GPC3 derived antigen peptide vaccine, mRNA vaccine, saRNA vaccine and preparation method and application thereof

Abstract

The invention discloses a GPC3 derived antigen peptide vaccine, an mRNA vaccine, a saRNA vaccine and a preparation method and application thereof. The amino acid sequence of the antigen peptide vaccine is one or any combination of more of amino acid sequences shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 6. According to the invention, an efficient pGPC3 model antigen peptide is screened based on GPC protein, and pGPC3 mRNA and pGPC3 saRNA are designed according to a pGPC3 sequence example. The pGPC3 saRNA (at) Lipi vaccine is prepared through a liposome technology, and due to the fact that limiting conditions such as nonapeptide and high-affinity binding with H-2 I type molecules (H-2Kb) are adopted during pGPC3 polypeptide antigen screening, APC cells are facilitated to present antigens through an MHCI restrictive pathway and activate CD8 + T cells, and GPC3 specific CTL is triggered to efficiently kill liver cancer cells. Finally, the pGPC3 saRNA (at) Lipi vaccine can completely inhibit the growth of the liver cancer, the inhibition rate reaches 99.8%, and the liver cancer of 4 / 8 mice completely fades down. In addition, the method can be used for vaccine adjuvant and carrier research on a liver cancer subcutaneous transplantation tumor model.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biotechnology, and particularly relates to a GPC3-derived antigen peptide saRNA vaccine design, a preparation method and application. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is the sixth most diagnosed cancer and the third leading cause of cancer death worldwide ] In recent years, with the continuous development of immunology, immunotherapy has become a research hotspot, and the development of cancer immunotherapy has improved the prognosis of various human cancers and produced significant therapeutic effects on patients who are difficult to cure by other conventional treatment modes.

[0003] Carcinoembryonic antigen (CEA) is an acidic glycoprotein with human embryonic antigen characteristics, which exists on the surface of cancer cells differentiated from endoderm cells, is a structural protein of the cell membrane, and is also a broad-spectrum tumor marker. Glypican 3 (GPC3) is a carcinoembryonic glycoprotein attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor, which is composed of 580 amino acids and has a size of 70 kDa, and is believed to play a crucial role in regulating the proliferation of embryonic mesodermal tissue cells. GPC3 is widely expressed in the placenta, as well as the liver, lung and kidney of the embryo, but is hardly detected in most organs of adults. GPC3 is overexpressed in some tumors, especially hepatocellular carcinoma (HCC). Serum GPC3 levels and GPC3 immunoreactivity in tumor cells are of great significance for the prognosis of HCC patients.

[0004] Most of the current vaccine and adjuvant research is based on melanoma, and there are few studies on nano-vaccines for liver cancer. The reasons are as follows: (1) the existence of OVA gene modified melanoma cells (B16-OVA) on the market makes the immune cells of the body accurately recognize and kill melanoma cells carrying OVA after being stimulated by the "model antigen" OVA. 257-264 Currently, there is no commercially available OVA-modified liver cancer cell, and the process of artificially modifying liver cancer cells is tedious, so OVA antigen peptide cannot be used as a model antigen in the study of liver cancer vaccines. (2) Tyrosinase-related protein-2 (TRP-2) is a tumor-associated antigen widely expressed in melanoma, so TRP-2 180-188 is an ideal target for immunological treatment of melanoma and glioma. However, there is currently a lack of a model antigen for liver cancer, which limits the research and clinical transformation of liver cancer vaccines. Definitions: VEEV virus: Venezuelan encephalitis virus. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a design, preparation method, and application of a GPC3-derived antigenic peptide saRNA vaccine.

[0006] The objective of this invention is achieved through the following technical solution: A GPC3-derived antigenic peptide vaccine, wherein the amino acid sequence of the antigenic peptide vaccine is one or more of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, preferably SEQ ID NO:4.

[0007] A GPC3-derived antigenic peptide mRNA vaccine, wherein the nucleotide sequence of the CDS region of the GPC3-derived antigenic peptide mRNA vaccine encodes one of the antigenic peptide vaccines selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or the nucleotide sequence of the CDS region of the mRNA vaccine encodes a repeating unit formed by 6-14 P2A restriction enzyme regions-GPC3 antigenic peptide vaccine regions, wherein the amino acid sequence of the GPC3 antigenic peptide vaccine region is one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and the amino acid sequence of the P2A restriction enzyme region is shown in SEQ ID NO:7.

[0008] As a further improvement, the nucleotide sequence of the CDS region of the GPC3-derived antigenic peptide mRNA vaccine is shown in SEQ ID 8, and the nucleotide sequence of the GPC3-derived antigenic peptide mRNA vaccine is shown in SEQ ID 10.

[0009] A GPC3-derived antigenic peptide saRNA vaccine, wherein the nucleotide sequence of the CDS region of the saRNA vaccine encodes one of the antigenic peptide vaccines selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or the nucleotide sequence of the CDS region of the mRNA vaccine encodes a repeating unit encoding a region formed by multiple P2A restriction enzyme regions and GPC3 antigenic peptide vaccine regions, wherein the amino acid sequence of the GPC3 antigenic peptide vaccine region is one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the amino acid sequence of the P2A restriction enzyme region is shown in SEQ ID NO:7.

[0010] In a further improvement, the self-amplifying virus of the saRNA vaccine is one of VEEV virus, alpha virus, flavivirus, filamentous virus, or lentivirus; the nucleotide sequence of the CDS region of the mRNA vaccine encodes a repeating unit region formed by 6-14 P2A restriction enzyme regions-GPC3 antigen peptide vaccine regions.

[0011] In a further improvement, the self-amplifying virus of the GPC3-derived antigenic peptide saRNA vaccine is VEEV virus, the nucleotide sequence of the CDS region of the GPC3-derived antigenic peptide saRNA vaccine is shown in SEQ ID 9, the 5' end of the saRNA vaccine encodes the nucleotide coding region of four non-structural proteins of VEEV virus, and finally the CDS region of VEEV virus located after the subgenomic promoter is replaced with the coding region of the repeat unit.

[0012] One use of the above-mentioned vaccine is as a medicine for treating or preventing neoplastic diseases, including but not limited to liver cancer.

[0013] A method for designing a vaccine, wherein the vaccine is at least one of a GPC3-derived antigenic peptide vaccine, a GPC3-derived antigenic peptide mRNA vaccine, and a GPC3-derived antigenic peptide saRNA vaccine, specifically comprising the following steps: Step 1: Predict the antigenic epitopes of the polypeptide sequences in the complete amino acid sequence of GPC3 using IEDB software. The software scores the immunogenicity of the polypeptide sequences in the GPC3 protein sequence and arranges them from highest to lowest score. Step 2: The top n ranked peptide sequences are docked with H-2Kb molecules using the MHC-I binding prediction tool on the IEDB website to predict docking affinity. Peptide sequences with predicted docking affinity within a preset threshold range are selected as candidate peptides. Step 3: Incubate each candidate peptide with dendritic cells derived from bone marrow and splenic lymphocytes for 6 h, and detect the actual affinity of each candidate peptide epitope to H-2Kb molecules by flow cytometry. Step 4: Arrange the candidate peptides in descending order of actual affinity and design a control group. Prepare a vaccine by mixing the antigens of the first m candidate peptides with Freund's adjuvant and immunize mice twice, seven days apart. The first dose was prepared by mixing 100 μg of candidate peptide antigen with complete Freund's adjuvant, and the booster dose was prepared by mixing 50 μg of candidate peptide antigen with incomplete Freund's adjuvant. Seven days after the last immunization, spleen lymphocytes were extracted and stimulated again with 20 μg / mL of the corresponding peptide antigen. Two days later, they were co-incubated with Hepa1-6 liver cancer cells for 24 h. The killing efficiency of antigen peptide-specific cytotoxic T cells against Hepa1-6 liver cancer cells was detected by CCK-8 assay. The candidate peptides with higher killing efficiency than the control group were used as GPC3-derived antigen peptide vaccines. When the vaccine is one of a GPC3-derived antigenic peptide mRNA vaccine and a GPC3-derived antigenic peptide saRNA vaccine, the following steps are also included: Step 5: Design at least one of the pGPC3 mRNA and pGPC3 saRNA nucleotide sequences for translation to generate GPC3-derived antigenic peptide vaccines. Amplify plasmids containing pGPC3 mRNA or pGPC3 saRNA nucleotide sequences using E. coli, transcribe and purify them in vitro, and then verify whether the extracted RNA is consistent with the designed pGPC3 mRNA or pGPC3 saRNA nucleotide sequence after sequencing. If they are consistent, successfully transformed E. coli are obtained. Use the successfully transformed E. coli to produce pGPC3 mRNA or pGPC3 saRNA nucleotide sequences. Step 6: Load the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence onto liposomes to prepare GPC3-derived antigenic peptide mRNA vaccine or GPC3-derived antigenic peptide saRNA vaccine.

[0014] In a further improvement, in step one, the length of the polypeptide sequence is 8-10. The amino acid sequence of the antigen peptide vaccine is at least one of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0015] A vaccine, wherein the vaccine is a saRNA vaccine or an mRNA vaccine, wherein the CDS region of the vaccine contains multiple tandem repeat unit coding regions, each repeat unit coding region being formed by a P2A enzyme cleavage coding region plus an antigen peptide vaccine coding region.

[0016] The advantages of this invention are as follows: (1) A highly efficient GPC3 model antigen peptide was screened out for use as a vaccine adjuvant and vector in the C57BL / 6j mouse Hepa1-6 subcutaneous hepatocellular carcinoma xenograft model.

[0017] (2) The screening of GPC3 polypeptide antigens uses nonapeptide and high affinity binding to H-2 type I molecules (H-2Kb) as limiting conditions, which is conducive to APC cells presenting antigens through the MHC I-restricted pathway and activating CD8+ T cells, thereby triggering GPC3-specific CTLs to efficiently kill liver cancer cells.

[0018] (3) The unique design mode of GPC3-derived antigen peptide mRNA and saRNA vaccines: each mRNA and saRNA has 6-14 alternating repeating units of "P2A enzyme digestion-GPC3-derived antigen short peptide" formed alternately, that is, after a single translation, one mRNA and saRNA can be digested to obtain 6-14 pGPC3 short peptides.

[0019] (4) The GPC3 model antigen peptide saRNA vaccine is a liposomal vaccine. A single dose can induce sustained immunity, directly inhibit liver cancer and achieve complete regression of liver cancer carried by mice. It can effectively prevent the occurrence and development of liver cancer and can be used as an adjuvant treatment after surgery.

[0020] (5) The design, screening and preparation methods of the GPC3 model antigen peptide saRNA vaccine in this project are universal and can be used for research on adjuvants and vectors for human liver cancer vaccines. Attached Figure Description

[0021] The invention will be further illustrated with reference to the accompanying drawings, but the contents of the drawings do not constitute any limitation on the invention.

[0022] Figure 1 The docking model and docking fraction diagram of the candidate peptide with the MHC-Ⅰ molecule are shown.

[0023] Figure 2a To analyze the affinity of candidate peptides for MHC-I molecules on dendritic cells using flow cytometry; Figure 2b Flow cytometry was used to quantify the affinity of candidate peptides for MHC-I molecules on dendritic cells. Data are presented as mean ± standard deviation, n=3. ***P<0.001, ns: no significant difference.

[0024] Figure 3a To analyze the affinity of candidate peptides for MHC-I molecules on splenic lymphocytes using flow cytometry.

[0025] Figure 3b Flow cytometry was used to quantify the affinity of candidate peptides for MHC-I molecules on splenic lymphocytes. Data are presented as mean ± standard deviation, n=3. ***P<0.001, ns: no significant difference.

[0026] Figure 4 A diagram of an immunoassay protocol used to screen for the in vivo immunogenicity of peptides; Figure 5a To screen for GPC3-specific epitope peptides for the killing rate of CTLs on Hepa1-6 cells by CCK-8 assay.

[0027] Figure 5b To investigate the effects of CTLs stimulated with or without peptide stimulation on the viability of Hepa1-6 and L02 cells using a CCK-8 assay. Data are expressed as mean ± standard deviation, n=3. *P<0.05; **P<0.01; ***P<0.001, ns: no significant difference.

[0028] Figure 6 To observe the effects of specific CTLs induced by various candidate peptides on the morphology of Hepa1-6 cells under an optical microscope.

[0029] Figure 7 The IFN-γ content in the cell supernatant of CTLs stimulated with various candidate peptides and co-incubated with Hepa1-6 cells was detected by ELISA. Data are expressed as mean ± standard deviation, n=3. *P<0.05; **P<0.01; ***P<0.001, ns: no significant difference. Figure 8 This is a diagram of an immunization protocol used to study the antitumor efficacy of model antigen peptide vaccines.

[0030] Figure 9a The mean growth curves of Hepa1-6 tumors in tumor-bearing mice after receiving different treatments.

[0031] Figure 9b The graph shows the changes in body weight of mice in each group.

[0032] Figure 9c The mean tumor weight removed from mice in each group on day 21. Data are expressed as mean ± standard deviation, n=5. *P<0.05; **P<0.01; ***P<0.001, ns: no significant difference.

[0033] Figure 10Photograph of a tumor removed after 21 days of treatment with pGPC3 short peptide.

[0034] Figure 11 The sequence design for pGPC3 mRNA and the sequence alignment results showed that the obtained sample sequence was completely consistent with the designed sequence.

[0035] Figure 12 The sequence design for pGPC3 saRNA and the sequence alignment results show that the obtained sample sequence is completely consistent with the designed sequence.

[0036] Figure 13 The hydrated particle size of the pGPC3 saRNA lipid vaccine under dynamic light scattering is 218 nm.

[0037] Figure 14 The morphological characteristics of the pGPC3 saRNA lipid vaccine under transmission electron microscopy show that it has a multi-compartment liposome structure.

[0038] Figure 15 The pGPC3@Lipi vaccine treatment regimen and mouse tumor growth curves are presented. Data are expressed as mean ± standard deviation, n=8. *P<0.05; **P<0.01; ***P<0.001.

[0039] Figure 16 Photograph of a tumor removed 30 days after treatment with pGPC3@Lipi vaccine.

[0040] Figure 17 This section describes the immunocellular typing of lymph nodes in treated mice, analyzed by flow cytometry. Data are presented as mean ± standard deviation, n=5. *P<0.05; **P<0.01; ***P<0.001.

[0041] Figure 18 This section describes the immunocellular typing of tumor cells in treated mice, analyzed by flow cytometry. Data are presented as mean ± standard deviation, n=5. *P<0.05; **P<0.01; ***P<0.001.

[0042] Figure 19 This section describes the immunocellular typing of spleen cells in treated mice, analyzed by flow cytometry. Data are presented as mean ± standard deviation, n=5. *P<0.05; **P<0.01; ***P<0.001.

[0043] Figure 20 This study presents a pGPC3@Lipi vaccine treatment regimen for the prevention of liver cancer and mouse tumor growth curves. Data are expressed as mean ± standard deviation, n=12. *P<0.05; **P<0.01; ***P<0.001.

[0044] Figure 21 This study presents a pGPC3@Lipi vaccine regimen for preventing liver cancer recurrence after surgery and mouse tumor growth curves. Data are expressed as mean ± standard deviation, n=12. *P<0.05; **P<0.01; ***P<0.001. Detailed Implementation

[0045] To make the purpose, technical solution, and advantages of the invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and examples.

[0046] Peptide screening and design (peptides were screened with the aid of the antigen screening website IEDB (http: / / tools.iedb.org / processing / ), that is, input the full-length mouse-derived GPC3 protein sequence, select the screening method, mouse MHC molecules, MHC alleles (H-2Kb, H-2Db) and amino acid length, and submit to obtain the screening results, as shown in Table 1.

[0047] MHC-I binding prediction of peptides was assisted by the MHC-I binding prediction tool on the IEDB website (http: / / tools.iedb.org / mhci / ). The top ten peptide sequences with the highest screening scores were input as shown in Table 1. The prediction method, mouse MHC molecule, MHC alleles (H-2Kb, H-2Db), and amino acid length were selected, and the prediction results were submitted. IC50 was then calculated. 50 A value between 0 and 300 indicates that the selected peptide has a strong affinity for mouse MHC-Ⅰ (H-2Ⅰ) molecules and can be used as a candidate vaccine antigen, as shown in Table 2.

[0048] Table 1. Top 10 GPC3-specific epitope peptides in predicted immunogenicity scores

[0049] Table 2. Predicted MHC-Ⅰ affinity IC50 of peptides using different prediction methods 50 value

[0050] 2. Docking score prediction for peptide and protein models The mouse H-2Kb molecular sequence and model were retrieved from Uniprot (https: / / www.uniprot.org / ), and the α-chain model was created using Pymol software. The protein model and peptide sequence were then input into Hpepdock 2.0 (http: / / huanglab.phys.hust.edu.cn / hpepdock / ), and the docking score was calculated as follows: Figure 1Table 3 shows the top 6 sequences with high docking scores, predicting the affinity of each candidate antigen peptide for H-2Kb. Figure 1 The PC (positive control) sequence is FAPGNYPAL, derived from Sendai virus nucleoprotein SV9, a polypeptide proven to have a high affinity for H-2Kb. The NC (negative control) sequence is EYILSLEEL, a polypeptide derived from human GPC3 protein, which has a low affinity for H-2Kb molecules.

[0051] Table 3 Candidate polypeptide sequences with high affinity

[0052] 3. Lymphocyte extraction and culture Four to six-week-old C57BL / 6j mice were euthanized by cervical dislocation and immersed in alcohol for at least 10 min. In a laminar flow hood, the skin on the left side of the mouse was cut open, and the spleen was removed and placed in a 70-micron (200-mesh) cell sieve. The spleen was gently ground using a 5 mL syringe plunger while rinsing with PBS to collect the cell suspension. The cells were centrifuged at 500 g / min for 5 min, and the cell pellet was lysed with 1 mL of erythrocyte lysis buffer for 2 min. The cells were resuspended and washed with an appropriate amount of PBS, and then centrifuged at 500 g / min for 5 min. The supernatant was discarded, and the cells were seeded into 6-well plates with complete culture medium containing stimulating factors (45 mL RPMI 1640 medium + 5 mL fetal bovine serum + 0.5 mL penicillin antibody + 10 μL IL-2 (final IL-2 concentration of 20 ng / mL)).

[0053] 4. Extraction and culture of bone marrow-derived dendritic cells (BMDCs) Four to six-week-old C57BL / 6j infants were euthanized by cervical dislocation, and the tibia and femur of the hind limbs were harvested. The muscles and fibula were removed using scissors and forceps. The tibia and femur were immediately immersed in alcohol and repeatedly rinsed with PBS in a clean bench. The liquid in the centrifuge tubes was collected, passed through a 70-micron (200-mesh) cell sieve, and centrifuged at 1000-1200 r / min for 5 min. The supernatant was discarded, and 1 mL of erythrocyte lysis buffer was added. The cells were lysed for 5 min, washed with PBS, and centrifuged again at 1000-1200 r / min for 5 min. The cells were then seeded into 6-well plates with complete culture medium containing stimulating factors (45 mL RPMI 1640 medium + 5 mL fetal bovine serum + 0.5 mL penicillin-dextrin antibiotics + 1 μL GM-CSF + 1 μL IL-4 (final concentration of GM-CSF and IL-4 is 20 ng / mL)). Half of the medium was changed every other day. The cells were considered immature BMDCs on the sixth day.

[0054] 5. Peptide-induced shock to BMDCs and lymphocytes BMDCs cultured for 6 days and spleen lymphocytes cultured for 2 days were re-seeded into 24-well plates in serum-free RPMI-1640 medium. Positive peptides, negative peptides, and 6 candidate peptides were added at 10 μg / well, and the plates were incubated at 37°C for 6 h. After incubation, the cells were gently aspirated and washed with PBS, blocked with PBS containing 1% BSA for 30 min, and then incubated with FITC Anti-H-2Kb for 30 min. The expression of H-2Kb molecules on DCs and lymphocytes was detected by flow cytometry. Figure 3a and 3b As shown.

[0055] 6. Preparation and injection of immunotherapy drugs The candidate peptide was blended with either complete or incomplete Freund's adjuvant (100 μg / mouse for the initial immunization with complete Freund's adjuvant; 50 μg / mouse for the booster dose with incomplete Freund's adjuvant). The mixture was homogenized and sonicated until completely emulsified. The emulsion was then dropped into ice-cold PBS and allowed to stand for 5-10 minutes to observe its emulsion state. A qualified water-in-oil emulsion would maintain intact droplets floating on the surface; an emulsion that disperses into flakes was considered unqualified. One day prior to the treatment, 6-8 week old normal C57BL / 6j mice were shaved from both groins and injected with the immunization agent. Two immunizations were administered. One week after the final treatment, splenic lymphocytes were extracted for subsequent experiments.

[0056] 7. CTL in vitro killing experiment Splenic lymphocytes were extracted from immunized mice and restimulated with 20 μg / mL peptide for 48 h. Hepa1-6 and LO2 cells were seeded into 96-well plates at a cell density of 5 x 10⁻⁶ cells / well. 3 Set up experimental wells, effector cell wells, target cell wells, and background wells, and allow them to adhere overnight. Discard the net cell culture medium. Co-culture Hepa1-6 cells with peptide-stimulated and peptide-free spleen lymphocytes in serum-free 1640 medium at effector-to-target ratios of 10:1, 20:1, and 40:1 for 24 h, with a total culture volume of 100 μL per well. After culture, add 10 μL of CCK-8 to each well and incubate at 37°C for 2 h. Detect the cells using a microplate reader at 450 nm. Calculate the CTL killing rate using the following formula.

[0057] CTL kill rate as Figure 5a and 5b As shown.

[0058] 8. Construction of animal models Collect Hep1-6 cells in good growth condition, resuspend them in PBS, and adjust the cell number to an appropriate concentration. At 6-8 weeks, subcutaneously inoculate 0.1 mL of cell suspension into the right buttock of male C57BL / 6 tumors; the day of tumor inoculation is recorded as day -7.

[0059] 9. Preliminary evaluation of the efficacy of polypeptide vaccines against liver cancer On day 0, tumor-bearing mice were randomly divided into two groups of five each for the tumor suppression experiment (n=5). Saline was used as the negative control group. On days 0, 7, and 14 after tumor implantation, saline and pGPC3 (i.e., the P4 sequence in Table 3) were injected intradermally into the ipsilateral groin, respectively. Each injection contained 25 μg of the antigenic peptide (pGPC3 = 25 μg). Starting from day 0, the tumor size of each mouse was measured every other day using digital calipers, and the tumor volume was calculated using the following formula. The weight of each mouse was also recorded. On day 21, the mice were dissected, and the tumor and inguinal lymph nodes were measured and their volumes calculated.

[0060] Tumor and lymph node volume = A × B² / 2 Tumor inhibition rate (%) = (M1 - M2) / M1 × 100% In the formula: A is the long diameter of the tumor, B is the short diameter of the tumor, M1 is the tumor weight in the saline group, and M2 is the tumor weight in the experimental group. The results are as follows: Figure 10 As shown.

[0061] 10. Design, transcription, and sequence validation of mRNA and saRNA The designed mRNA and saRNA included a 5' Cap, 5' UTR, 3' UTR, and a Poly A tail. The CDS region of the mRNA contained the pGPC3 nucleotide sequence (SEQ ID 8, where the U base was replaced with T to meet sequence listing requirements, although it is known that the T base in mRNA corresponds to the U base). The 5' end of the saRNA encoded four non-structural proteins (NSPs) from Venezuelan encephalitis virus (VEEV) (see the relevant structures in "LNP-RNA-engineered adipose stem cells for accelerated diabetes wound healing" and "Safety and immunogenicity of a self-amplifying RNAvaccine against COVID-19: COVAC1, a phase I, dose-ranging trial") and the pGPC3 nucleotide sequence of the CDS region. Plasmid DNA templates and E. coli strains were constructed based on the mRNA and saRNA.

[0062] 11. Extraction and purification of mRNA and saRNA (1) Plasmid DNA template acquisition and DNA linearization Prepare the reaction solution according to the table below: Table 4 DNA linearization components

[0063] After gently mixing, centrifuge briefly and incubate at 37°C for 30 min.

[0064] (2) DNA purification Transfer 2000 μL of the enzymatic reaction solution to a clean 15 mL centrifuge tube, add 10 mL of Buffer B3, and mix thoroughly. Pass the entire mixture through an adsorption column and centrifuge at 8,000 x g for 30 s. Discard the liquid in the collection tube and place the adsorption column in the same collection tube. Use 750 μL each time, loading the column multiple times. Add 500 μL of Wash Solution to the adsorption column and centrifuge at 9,000 x g for 30 s. Discard the liquid in the collection tube and place the adsorption column in the same collection tube. Repeat once. Place the empty adsorption column and collection tube in a centrifuge and centrifuge at 9,000 x g for 1 min. Add 30 μL of Elution Buffer preheated to 60 °C to the center of the adsorption membrane. Incubate at room temperature for 2 min, then centrifuge at 9,000 x g for 1 min. Store the obtained DNA solution at -20 °C or use it for subsequent experiments.

[0065] 12. In vitro transcription and purification of mRNA and saRNA The in vitro transcription components were prepared according to the following composition: Table 5 In vitro transcription components

[0066] Incubate at 37℃ for 2 h to transcribe mRNA or saRNA.

[0067] mRNA and saRNA prepurification Lithium chloride precipitation method: The RNA length must be greater than 300 nt, and the concentration must not be less than 100 ng / uL. Add 30 μL of RNase-free H2O and 30 μL of 7.5 M lithium chloride to 20 μL of the reaction mixture. After mixing thoroughly, incubate at -20℃ for at least 30 min, then centrifuge at maximum speed (4℃) for 15 min and collect the precipitate. Wash the RNA precipitate with 500 μL of ice-cold 70% ethanol. Dissolve the RNA precipitate with 20 μL of RNase-free H2O. Store the purified RNA solution at -20℃.

[0068] Magnetic bead purification method: Remove VAHTS Clean Beads from 2-8℃ 30 min in advance, equilibrate to room temperature, and invert or vortex to thoroughly mix the beads. Add 36 μL of RNA Clean Beads to 20 μL of sample, pipette 10 times to mix thoroughly, and incubate at room temperature for 5 min to allow RNA to bind to the beads. Place the sample on a magnetic rack for 5 min, and after the solution becomes clear, carefully remove the supernatant. Keeping the sample on the magnetic rack, add 200 μL of 70% ethanol to rinse the beads, being careful not to disperse them, and repeat the previous step. Keeping the sample on the magnetic rack, add 378 μL of freshly prepared 70% ethanol (prepared with RNase-free water) to rinse the beads, being careful not to disperse them. Keeping the sample on the magnetic rack, open the cap for 5 min to allow the beads to dry. Remove the sample from the magnetic rack, add 210 μL of RNase-free water, and pipette 10 times to mix thoroughly. Let stand at room temperature for 5 min, place the sample on a magnetic rack for 5 min, and after the solution becomes clear, collect the supernatant into a new 1.5 mL EP tube.

[0069] 13. Preparation of saRNA vaccines The liposome formulation includes: DSPE-PEG, cholesterol, and egg yolk lecithin. The saRNA is thoroughly mixed with the liposomes in the solution, and the saRNA is extruded through a liposome extruder to obtain the saRNA vaccine.

[0070] 14. Preliminary evaluation of the efficacy of saRNA vaccine against liver cancer On day -5, tumor-bearing mice were randomly divided into 3 groups of 8 mice each for the tumor suppression experiment (n=8). Saline was used as the negative control group. On day -4, tumors were implanted, and mice were intradermally injected into the ipsilateral groin with saline, pGPC3 polypeptide (i.e., the P4 sequence in Table 3), or pGPC3 saRNA lipids, with each injection containing either 25 μg of antigen polypeptide (pGPC3 = 25 μg) or 10 μg of pGPC3 saRNA (pGPC3 saRNA = 10 μg). Starting from day 0, the tumor size of each mouse was measured every other day using digital calipers, and the tumor volume was calculated according to the following formula. The weight of each mouse was also recorded. On day 30, the mice were dissected, and the tumor volume was measured and calculated. The results are as follows: Figure 11 As shown.

[0071] 15. Immune cell analysis After treatment, lymph nodes, spleen, and tumors were removed from the mice. After filtration through a grinding sieve, the samples were washed with PBS, centrifuged, and erythrocytes were removed using erythrocyte lysis buffer. The samples were then incubated with the corresponding flow cytometry antibodies in 1% BSA solution. Dendritic cells were labeled with CD11c, CD80, and CD86, while T cells were labeled with CD3, CD4, CD8, CD44, and CD62L. Five samples from each group were analyzed using a flow cytometer. The results are as follows: Figures 13-15 As shown. From Figure 15 The results showed that pGPC3 saRNA6 @Lipi (which has six repeat units) was significantly better than pGPC3 saRNA1 @Lipi (which has only one repeat unit).

[0072] 16. Preliminary evaluation of saRNA vaccine in preventing liver cancer On day -7, tumor-bearing mice were randomly divided into 3 groups of 12 mice each (n=12) for the experiment. Saline served as the negative control group. On day -4, tumors were implanted, and mice were intradermally injected into the ipsilateral groin with saline, pGPC3 polypeptide (i.e., the P4 sequence in Table 3), or pGPC3 saRNA lipids, each injection containing either 25 μg of antigen polypeptide (pGPC3 = 25 μg) or 10 μg of pGPC3 saRNA (pGPC3 saRNA = 10 μg). On day 0, Hepa1-6 cells were inoculated into the right buttock of each mouse. Starting from day 0, the tumor size of each mouse was measured every other day using digital calipers, and the tumor volume was calculated according to the following formula. The weight of each mouse was also recorded. On day 30, the mice were dissected, and the tumor volume was measured and calculated. The results are as follows: Figure 16 As shown.

[0073] 17. Preliminary evaluation of the efficacy of saRNA vaccine in preventing recurrence of liver cancer after surgery. On day -12, tumor-bearing mice were randomly divided into three groups of 12 mice each (n=12) for the experiment. Hepa1-6 cells were inoculated into the right buttock of each mouse. On day -5, a subcutaneous hepatocellular carcinoma tissue of approximately 300 mm³ was surgically removed. Saline was used as the negative control group. On day 0, saline, pGPC3 polypeptide (i.e., the P4 sequence in Table 3), and pGPC3saRNA lipids were injected intradermally into the ipsilateral groin. Each injection contained either 25 μg of antigen polypeptide (pGPC3 = 25 μg) or 10 μg of pGPC3 saRNA (pGPC3 saRNA = 10 μg). Starting from day 0, the tumor size of each mouse was measured every other day using digital calipers, and the tumor volume was calculated according to the following formula. The weight of each mouse was also recorded. On day 30, the mice were dissected, and the tumor volume was measured and calculated. The results are as follows: Figure 17 As shown.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A GPC3-derived antigenic peptide vaccine, characterized in that, The amino acid sequence of the antigen peptide vaccine is one or more of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:6, and any combination thereof.

2. A GPC3-derived antigenic peptide mRNA vaccine, characterized in that, The nucleotide sequence of the CDS region of the GPC3-derived antigenic peptide mRNA vaccine encodes one of the antigenic peptide vaccines in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or the nucleotide sequence of the CDS region of the mRNA vaccine encodes a repeating unit formed by 6-14 P2A restriction enzyme regions-GPC3 antigenic peptide vaccine regions. The amino acid sequence of the GPC3 antigenic peptide vaccine region is one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and any combination thereof. The amino acid sequence of the P2A restriction enzyme region is shown in SEQ ID NO:

7.

3. The GPC3-derived antigenic peptide mRNA vaccine as described in claim 2, characterized in that, The nucleotide sequence of the CDS region of the GPC3-derived antigenic peptide mRNA vaccine is shown in SEQ ID 8, and the nucleotide sequence of the GPC3-derived antigenic peptide mRNA vaccine is shown in SEQ ID 10.

4. A GPC3-derived antigenic peptide saRNA vaccine, characterized in that, The nucleotide sequence of the CDS region of the saRNA vaccine or the mRNA vaccine encodes one of the antigen peptide vaccines in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or the nucleotide sequence of the CDS region of the mRNA vaccine encodes a repeating unit encoding a region formed by multiple P2A restriction enzyme regions-GPC3 antigen peptide vaccine regions. The amino acid sequence of the GPC3 antigen peptide vaccine region is one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and any combination thereof. The amino acid sequence of the P2A restriction enzyme region is shown in SEQ ID NO 7.

5. The GPC3-derived antigenic peptide saRNA vaccine as described in claim 4, characterized in that, The self-amplifying virus of the saRNA vaccine is one of VEEV virus, alpha virus, flavivirus, filamentous virus or lentivirus; the nucleotide sequence of the CDS region of the mRNA vaccine encodes a region encoding a repeating unit formed by 6-14 P2A restriction enzyme regions-GPC3 antigen peptide vaccine regions.

6. The use of the vaccine according to any one of claims 1-5, characterized in that, The vaccine is used to prepare a drug for the treatment or prevention of neoplastic diseases, including but not limited to liver cancer.

7. A method for designing a vaccine, characterized in that, The vaccine is at least one of GPC3-derived antigenic peptide vaccine, GPC3-derived antigenic peptide mRNA vaccine, and GPC3-derived antigenic peptide saRNA vaccine, and specifically includes the following steps: Step 1: Predict the antigenic epitopes of the polypeptide sequences in the complete amino acid sequence of GPC3 using IEDB software. The software scores the immunogenicity of the polypeptide sequences in the GPC3 protein sequence and arranges them from highest to lowest score. Step 2: The top n ranked peptide sequences are docked with H-2Kb molecules using the MHC-I binding prediction tool on the IEDB website to predict docking affinity. Peptide sequences with predicted docking affinity within a preset threshold range are selected as candidate peptides. Step 3: Incubate each candidate peptide with dendritic cells derived from bone marrow and splenic lymphocytes for 6 h, and detect the actual affinity of each candidate peptide epitope to H-2Kb molecules by flow cytometry. Step 4: Arrange the candidate peptides in descending order of actual affinity and design a control group. Prepare a vaccine by mixing the antigens of the first m candidate peptides with Freund's adjuvant and immunize mice twice, seven days apart. The first dose was prepared by mixing 100 μg of candidate peptide antigen with complete Freund's adjuvant, and the booster dose was prepared by mixing 50 μg of candidate peptide antigen with incomplete Freund's adjuvant. Seven days after the last immunization, spleen lymphocytes were extracted and stimulated again with 20 μg / mL of the corresponding peptide antigen. Two days later, they were co-incubated with Hepa1-6 liver cancer cells for 24 h. The killing efficiency of antigen peptide-specific cytotoxic T cells against Hepa1-6 liver cancer cells was detected by CCK-8 assay. The candidate peptides with higher killing efficiency than the control group were used as GPC3-derived antigen peptide vaccines. When the vaccine is one of a GPC3-derived antigenic peptide mRNA vaccine and a GPC3-derived antigenic peptide saRNA vaccine, the following steps are also included: Step 5: Design at least one of the pGPC3 mRNA and pGPC3 saRNA nucleotide sequences for translation to generate GPC3-derived antigenic peptide vaccines. Amplify plasmids containing pGPC3 mRNA or pGPC3 saRNA nucleotide sequences using E. coli, transcribe and purify them in vitro, and then verify whether the extracted RNA is consistent with the designed pGPC3 mRNA or pGPC3 saRNA nucleotide sequence after sequencing. If they are consistent, successfully transformed E. coli are obtained. Use the successfully transformed E. coli to produce pGPC3 mRNA or pGPC3 saRNA nucleotide sequences. Step 6: Load the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence onto liposomes to prepare GPC3-derived antigenic peptide mRNA vaccine or GPC3-derived antigenic peptide saRNA vaccine.

8. The vaccine design method as described in claim 7, characterized in that, In step one, the length of the polypeptide sequence is 8-10. The amino acid sequence of the antigen peptide vaccine is at least one of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

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