid ethanol solution: a) The components and molecular weights of LNP are shown in the following table: Name Molar ratio% Molecular weight Mass DLIN-MC3 50 642.09 130 mg DSPC 10 790.2 33 mg PEG-2000 1.5 2509.2 16 mg CHO 38.5 386.654 62 mg Absolute ethanol (AR) 100 ml b) Configure the mRNA to 33.3 μg / mL; Name Weight or volume Glacial acetic acid 8 ml Sodium acetate trihydrate 3.2 g Purified water 1000 ml S3.2. Calculate the mRNA concentration: a) Calculate the required RNA concentration based on N / P = 6 and FRR = 3; b) The average molecular weight of the bases of RNA is 340, and each base carries 1 phosphate, so the phosphorus content in RNA is 3.1 nmol / μg. c) When calculating the nitrogen-phosphorus ratio, only calculate the number of nitrogen atoms in the main lipids, so there is 0.5 mole of N in each mole of the mixed lipids; S3.3. Prepare the mRNA-acetate buffer: Weigh 3.2 g of sodium acetate trihydrate and 8 ml of glacial acetic acid, and make up the volume to 1000 ml; add the corresponding gradient of mRNA S3.4. Microfluidic mixing: a. Pass the lipid-ethanol solution through a 0.22 μm hydrophobic polytetrafluoroethylene membrane, and pass the mRNA-citrate buffer through a 0.22 μm hydrophilic polytetrafluoroethylene membrane. After passing through the membrane, load them into a microsyringe; b. With FRR = 3, the phospholipids and the buffer pass through the nexstarnano1 chip at 0.2 ml / min and 0.6 ml / min respectively and are mixed; S3.5. Dialysis and storage: a) After preparation, put it into a dialysis tube (Slide-A-Lyzer™ MINI Dialysis Device, 10K MWCO, 0.5 mL), add 14 ml of PBS and dialyze on a shaker. Change the solution after 2 hours until the dialysis ends after 18 hours; b) Store at 4°C for future use; S3.6. Characterization and identification of LNP-mRNA The Quant-iT™ RiboGreen® RNA reagent is a highly sensitive fluorescent nucleic acid stain that can detect 1 - 200 ng of nucleic acid in solution. This nucleic acid dye cannot penetrate LNP, so only the free, unencapsulated nucleic acid can be bound; Triton-100, as a surfactant, is often used as a demulsifier. The LNP-mRNA treated with 1% Triton-100 can release the encapsulated nucleic acid to obtain the total nucleic acid amount; the drug loading amount is obtained by calculating the difference in nucleic acid amount before and after demulsification, and then dividing by the total nucleic acid amount to get the encapsulation efficiency, that is: Encapsulation efficiency (%) = (Quantification after demulsification - Quantification before demulsification) / Quantification after demulsification; Dilute 20xTE to 1x with DEPC water; Dilute the standards in the kit with the diluted 1XTE to two final concentrations of 2 μg / mL and 100 ng / mL; Dilute the QuantiT™ RiboGreen® Reagen in the kit with 1xTE to two final concentrations of 200-fold and 2000-fold; Perform the detection according to the kit instructions, making duplicate wells; Use SPARK to read the fluorescence intensity, set the excitation light to 480 nm and the emission light to 520 nm; Calculate the encapsulation efficiency.

[0007] As a preferred embodiment of the present invention, S4. Construct a mouse tumor model (taking hepatocarcinoma H-22-LUC cells as an example). Establish a subcutaneous transplantation model in mice by the cell inoculation method (H22-luc cell line): culture with 90% 1640 medium + 10% FBS, and change the medium daily; culture and collect H22-luc cells in the logarithmic growth phase, resuspend them in RPMI1640 medium after counting, and adjust the cell suspension concentration to 5×106 cells / mL. Subcutaneously inoculate tumor cells in the upper right groin of mice with a 1 mL syringe (No. 4 needle), 1×106 cells / 0.2 mL / mouse, and a total of 35 C57BL / 6J mice are inoculated. When the average tumor volume of mice reaches 50-100 mm3 after H22-luc cell inoculation, that day is recorded as Day0. Exclude mice with too large or too small tumor volumes, and 30 mice are enrolled. Randomly divide the mice into a PBS group, an S-mRNA-LNP vaccine group without neoantigen peptides, and an Sm-mRNA-LNP vaccine group loaded with neoantigen peptides, with 10 mice in each group; each group of mice is administered on D0, D7, D14, D21, and D28; the PBS group is injected with 100 μL of PBS, and the S-mRNA-LNP group and the Sm-mRNA-LNP group are injected with 100 μL of the corresponding drug; after the mice are injected with tumor cells, the long and short diameters of the tumors are measured with vernier calipers once every 3 days, and the body weight of the mice is measured once every 3 days. The tumor volume calculation formula: calculate the tumor volume as 0.5×long diameter (mm)×short diameter2 (mm); perform in vivo imaging on all mice before and after drug administration. Inject each mouse intraperitoneally with D fluorescein potassium / sodium salt solution (15 mg / mL), and the dosage is 10 μL / g; perform in vivo imaging 10-15 minutes after injection; on D30, randomly select 5 mice from each group for euthanasia; observe the survival period of the remaining mice. When the tumor volume is greater than 2000 mm3, euthanize the mice without other treatment; if the tumor shrinks or disappears, the maximum period is 60 days; collect blood from the eyes of 5 randomly sacrificed mice in each group and drop it into an anticoagulant tube. Grind the spleen into a single-cell suspension, filter it through a 40 μm cell sieve, completely peel off the tumor tissue, take pictures and save them, then divide the tumor tissue into two halves, fix one half in formalin, and store the remaining half in Hanks balanced salt solution, digest it into a single-cell suspension, and perform flow cytometry detection. The cell types detected include CD4+ T cells, CD8+ T cells, Treg cells, and TAM cells, and further analyze the typing of TAM cells; S4.1. The LNP-mRNA vaccine loaded with neoantigen peptides (Sm-mRNA-LNP) induces neoantigen-specific T cell responses in tumor-bearing mice; Compared with the S-mRNA-LNP vaccine group, the neoantigen-loaded Sm-mRNA-LNP vaccine group could induce stronger T cell responses, and there were significant differences in the ability to induce IFN-γ secretion (P < 0.0001); To further explore the cytotoxicity of NRT cells induced by the Sm-mRNA-LNP vaccine and NRT cells induced by the S-mRNA-LNP vaccine, the killing ability of the two effector T cells against H-22 cells was detected under the effector-to-target ratio of 50:1; A: Flow chart of the experiment for detecting IFN-γ secretion by ELISPOT; B: Typical graph of IFN-γ secretion detected by ELISPOT (the dots in the figure are IFN-γ secreted by individual T cells); C: Cytotoxicity evaluation of NRT cells induced by the Sm-mRNA-LNP vaccine and NRT cells induced by the S-mRNA-LNP vaccine against the target cell H-22. **P < 0.01, ***P < 0.001, ns: no significant difference; S4.2, Inhibitory effect of the LNP-mRNA vaccine loaded with neoantigen peptides on tumor growth in tumor-bearing mice; Observing tumor-bearing mice treated with the LNP-mRNA vaccine loaded with neoantigen peptides, it was found that compared with the S-mRNA-LNP vaccine group without neoantigen peptides, the immune response induced by the Sm-mRNA-LNP vaccine group loaded with neoantigen peptides could significantly inhibit the tumor growth of tumor-bearing mice; Body weight is an important health indicator that can reflect the impact of treatment on the overall health of animals; No obvious changes in body weight were observed in all immunotherapy group mice, indicating that the vaccine immunotherapy has good tolerance; S4.3, Effect of the LNP-mRNA vaccine loaded with neoantigen peptides on the tumor microenvironment of tumor-bearing mice; From the statistical results, it can be seen that compared with the PBS group and the S-mRNA-LNP vaccine group without neoantigen peptides, the Sm-mRNA-LNP vaccine group could significantly increase the proportion of effector T cells in the blood and spleen of mice, promote the infiltration of CD8+ T cells in tumor tissues, and enhance their ability to secrete INF-γ to kill tumor cells.

[0008] The working principle and beneficial effects of the present invention are as follows: The present invention prepares an mRNA vaccine carrying tumor neoantigens, and verifies its significant anti-tumor effect through in vitro and in vivo experiments. It is designed and synthesized according to newly screened neoantigen mutation sequences, with high specificity and pertinence. The experimental results show that this vaccine can significantly enhance the immune response of specific CTLs cells, induce neoantigen-specific T cell responses. In the tumor-bearing mouse model, this vaccine has a significant inhibitory effect on tumor growth and has no significant impact on the body weight of mice, indicating its good safety and tolerance. In addition, this vaccine can also significantly increase the proportion of effector T cells in the blood and spleen of tumor-bearing mice, promote the infiltration of CD8+ T cells into tumor tissues, and enhance their ability to secrete INF-γ to kill tumor cells, thereby effectively improving the tumor microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present invention will be further described in detail below in conjunction with the drawings and specific embodiments.

[0010] Figure 1 It is the high-frequency mutation distribution map of tissue specimens of tumor patients of the present invention; Figure 2 It is the long peptide sequence map of neoantigens of the present invention; Figure 3 It is the diagram of the influence of the neoantigen polypeptide-specific DC vaccine of the present invention on CD8+ effector T cells and the diagram of the influence of the neoantigen polypeptide-specific DC vaccine on CD4+ effector T cells; Figure 4 It is the mRNA quality identification map of the present invention; Figure 5 In 5A is the flow chart of the ELISPOT experiment of the present invention, 5B is the detection result diagram of the present invention, and 5C is the comparison schematic diagram of the present invention; Figure 6 It is the schematic diagram of the treatment plan of the present invention; Figure 7 In 7A is the bioluminescence in vivo imaging of tumor-bearing mice on the first day of tumor formation and before euthanasia, 7B is the anatomical diagram of mouse tumors, and 7C is the curve of the change in the tumor volume of mice after treatment of the present invention; Figure 8 It is the schematic diagram of the influence on the body weight of mice of the present invention; Figure 9 It is the diagram of the change in the proportion of CD4+ and CD8+ T cells in the blood and spleen of tumor-bearing mice in each group detected by flow cytometry of the present invention; Figure 10 It is the diagram of the cytokine secretion of specific CTLs cells stimulated by the neoantigen-specific DC vaccine of the present invention; Figure 11 It is the schematic diagram of the encapsulation efficiency calculation of the present invention. SPECIFIC EMBODIMENTS

[0011] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0012] As Figures 1 to 11 shown, 1. Specific neoantigen prediction: We performed bioinformatics analysis on the next-generation sequencing database of tumor patients in our hospital and selected tumor-specific neoantigens through the following steps: (1) Remove low-quality reads through a filtering step; (2) Detect single nucleotide variations using the Mutect and VarScan methods. (3) Annotate missense mutations using the ANNOVAR software; (4) Evaluate the MHC I affinity of tumor neoantigens and the corresponding WT sequences using the PSSMHCpan algorithm; (5) Analyze the expression levels of neoantigens in the transcriptome; (6) Determine the coding sequence (CDS) of the target gene. A gene high-frequency mutation map (Figure 1) was listed, 85 neoantigen epitopes were predicted and identified, hot mutations from 5 high-frequency driver mutation genes (TP53, PIK3CA, ERBB2, ARID1A, and CDKN2A genes) were screened out, and neoantigen long peptides were designed and synthesized (Figure 2), and anti-tumor immunity research was carried out.

[0013] We verified that mature DC cells loaded with neoantigen peptides can stimulate the immunogenic characteristics of specific CTLs: Flow cytometry results showed that DC cells loaded with neoantigen peptides could significantly increase the secretion of IFNγ, IL-2, TNF-α, IL-6, and IL-5 by CTLs cells and reduce the secretion level of IL-1beta (Table 1). Neoantigen polypeptides or neoantigen-specific DC vaccines have a good sensitizing effect on T cells, promote the proliferation of CD3+ T cells, and promote the increase in the total number of effector T cells, especially a significant increase in CD8+ and CD4+ effector T cells (Teff). (See Figure 3 and Figure 4 ) Table 1 Secretion of cytokines by neoantigen-specific DC vaccines stimulating specific CTLs Figure 3 : The left figure shows the effect of the neoantigen polypeptide-specific DC vaccine on CD8+ effector T cells; the right figure shows the effect of the neoantigen polypeptide-specific DC vaccine on CD4+ effector T cells 2. Design and preparation of tumor neoantigen mRNA vaccines The pUC57 plasmid cassette was selected as the vector. First, the designed DNA sequence of the RNA vaccine was inserted between the MluI and XhoI restriction sites of the pUC57 plasmid, and the plasmid carrying the RNA vaccine sequence was obtained by amplification. The linearized plasmid was subjected to in vitro transcription using an in vitro transcription kit (mMESSAGE mMACHINETM T7 ULTRA Transcription Kit), and the transcription product was further processed with a purification kit (MEGAclearTM Transcription Clean-Up Kit) to obtain the RNA vaccine sequence for subsequent experiments. The synthesized RNA vaccine was detected for concentration, purity, and gel electrophoresis ( Figure 4 ). The absorbance at A260 of the synthesized RNA product was 23.646, and the absorbance at A280 was 12.38. The calculated A260 / A280 value was 1.91, indicating good purity of the synthesized product. The RNA concentration was calculated to be 0.83 mg / ml. At the same time, the synthesized RNA was sequenced, and the sequencing results confirmed the correctness of the RNA sequence obtained by IVT, which could be used for further RNA vaccine experiments.

[0014] Figure 4 mRNA Quality Identification 3. Encapsulation of mRNA by LNP and Characterization and Identification of LNP-mRNA Since mRNA molecules are negatively charged, difficult to pass through cell membranes, and easily degraded by nucleases in the body, delivery technology is the key to the success of mRNA therapy. Lipid nanoparticles (LNPs) are a commonly used delivery system, usually composed of ionizable lipids, helper lipids, cholesterol, and polyethylene glycol-modified lipids, which can effectively encapsulate and protect mRNA and promote its entry into cells. There are various methods for preparing LNPs, and among them, the microfluidic mixing method has become the most popular preparation method in clinical research due to its high encapsulation efficiency, precise control of particle size and PDI, and good repeatability.

[0015] 3.1 Preparation of Lipid Ethanol Solution: a) The components and molecular weights of LNP are shown in the following table: Name Molar Ratio % Molecular Weight Mass DLIN-MC3 50 642.09 130 mg DSPC 10 790.2 33 mg PEG-2000 1.5 2509.2 16 mg CHO 38.5 386.654 62 mg Absolute ethanol (AR) 100 ml b) Prepare the mRNA to a concentration of 33.3 μg / mL.

[0016] Name Weight or volume Glacial acetic acid 8 ml Sodium acetate trihydrate 3.2 g Purified water 1000 ml 3.2 Calculate the mRNA concentration: a) Calculate the required RNA concentration based on N / P = 6 and FRR = 3.

[0017] b) The average molecular weight of the bases of RNA is 340, and each base carries 1 phosphate, so the phosphorus content in RNA is 3.1 nmol / μg. c) When calculating the nitrogen-phosphorus ratio, only calculate the number of nitrogen atoms in the main lipids, so each mole of the mixed lipids contains 0.5 moles of N.

[0018] 3.3 Prepare the mRNA-acetate buffer: Weigh 3.2 g of sodium acetate trihydrate and 8 ml of glacial acetic acid, make up the volume to 1000 ml. Add the corresponding gradient of mRNA 3.4 Microfluidic mixing: a. Pass the lipid-ethanol solution through a 0.22 μm hydrophobic polytetrafluoroethylene membrane, and pass the mRNA-citrate buffer through a 0.22 μm hydrophilic polytetrafluoroethylene membrane. After passing through the membrane, load them into a microsyringe.

[0019] b. With FRR = 3, the phospholipids and the buffer are mixed at 0.2 ml / min and 0.6 ml / min through the nexstarnano1 chip.

[0020] 3.5 Dialysis and storage: a) After preparation, put it into a dialysis tube (Slide-A-Lyzer™ MINI Dialysis Device, 10K MWCO, 0.5 mL), add 14 ml of PBS, and dialyze on a shaker. Change the solution after 2 hours until the dialysis is completed after 18 hours.

[0021] b. Store at 4°C for later use.

[0022] 3.6 Characterization and identification of LNP-mRNA Quant-iT™ RiboGreen® RNA reagent is a super-sensitive fluorescent nucleic acid stain that can detect 1-200 ng of nucleic acid in solution. This nucleic acid dye cannot penetrate LNP, so only free, unencapsulated nucleic acid can be bound. Triton-100, a surfactant, is often used as a demulsifier. LNP-mRNA treated with 1% Triton-100 can release the encapsulated nucleic acid to obtain the total nucleic acid amount. The drug loading is obtained by calculating the difference in nucleic acid amount before and after demulsification, and the encapsulation efficiency can be obtained by dividing it by the total nucleic acid amount, that is: Encapsulation efficiency (%) = (Quantification after demulsification - Quantification before demulsification) / Quantification after demulsification. Dilute 20xTE to 1x with DEPC water; dilute the standards in the kit with the diluted 1XTE to two final concentrations of 2 μg / mL and 100 ng / mL; dilute QuantiT™ RiboGreen® Reagen in the kit with 1xTE to two final concentrations of 200-fold and 2000-fold; perform detection according to the kit instructions and make duplicate wells. Use SPARK to read the fluorescence intensity, set the excitation light to 480 nm, and the emission light to 520 nm. Calculate the encapsulation efficiency.

[0023] 4. In vivo experiments were conducted to verify the anti-tumor immune therapy effect of specific LNP-mRNA vaccine pulsed DC cells.

[0024] Construct a mouse tumor model (taking liver cancer H-22-LUC cells as an example). Establish a subcutaneous transplantation model in mice by the cell inoculation method (H22-luc cell line): Culture with 90% 1640 medium + 10% FBS and change the medium daily. Collect H22-luc cells in the logarithmic growth phase, resuspend them in RPMI1640 medium after counting, and adjust the cell suspension concentration to 5×106 cells / mL. Subcutaneously inoculate tumor cells in the mice above the right groin with a 1 mL syringe (No. 4 needle), 1×106 cells / 0.2 mL / mouse, and a total of 35 C57BL / 6J mice are inoculated. When the average tumor volume of the mice reaches 50-100 mm3 after the inoculation of H22-luc cells, that day is recorded as Day0. Exclude mice with too large or too small tumor volumes, and 30 mice are included in the group. Randomly divide the mice into a PBS group, an S-mRNA-LNP vaccine group without neoantigen peptides, and an Sm-mRNA-LNP vaccine group loaded with neoantigen peptides, with 10 mice in each group. Administer drugs to the mice on D0, D7, D14, D21, and D28. Inject 100 μL of PBS into the PBS group, and inject 100 μL of the corresponding drug into the S-mRNA-LNP group and the Sm-mRNA-LNP group. After injecting the tumor cells into the mice, measure the long and short diameters of the tumor once every 3 days with a vernier caliper, and measure the body weight of the mice once every 3 days. The tumor volume calculation formula: Calculate the tumor volume as 0.5×long diameter (mm)×short diameter2 (mm). Perform in vivo imaging on all mice before and after drug administration. Intraperitoneally inject D fluorescein potassium / sodium salt solution (15 mg / mL) into each mouse, and the dosage is 10 μL / g. Perform in vivo imaging 10-15 minutes after injection. Euthanize 5 randomly selected mice in each group on D30. Observe the survival period of the remaining mice. When the tumor volume is greater than 2000 mm3, euthanize the mice without other treatments. If the tumor shrinks or disappears, the maximum period is 60 days. Collect blood from the eyes of 5 randomly sacrificed mice in each group and drop it into an anticoagulant tube. Grind the spleen into a single cell suspension, filter it through a 40 μm cell sieve, completely peel the tumor tissue, take pictures and save them, then divide the tumor tissue into two parts, fix one half in formalin, store the remaining half in Hanks balanced salt solution, digest it into a single cell suspension, and perform flow cytometry detection. The cell types detected include CD4+ T cells, CD8+ T cells, Treg cells, and TAM cells, and further analyze the typing of TAM cells.

[0025] 4.1 The LNP-mRNA vaccine (Sm-mRNA-LNP) loaded with neoantigen peptides induces neoantigen-specific T cell responses in tumor-bearing mice The ELISPOT experimental procedure is shown in Figure ( Figure 5 A), and the detection results ( Figure 5B) The results showed that, compared with the S-mRNA-LNP vaccine group, the neoantigen-loaded Sm-mRNA-LNP vaccine group could induce stronger T cell responses, and there were significant differences in the ability to induce IFN-γ secretion (P < 0.0001). To further explore the cytotoxicity of NRT cells induced by the Sm-mRNA-LNP vaccine and NRT cells induced by the S-mRNA-LNP vaccine, the killing ability of the two effector T cells against H-22 cells was detected under the effector-to-target ratio of 50:1. The results showed that the ability of NRT cells induced by the Sm-mRNA-LNP vaccine to kill H-22 cells was significantly higher than that of NRT cells induced by the S-mRNA-LNP vaccine ( Figure 5 C).

[0026] Figure 5 A: Flow chart of the IFN-γ secretion detection experiment by ELISPOT; B: Typical graph of IFN-γ secretion detection by ELISPOT (the dots in the figure are IFN-γ secreted by single T cells); C: Cytotoxicity evaluation of NRT cells induced by the Sm-mRNA-LNP vaccine and NRT cells induced by the S-mRNA-LNP vaccine against the target cell H-22. **P < 0.01, ***P < 0.001, ns: no statistically significant difference.

[0027] 4.2 Inhibitory effect of the LNP-mRNA vaccine loaded with neoantigen peptides on tumor growth in tumor-bearing mice As Figure 6 shown in the treatment protocol, tumor-bearing mice treated with the neoantigen peptide-loaded LNP-mRNA vaccine were observed. The results showed that, compared with the S-mRNA-LNP vaccine group without neoantigen peptides, the immune response induced by the Sm-mRNA-LNP vaccine group loaded with neoantigen peptides could significantly inhibit the tumor growth of tumor-bearing mice ( Figure 7 ). Body weight is an important health indicator that can reflect the impact of treatment on the overall health status of animals. As Figure 8 shown, no obvious changes in body weight were observed in all immunotherapy group mice, indicating that the vaccine immunotherapy had good tolerance.

[0028] Figure 7 A: Bioluminescence in vivo imaging of tumor-bearing mice on the first day of tumor formation and before euthanasia. It can be seen from the figure that the tumor activity in the Sm-mRNA group loaded with neoantigen peptides was significantly reduced. B: Dissected mouse tumors. C: Curve of changes in mouse tumor volume after treatment. **P < 0.01, ***P < 0.001, ns: no statistically significant difference.

[0029] Figure 8: Effects of different treatments on the body weight of mice. **P < 0.01, ***P < 0.001, ns: no statistically significant difference.

[0030] 4.3 Effects of the LNP-mRNA vaccine loaded with neoantigen peptides on the tumor microenvironment of tumor-bearing mice.

[0031] As Figure 9 shown, flow cytometry was used to detect the changes in the tumor microenvironment of tumor-bearing mice after vaccine treatment. From the statistical results, it can be seen that compared with the PBS group and the S-mRNA-LNP vaccine group without neoantigen peptides, the Sm-mRNA-LNP vaccine group can significantly increase the proportion of effector T cells in the blood and spleen of mice, promote the infiltration of CD8+ T cells in tumor tissues, and enhance their ability to secrete INF-γ to kill tumor cells.

[0032] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A novel mRNA tumor vaccine for treating mouse liver cancer, characterized in that: include S1. Prediction of specific neoantigens: We performed bioinformatics analysis on the next-generation sequencing database of tumor patients in our hospital and selected tumor-specific neoantigens through the following steps; S11, remove low-quality reads through filtering steps; S12, detection of single nucleotide variations using Mutect and VarScan methods; S13, annotate missense mutations using ANNOVAR software; S14, using the PSSMHCpan algorithm to assess the MHC I affinity of tumor neoantigens and corresponding WT sequences; S15, analysis of neoantigen expression levels in the transcriptome; S16. Determine the target gene coding sequence (CDS), list the high-frequency mutation map of the gene, predict and identify 85 new antigen epitopes, screen out hotspot mutations from 5 high-frequency driver mutation genes (TP53, PIK3CA, ERBB2, ARID1A and CDKN2A genes), design and synthesize new antigen long peptides, and conduct anti-tumor immunity research.

2. The design and preparation of a novel mRNA tumor vaccine for treating mouse liver cancer according to claim 1, characterized in that: S2. mRNA sequence design mainly includes the design of 5 elements: 5' cap (cap structure), 5' UTR, ORF, 3' UTR and poly A tail (polyadenylic acid tail); pUC57 plasmid cassette was selected as the vector. The DNA sequence of the designed RNA vaccine was first inserted into the pUC57 plasmid: between the MluI and XhoI restriction sites, and the plasmid loaded with the RNA vaccine sequence was amplified. The linearized plasmid was transcribed in vitro using an in vitro transcription kit (mMESSAGE mMACHINETMT7 ULTRA Transcription Kit). The transcription product was then treated with a purification kit (MEGAclear TMT transcription Clean-Up Kit) to obtain the RNA vaccine sequence for subsequent experiments. The synthesized RNA vaccine was tested for concentration, purity and gel electrophoresis. The A260 absorbance of the synthesized RNA product was 23.646, and the A280 absorbance was 12.

38. The calculated A260 / A280 value was 1.91, indicating that the purity of the synthesized product was good. The calculated RNA concentration was 0.83 mg / ml. At the same time, the synthesized RNA was sequenced and tested. The sequencing results confirmed the correctness of the RNA sequence obtained by IVT and it can be used for further RNA vaccine experiments.

3. The novel mRNA tumor vaccine LNP encapsulated mRNA and LNP-mRNA characterization and identification for treating mouse liver cancer according to claim 1, characterized in that: S3. Since mRNA molecules are negatively charged and difficult to pass through the cell membrane, and are easily degraded by nucleases in the body, the delivery technology, LNP (lipid nanoparticle) system, composed of ionizable lipids, auxiliary lipids, cholesterol and polyethylene glycol-modified lipids, can effectively encapsulate and protect mRNA and promote its entry into cells; S3.

1. Prepare lipid ethanol solution: a) The composition and molecular weight of LNP are shown in the following table: Name Mole Ratio % Molecular Weight Mass DLIN-MC3 50 642.09 130mg DSPC 10 790.2 33mg PEG-2000 1.5 2509.2 16mg CHO 38.5 386.654 62mg Anhydrous ethanol (AR) 100ml b) mRNA was prepared to 33.3 ug / mL; Name Weight or Volume Glacial acetic acid 8ml Sodium acetate trihydrate 3.2g Purified water 1000ml S3.

2. Calculate mRNA concentration: a) Calculate the required RNA concentration based on N / P=6 and FRR=3; b) The average molecular weight of RNA bases is 340, and each base carries 1 phosphate, so the phosphorus content in RNA is 3.1 nmol / μg. c) When calculating the nitrogen-to-phosphorus ratio, only the number of nitrogen atoms in the main lipids is counted, so each mole of mixed lipids contains 0.5 mol of N; S3.

3. Prepare mRNA-acetate buffer: Weigh 3.2g sodium acetate trihydrate and 8ml glacial acetic acid, dilute to 1000ml; add the corresponding gradient of mRNA S3.4, Microfluidic Mixing: a. Pass the lipid-ethanol solution through a 0.22 μm hydrophobic polytetrafluoroethylene membrane, and pass the mRNA-citrate buffer through a 0.22 μm hydrophilic polytetrafluoroethylene membrane, and then load into a micro-syringe; b. At FRR = 3, phospholipids and buffer were mixed at 0.2 ml / min and 0.6 ml / min through the NEXSTAR nano1 chip; S3.5, Dialysis and Storage: a) After preparation, place the sample in a dialysis tube (Slide-A-Lyzer™ MINI Dialysis Device, 10K MWCO, 0.5 mL) and add 14 ml PBS. Perform dialysis on a shaker. Change the solution after 2 hours until the dialysis is completed after 18 hours. b) Store at 4°C until use; S3.

6. LNP-mRNA characterization and identification Quant-iT™ RiboGreen® RNA reagent is an ultra-sensitive fluorescent nucleic acid stain that can detect 1-200 ng of nucleic acid in solution. This nucleic acid dye cannot penetrate LNP, so only free nucleic acid not encapsulated by LNP can be bound; Triton-100 is often used as a demulsifier as a surfactant. LNP-mRNA obtained by treating with 1% Triton-100 can release the encapsulated nucleic acid and obtain the total nucleic acid amount; the drug loading amount is obtained by calculating the difference in the amount of nucleic acid before and after demulsification, and then divided by the total amount of nucleic acid to obtain the encapsulation rate, that is: Encapsulation rate (%) = (quantification after demulsification - quantification before demulsification) / quantification after demulsification; dilute 20xTE to 1x with DEPC water; dilute the standard in the kit with the diluted 1XTE to two final concentrations of 2 μg / mL and 100 ng / mL; dilute the QuantiT™ RiboGreen® Reagen in the kit with 1xTE to 200 times and 2000 times times of two final concentrations; perform the test according to the instructions of the kit and make duplicate wells; use SPARK to read the fluorescence intensity, with the excitation light set to 480 nm and the emission light set to 520 nm; calculate the encapsulation efficiency.

4. The experimental verification of a novel mRNA tumor vaccine for treating mouse liver cancer according to claim 3, characterized in that: S4. Construct a mouse tumor model (take liver cancer H-22-LUC cells as an example), and establish a mouse subcutaneous transplantation model by cell inoculation method (H22-luc cell line): 90% 1640 medium + 10% FBS culture, change the medium daily; collect H22-luc cells in the logarithmic growth phase, count and resuspend in the culture medium RPMI1640, and adjust the cell suspension concentration to 5×106 cells / mL. Use a 1 mL syringe (No. 4 needle) to subcutaneously inoculate tumor cells above the right groin of the mouse, 1×106 cells / 0.2 mL / mouse, and inoculate a total of 35 C57BL / 6J mice. When the average tumor volume of mice reached 50-100 mm3 after H22-luc cell inoculation, the diary was Day 0, and mice with too large or too small tumor volume were eliminated. 30 mice were randomly divided into PBS group, S-mRNA-LNP vaccine group without neoantigen peptide, and Sm-mRNA-LNP vaccine group loaded with neoantigen peptide, with 10 mice in each group; mice in each group were administered on D0, D7, D14, D21, and D28; the PBS group was injected with 100 μL of PBS, and the S-mRNA-LNP group and Sm-mRNA-LNP group were injected with 100 μL of the corresponding drug; after the mice were injected with tumor cells, the long and short diameters of the tumor were measured once every 3 days with a vernier caliper, and the weight of the mice was measured once every 3 days. The tumor volume was calculated by the formula: 0.5×long diameter (mm)×short diameter 2 (mm); all mice were imaged in vivo before and after medication. Each mouse was intraperitoneally injected with D-luciferin potassium / sodium salt solution (15 mg / mL) at a dosage of 10 μL / g; live imaging was performed 10 to 15 minutes after injection; 5 mice were randomly selected from each group and euthanized on D30; the survival of the remaining mice was observed, and when the tumor volume was greater than 2000 mm3, the mice were euthanized without other treatment; if the tumor shrank or disappeared, the maximum period was 60 days; the eyeballs of the 5 mice randomly killed in each group were removed to collect blood, which was dripped into an anticoagulant tube, the spleen was ground into a single cell suspension, filtered with a 40 μm cell sieve, the tumor tissue was completely removed, and after taking pictures and keeping them, the tumor tissue was divided into two, one half was fixed in formalin, and the remaining half was stored in Hanks balanced solution, digested into a single cell suspension, and subjected to flow cytometry detection. The cell classifications detected included CD4+ T cells, CD8+ T cells, Treg cells, and TAM cells, and the typing of TAM cells was further analyzed; S4.1, LNP-mRNA vaccine loaded with neoantigen peptides (Sm-mRNA-LNP) induced neoantigen-specific T cell responses in tumor-bearing mice; Compared with the S-mRNA-LNP vaccine group, the Sm-mRNA-LNP vaccine group loaded with new antigens induced stronger T cell responses, and the ability to induce IFN-γ secretion was significantly different (P<0.0001). In order to further explore the cytotoxicity of NRT cells induced by Sm-mRNA-LNP vaccines and NRT cells induced by S-mRNA-LNP vaccines, the killing ability of the two effector T cells on H-22 cells was detected under the condition of an effector-target ratio of 50:

1. Flow chart of ELISPOT assay for IFN-γ secretion; B: Typical graph of ELISPOT detection of IFN-γ secretion (the dots in the figure are IFN-γ secreted by a single T cell); C: Evaluation of the cytotoxicity of NRT cells induced by Sm-mRNA-LNP vaccine and NRT cells induced by S-mRNA-LNP vaccine on target cells H-22. **P < 0.01, ***P < 0.001, ns: no statistically significant difference; S4.

2. Inhibitory effect of LNP-mRNA vaccine loaded with neoantigen peptide on tumor growth in tumor-bearing mice; The results showed that the immune response induced by the Sm-mRNA-LNP vaccine loaded with neoantigen peptides could significantly inhibit the tumor growth of tumor-bearing mice compared with the S-mRNA-LNP vaccine group without neoantigen peptides. Body weight is an important health indicator that can reflect the effect of treatment on the overall health of animals. No significant changes in body weight were observed in all immunotherapy groups, indicating that vaccine immunotherapy is well tolerated. S4.

3. Effects of LNP-mRNA vaccines loaded with neoantigen peptides on the tumor microenvironment of tumor-bearing mice; From the statistical results, it can be seen that compared with the PBS group and the S-mRNA-LNP vaccine group without new antigen peptides, the Sm-mRNA-LNP vaccine group can significantly increase the proportion of effector T cells in the blood and spleen of mice, promote the infiltration of CD8+T cells in tumor tissues, and enhance their ability to secrete INF-Y to kill tumor cells.

Images