GPC3-derived antigen peptide vaccine, mRNA vaccine, saRNA vaccine, and preparation methods and applications thereof

Abstract

The present 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 more arbitrary combinations 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. The present invention screens out an efficient pGPC3 model antigen peptide based on GPC protein, and obtains pGPC3mRNA and pGPC3saRNA according to the pGPC3 sequence design. The pGPC3saRNA@Lipi vaccine is prepared by liposome technology. Due to the restriction conditions such as the use of nine peptides and high affinity binding to H-2 type molecule (H-2Kb) when screening pGPC3 polypeptide antigens, it is beneficial for APC cells to present antigens through the MHC I restriction pathway and activate CD8+T cells, thereby triggering GPC3-specific CTL to efficiently kill liver cancer cells. Finally, the pGPC3saRNA@Lipi vaccine completely inhibited liver cancer growth by 99.8%, and completely regressed liver cancer in 4 / 8 mice. Furthermore, it can be used to study vaccine adjuvants and vectors in a subcutaneous liver cancer xenograft model.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the design, preparation method and application of a GPC3-derived antigen peptide saRNA vaccine. Background Art

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

[0003] Carcinoembryonic antigen (CEA) is an acidic glycoprotein with human embryonic antigenic properties. It is present on the surface of cancer cells differentiated from endoderm cells. It is a structural protein of the cell membrane and also serves as a broad-spectrum tumor marker. Glypican 3 (GPC3) is an oncofetal glycoprotein attached to the cell membrane via a glycophosphatidylinositol (GPI) anchor. It consists of 580 amino acids and is 70 kDa in size. It is believed to play a crucial role in regulating cell proliferation in embryonic mesodermal tissues. GPC3 is widely expressed in the placenta, as well as in the embryonic liver, lung, and kidney, but is virtually undetectable in most organs in adults. GPC3 is overexpressed in certain tumors, particularly hepatocellular carcinoma (HCC). Serum GPC3 levels and GPC3 immunoreactivity in tumor cells are important for the prognosis of HCC patients.

[0004] Currently, most of the research on vaccines and adjuvants is based on melanoma, and there are few studies on nano-vaccines for liver cancer. There are two reasons for this: (1) There are OVA gene-modified melanoma cells (B16-OVA) on the market, which makes the body's immune cells 257-264 After stimulation, the peptide can accurately identify and kill melanoma cells carrying OVA. Currently, there is a lack of commercial OVA-modified liver cancer cells. The process of artificially modifying liver cancer cells is cumbersome, which makes it impossible to use OVA antigen peptide as a model antigen in the research of liver cancer vaccines. (2) Tyrosinase-related protein-2 (TRP-2) is a tumor-associated antigen widely expressed in melanoma. Therefore, TRP-2 180-188 It is an ideal target for immunotherapy of melanoma and glioma. However, the lack of a model antigen for liver cancer currently limits the research and clinical transformation of liver cancer vaccines.

[0005] Glossary:

[0006] VEEV virus: Venezuelan encephalitis virus. Summary of the Invention

[0007] To solve the above technical problems, the present invention proposes a GPC3-derived antigen peptide saRNA vaccine design, preparation method and application.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A GPC3-derived antigen peptide vaccine, wherein the amino acid sequence of the antigen peptide vaccine is any combination of 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.

[0010] A GPC3-derived antigen peptide mRNA vaccine, wherein the nucleotide sequence of the CDS region of the GPC3-derived antigen peptide mRNA vaccine encodes an antigen peptide vaccine 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 cleavage regions-GPC3 antigen peptide vaccine regions, and the amino acid sequence of the GPC3 antigen peptide vaccine region is any combination of 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; the amino acid sequence of the P2A cleavage region is shown in SEQ ID NO 7.

[0011] As a further improvement, the nucleotide sequence of the CDS region of the GPC3-derived antigen peptide mRNA vaccine is shown as SEQ ID 8, and the nucleotide sequence of the GPC3-derived antigen peptide mRNA vaccine is shown as SEQ ID 10.

[0012] A GPC3-derived antigen peptide saRNA vaccine, the nucleotide sequence of the CDS region of the saRNA vaccine or the mRNA vaccine encodes an antigen peptide vaccine of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or the nucleotide sequence of the CDS region of the mRNA vaccine encodes a repeating unit coding region formed by multiple P2A enzyme cleavage regions-GPC3 antigen peptide vaccine regions, and the amino acid sequence of the GPC3 antigen peptide vaccine region is any combination of 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; the amino acid sequence of the P2A enzyme cleavage region is shown in SEQ ID NO 7.

[0013] As a further improvement, the self-amplifying virus of the saRNA vaccine is one of VEEV virus, alpha virus, flavivirus, filovirus or lentivirus; the nucleotide sequence of the CDS region of the mRNA vaccine encodes a repeating unit coding region formed by 6-14 P2A enzyme cleavage regions-GPC3 antigen peptide vaccine regions.

[0014] As a further improvement, the self-amplifying virus of the GPC3-derived antigen peptide saRNA vaccine is VEEV virus, the nucleotide sequence of the CDS region of the GPC3-derived antigen peptide saRNA vaccine is shown in SEQ ID 9, the 5' end of the saRNA vaccine encodes the nucleotide coding region of the four non-structural proteins of the VEEV virus, and finally the CDS region of the VEEV virus located behind the subgenomic promoter is replaced with the repeat unit coding region.

[0015] A use of the above vaccine, wherein the vaccine is used as a drug for treating or preventing tumor diseases, including but not limited to liver cancer.

[0016] A method for designing a vaccine, wherein the vaccine is at least one of a GPC3-derived antigen peptide vaccine, a GPC3-derived antigen peptide mRNA vaccine, and a GPC3-derived antigen peptide saRNA vaccine, specifically comprising the following steps:

[0017] Step 1: Use IEDB software to predict antigenic epitopes of peptide sequences in the full amino acid sequence of GPC3. The software then scores the immunogenicity of peptide sequences in the GPC3 protein sequence and arranges them in descending order according to the scores.

[0018] Step 2: The top n ranked peptide sequences were docked with the H-2Kb molecule using the MHC-I binding prediction tool on the IEDB website to predict the docking affinity. The peptide sequences with the predicted docking affinity within the preset threshold range were selected as candidate peptides.

[0019] Step 3: Co-incubate each candidate polypeptide with bone marrow-derived dendritic cells and spleen lymphocytes for 6 hours, and detect the actual affinity of each candidate polypeptide epitope to the H-2Kb molecule by flow cytometry;

[0020] Step 4: Arrange the candidate polypeptides according to their actual affinity from large to small and design a control group. Mix the antigens of the first m candidate polypeptides with Freund's adjuvant to prepare a vaccine to immunize mice twice, with a seven-day interval; the first shot is made by mixing 100 μg of the candidate polypeptide antigen with complete Freund's adjuvant, and the booster shot is made by mixing 50 μg of the candidate polypeptide antigen with incomplete Freund's adjuvant. Seven days after the last immunization, spleen lymphocytes are extracted and restimulated with 20 μg / mL of the corresponding polypeptide antigen. Two days later, the lymphocytes are co-incubated with Hepa1-6 liver cancer cells for 24 hours. The killing efficiency of antigen peptide-specific cytotoxic T cells against Hepa1-6 liver cancer cells is detected by CCK-8. The candidate polypeptide with a killing efficiency higher than that of the control group is used as the GPC3-derived antigen peptide vaccine;

[0021] When the vaccine is one of a GPC3-derived antigen peptide mRNA vaccine and a GPC3-derived antigen peptide saRNA vaccine, the following steps are also included:

[0022] Step 5. Design at least one of the pGPC3 mRNA and pGPC3 saRNA nucleotide sequences for translation to generate a GPC3-derived antigen peptide vaccine, amplify a plasmid containing the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence using Escherichia coli, purify after in vitro transcription, and then verify by sequencing whether the extracted RNA is consistent with the designed pGPC3 mRNA or pGPC3 saRNA nucleotide sequence. If they are consistent, successfully transformed Escherichia coli is obtained, and the successfully transformed Escherichia coli is used to produce the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence;

[0023] Step 6: Load the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence onto liposomes to prepare a GPC3-derived antigen peptide mRNA vaccine or a GPC3-derived antigen peptide saRNA vaccine.

[0024] Further improvement, in step 1, the length of the polypeptide sequence is 8-10;

[0025] 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.

[0026] A vaccine, which is a saRNA vaccine or an mRNA vaccine, wherein the CDS region of the vaccine comprises multiple tandem repeat unit coding regions, each repeat unit coding region being formed by a P2A enzyme cleavage coding region + an antigen peptide vaccine coding region.

[0027] The advantages of the present invention are as follows:

[0028] (1) A highly effective GPC3 model antigen peptide was screened and used for vaccine adjuvant and carrier research in the Hepa1-6 liver cancer subcutaneous transplant tumor model in C57BL / 6j mice.

[0029] (2) When screening GPC3 polypeptide antigens, the use of restrictive conditions such as nonapeptides and high-affinity binding to H-2 type I molecules (H-2Kb) 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.

[0030] (3) The unique design pattern of GPC3-derived antigen peptide mRNA and saRNA vaccines, each mRNA and saRNA has 6-14 "P2A enzyme-digested-GPC3-derived antigen short peptide" nucleotide alternating repeating units, that is, an mRNA and saRNA can be enzymatically digested to obtain 6-14 pGPC3 short peptides after a single translation.

[0031] (4) The GPC3 model antigen peptide saRNA vaccine is a liposome vaccine. A single dose injection can induce sustained immunity, directly inhibit liver cancer and achieve complete regression of liver cancer in mice. It can effectively prevent the occurrence and development of liver cancer and can be used as an auxiliary treatment after surgery.

[0032] (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 the research of human liver cancer vaccine adjuvants and carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The present invention is further described with reference to the accompanying drawings, but the contents in the drawings do not constitute any limitation to the present invention.

[0034] Figure 1 The docking model and docking score diagram of the candidate peptide and MHC-Ⅰ molecule.

[0035] Figure 2aFlow cytometry analysis of the affinity of candidate peptides to MHC-I molecules on dendritic cells;

[0036] Figure 2b Flow cytometry quantification of 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: not significantly different.

[0037] Figure 3a Flow cytometry analysis of the affinity of candidate peptides to MHC-I molecules on spleen lymphocytes.

[0038] Figure 3b Flow cytometry quantification of 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: not significantly different.

[0039] Figure 4 Figure 1 is a diagram of the immunization scheme used to screen the in vivo immunogenicity of peptides;

[0040] Figure 5a GPC3-specific epitope peptides were screened to detect the killing rate of CTLs on Hepa1-6 cells using CCK-8.

[0041] Figure 5b The effect of CTLs stimulated with or without P4 peptide on the activity of Hepa1-6 cells and L02 cells was detected by CCK-8.

[0042] Data are expressed as mean ± standard deviation, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001, ns: no significant difference.

[0043] Figure 6 The effects of specific CTLs induced by each candidate peptide on the morphology of Hepa1-6 cells were observed under an optical microscope.

[0044] Figure 7 ELISA was used to measure the IFN-γ content in the cell supernatant after co-incubation of Hepa1-6 cells with CTLs stimulated with each candidate peptide. Data are expressed as mean ± SD, n = 3. *P < 0.05; **P < 0.01; ***P < 0.001, ns: no significant difference.

[0045] Figure 8 Figure 2 is a diagram of the immunization scheme used to study the anti-tumor efficacy of the model antigen peptide vaccine.

[0046] Figure 9a Figure 2 shows the average growth curve of Hepa1-6 tumors in tumor-bearing mice after receiving different treatments.

[0047] Figure 9b The graph shows the body weight changes of mice in each group.

[0048] Figure 9c The mean tumor weight of the tumors removed on day 21 in each group of mice. Data are expressed as mean ± SD, n = 5. *P < 0.05; **P < 0.01; ***P < 0.001, ns: no significant difference.

[0049] Figure 10 This is a photo of a tumor removed after 21 days of treatment with pGPC3 short peptide.

[0050] Figure 11 The designed sequence of pGPC3 mRNA was compared with the sample sequence. The obtained sample sequence was completely consistent with the designed sequence.

[0051] Figure 12 The pGPC3 saRNA design sequence and sample sequence alignment results show that the sample sequence is completely consistent with the design sequence.

[0052] Figure 13 This is the hydrated particle size of pGPC3 saRNA lipid vaccine under dynamic light scattering, and the average hydrated particle size is 218 nm.

[0053] Figure 14 This is the morphological feature of the pGPC3 saRNA lipid vaccine under transmission electron microscopy, which is a multi-compartment liposome structure.

[0054] Figure 15 Figure 3. Treatment regimen with the pGPC3@Lipi vaccine and tumor growth curves in mice. Data are expressed as mean ± SD, n = 8. *P < 0.05; **P < 0.01; ***P < 0.001.

[0055] Figure 16 This is a photo of a tumor removed 30 days after treatment with the pGPC3@Lipi vaccine.

[0056] Figure 17 Figure 3. Flow cytometric analysis of immune cell types in the lymph nodes of treated mice. Data are expressed as mean ± standard deviation, n = 5. *P < 0.05; **P < 0.01; ***P < 0.001.

[0057] Figure 18 Figure 2 shows the immune cell profiling in tumors of treated mice analyzed by flow cytometry. Data are expressed as mean ± SD, n = 5. *P < 0.05; **P < 0.01; ***P < 0.001.

[0058] Figure 19Figure 3. Flow cytometric analysis of immune cell types in the spleens of treated mice. Data are expressed as mean ± standard deviation, n = 5. *P < 0.05; **P < 0.01; ***P < 0.001.

[0059] Figure 20 Figure 3. The pGPC3@Lipi vaccine prevents liver cancer and shows the tumor growth curve in mice. Data are expressed as mean ± SD, n = 12. *P < 0.05; **P < 0.01; ***P < 0.001.

[0060] Figure 21 Figure 3. The pGPC3@Lipi vaccine regimen for preventing liver cancer recurrence after surgery and the tumor growth curve in mice. Data are expressed as mean ± SD, n = 12. *P < 0.05; **P < 0.01; ***P < 0.001. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of the invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and examples.

[0062] 1. Peptide Screening and Design (Peptides were screened using the antigen screening website IEDB (http: / / tools.iedb.org / processing / ). The full-length mouse GPC3 protein sequence was input, and the screening method, mouse MHC molecule, MHC allele (H-2Kb, H-2Db), and amino acid length were selected. The screening results were submitted, as shown in Table 1.

[0063] The peptide MHC-Ⅰ binding prediction was assisted by the MHC-Ⅰ binding prediction tool on the IEDB website (http: / / tools.iedb.org / mhci / ). That is, the top ten peptide sequences with high screening scores were input as shown in Table 1, and the prediction method, mouse MHC molecule, MHC allele (H-2Kb, H-2Db) and amino acid length were selected. The prediction results were obtained by submitting the results. 50 A value between 0 and 300 indicates that the screened polypeptide has a strong affinity with the mouse MHC-Ⅰ (H-2Ⅰ) molecule and can be used as a candidate vaccine antigen, as shown in Table 2.

[0064] Table 1 Top ten GPC3-specific epitope peptides with the highest predicted immunogenicity scores

[0065]

[0066] Table 2 IC values ​​of peptides and MHC-Ⅰ affinity predicted by different prediction methods 50 value

[0067]

[0068] 2. Peptide and protein model docking score prediction

[0069] The mouse H-2Kb molecular sequence and model were searched on Uniprot (https: / / www.uniprot.org / ), and the α chain model was created using the software Pymol. The protein model and peptide chain sequence were input into Hpepdock2.0 (http: / / huanglab.phys.hust.edu.cn / hpepdock / ), and the docking score was calculated as follows: Figure 1 Table 3 shows the top 6 sequences with higher docking scores, and predicts the affinity of each candidate antigen peptide with H-2Kb. Figure 1 The PC (positive control) sequence in the assay is FAPGNYPAL, derived from the Sendai virus nucleoprotein SV9, a peptide that has been shown to bind to H-2Kb with high affinity. The NC (negative control) sequence is EYILSLEEL, a peptide derived from the human GPC3 protein that has low affinity for H-2Kb.

[0070] Table 3 Candidate polypeptide sequences with higher affinity

[0071]

[0072]

[0073] 3. Lymphocyte extraction and culture

[0074] 4-6 week old C57BL / 6j mice were killed by cervical dislocation and soaked in alcohol for at least 10 min. The skin on the middle left side of the mouse was cut open in a clean bench, and the spleen was removed and placed in a 70 μm (200 mesh) cell sieve. The spleen was gently ground using a 5 mL syringe piston and the cell suspension was collected by rinsing with PBS. The cell suspension was centrifuged at 500 g / min for 5 min, and the cell pellet was lysed with 1 mL of red blood cell lysis buffer for 2 min. An appropriate amount of PBS was added to resuspend and wash, and then centrifuged at 500 g / min for 5 min. The supernatant was discarded and the cells were plated in a 6-well plate with complete culture medium containing stimulating factors (45 mL RPMI1640 culture medium + 5 mL fetal bovine serum + 0.5 mL double antibody + 10 μL IL-2 (the final concentration of IL-2 was 20 ng / mL)).

[0075] 4. Extraction and culture of bone marrow-derived dendritic cells (BMDCs)

[0076] 4-6 week old C57BL / 6j were killed by cervical dislocation and the hind limb tibia and femur were removed. The muscles and fibula were removed with scissors and forceps, and the cells were immediately immersed in alcohol. The tibia and femur were repeatedly rinsed with PBS in a clean bench, and the liquid in the centrifuge tube was collected. The cells were passed through a 70-micron (200 mesh) cell sieve and centrifuged at 1000-1200 r / min for 5 min. The supernatant after centrifugation was discarded, 1 mL of red blood cell lysis buffer was added, lysed for 5 min, washed with PBS, and centrifuged at 1000-1200 r / min for 5 min. The cells were plated in a 6-well plate with complete culture medium containing stimulatory factors (45 mL RPMI1640 culture medium + 5 mL fetal bovine serum + 0.5 mL double antibody + 1 μL GM-CSF + 1 μL IL-4 (the final concentration of GM-CSF and IL-4 was 20 ng / mL)). Half of the cells were replaced with the medium every other day. Immature BMDCs were obtained on the sixth day.

[0077] 5. Peptide impact on BMDCs and lymphocytes

[0078] BMDCs cultured for 6 days and spleen lymphocytes cultured for 2 days were re-plated into 24-well plates in serum-free RPMI-1640 medium, and positive peptides, negative peptides, and 6 candidate peptides were added at 10 μg / well and cultured at 37°C for 6 hours. After the culture was completed, the cells were gently blown off and washed with PBS, and blocked with PBS containing 1% BSA for 30 minutes. Then, FITCAnti-H-2Kb was added and incubated for 30 minutes. The expression of H-2Kb molecules on DCs and lymphocytes was detected by flow cytometry. Figure 3a and 3b shown.

[0079] 6. Preparation and injection of immune injections

[0080] Complete Freund's adjuvant or incomplete Freund's adjuvant was mixed with the candidate peptide (100 μg of antigen per mouse for the first immunization, mixed with complete Freund's adjuvant; 50 μg of antigen per mouse for the booster, mixed with incomplete Freund's adjuvant). The mixture was homogenized and ultrasonicated until completely emulsified. The mixture was then dropped into ice-cold PBS and allowed to stand for 5-10 minutes to observe the emulsification state. A qualified water-in-oil emulsion should retain intact droplets floating on the water surface; a dispersible emulsion should be unqualified if it disperses into flakes. One day in advance, the groins of 6- to 8-week-old normal C57BL / 6j mice were depilated and injected with the immunization injection. Two immunizations were performed. One week after the final treatment, splenic lymphocytes were extracted for subsequent experiments.

[0081] 7. CTL killing experiment in vitro

[0082] Splenic lymphocytes were extracted from immunized mice and stimulated with 20 μg / mL peptide for 48 h. Hepa1-6 and LO2 cells were plated into 96-well plates at a cell density of 5×10 3 / well, set up experimental wells, effector cell wells, target cell wells and background wells, and let them adhere overnight. Discard the cell culture medium, and co-culture the spleen lymphocytes stimulated with or without peptide with Hepa1-6 cells in serum-free 1640 medium at effector-target ratios of 10:1, 20:1, and 40:1 for 24 hours. The total volume of culture medium in each well is 100 μL. After the culture is completed, 10 μL CCK-8 is added to each well, incubated at 37°C for 2 hours, and detected at a wavelength of 450 nm using an enzyme reader. The CTL killing rate is calculated according to the following formula

[0083]

[0084] CTL killing rate Figure 5a and 5b shown.

[0085] 8. Construction of animal models

[0086] Hep1-6 cells were harvested and resuspended in PBS to adjust the cell number to an appropriate concentration. 6-8 week old male C57BL / 6 mice were subcutaneously inoculated with 0.1 mL of the cell suspension on the right buttocks. The day of tumor inoculation was designated as day -7.

[0087] 9. Preliminary evaluation of the efficacy of peptide vaccines against liver cancer

[0088] On day 0, tumor-bearing mice were randomly divided into two groups, with 5 mice in each group for tumor inhibition experiments (n=5). Normal saline was used as the negative control group. On days 0, 7, and 14 after tumor inoculation, normal saline and pGPC3 (i.e., the P4 sequence in Table 3) were intradermally injected into the ipsilateral groin, respectively. Each vaccine injection contained 25 μg of antigen polypeptide (pGPC3=25 μg). Starting from day 0, the tumor size of each mouse was measured every other day with a digital vernier caliper, and the tumor volume was calculated according to the following formula. At the same time, the weight of each mouse was recorded. On day 21, the mice were dissected, and the tumors and inguinal lymph nodes of the mice were measured and the volumes were calculated.

[0089] Tumor and lymph node volume = A × B2 / 2

[0090] Tumor inhibition rate (%) = (M1-M2) / M1×100%

[0091] Where: A is the long diameter of the tumor, B is the short diameter of the tumor, M1 is the tumor weight of the saline group, and M2 is the tumor weight of the experimental group. Figure 10 shown.

[0092] 10. Design, transcription and sequence verification of mRNA and saRNA

[0093] The designed mRNA and saRNA include 5'Cap, 5'UTR, 3'UTR, and PolyA tail, wherein the CDS region of the mRNA is the pGPC3 nucleotide sequence (i.e., SEQ ID 8, wherein, in order to meet the requirements of the sequence list, the base U is replaced by T, but it is known that the base T in the mRNA corresponds to the base U); the saRNA 5' end encodes four non-structural proteins (NSPs) from the Venezuelan encephalitis virus (VEEV) (for details, see the relevant structures in "LNP-RNA-engineered adipose stem cells for accelerated diabetic wound healing" and "Safety and immunogenicity of a self-amplifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging trial") and the pGPC3 nucleotide sequence in the CDS region. And construct respective plasmid DNA templates and Escherichia coli strains based on mRNA and saRNA.

[0094] 11. Extraction and purification of mRNA and saRNA

[0095] (1) Plasmid DNA template acquisition and DNA linearization

[0096] Prepare the reaction solution according to the following table:

[0097] Table 4 DNA linearization components

[0098]

[0099] After gentle mixing, centrifugation was performed and the mixture was kept at 37°C for 30 min.

[0100] (2) DNA purification

[0101] 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 the adsorption column and centrifuge at 8,000 x g for 30 seconds. Discard the liquid in the collection tube and place the adsorption column in the same collection tube. Use 750 μL each time and apply the mixture multiple times. Add 500 μL of Wash Solution to the adsorption column and centrifuge at 9,000 x g for 30 seconds. Discard the liquid in the collection tube and place the adsorption column in the same collection tube. Repeat this process. Place the empty adsorption column and collection tube into a centrifuge and centrifuge at 9,000 x g for 1 minute. Add 30 μL of Elution Buffer, preheated to 60°C, to the center of the adsorption membrane. Let stand at room temperature for 2 minutes and then centrifuge at 9,000 x g for 1 minute. Store the resulting DNA solution at -20°C or use it for subsequent experiments.

[0102] 12. In vitro transcription and purification of mRNA and saRNA

[0103] Prepare the in vitro transcription components as follows:

[0104] Table 5 In vitro transcription components

[0105]

[0106] Incubate at 37°C for 2 h and transcribe to obtain mRNA or saRNA.

[0107] mRNA and saRNA pre-purification

[0108] Lithium chloride precipitation: RNA must be greater than 300 nt in length and have a concentration of at least 100 ng / µL. Add 30 µL of RNase-free HO and 30 µL of 7.5 M lithium chloride to a 20 µL reaction mixture. Mix thoroughly, incubate at -20°C for at least 30 minutes, and centrifuge at maximum speed at 4°C for 15 minutes to collect the precipitate. Wash the RNA precipitate with 500 µL of ice-cold 70% ethanol. Dissolve the RNA precipitate in 20 µL of RNase-free HO. Store the purified RNA solution at -20°C.

[0109] Magnetic bead purification method: Remove VAHTS Clean Beads from 2-8°C 30 minutes in advance, equilibrate to room temperature, and thoroughly mix by inversion or vortexing. Add 36 μL of RNA Clean Beads to 20 μL of sample and pipette up and down 10 times to mix thoroughly. Incubate at room temperature for 5 minutes to allow RNA to bind to the beads. Place the sample on a magnetic rack for 5 minutes. Once the solution has clarified, carefully remove the supernatant. While the sample is still on the magnetic rack, rinse the beads with 200 μL of 70% ethanol, taking care not to loosen them, and repeat the previous step. While the sample is still on the magnetic rack, rinse the beads with 378 μL of freshly prepared 70% ethanol (prepared with RNase-free water), taking care not to loosen them. While the sample is still on the magnetic rack, open the lid for 5 minutes. Allow the beads to dry, remove the sample from the magnetic rack, add 210 μL of RNase-free water, and mix thoroughly by pipetting up and down 10 times. Let it stand at room temperature for 5 minutes, place the sample on a magnetic stand for 5 minutes, and after the solution becomes clear, collect the supernatant and place it in a new 1.5 mL EP tube.

[0110] 13. saRNA vaccine preparation

[0111] The liposome formula includes: DSPE-PEG\cholesterol and egg yolk lecithin. The saRNA and liposomes are thoroughly mixed in the solution, and the saRNA is extruded through a liposome extruder to obtain the saRNA vaccine.

[0112] 14. Preliminary evaluation of the efficacy of saRNA vaccines against liver cancer

[0113] On day -5, the tumor-bearing mice were randomly divided into 3 groups, with 8 mice in each group for tumor inhibition experiments (n=8). Normal saline was used as the negative control group. On day -4, the tumor was implanted, and normal saline, pGPC3 polypeptide (i.e., P4 sequence in Table 3), and pGPC3 saRNA lipid were intradermally injected into the ipsilateral groin, respectively. Each injection contained 25 μg of antigen polypeptide (pGPC3=25 μg) or 10 μg of pGPC3 saRNA (pGPC3saRNA=10 μg). Starting from day 0, the tumor size of each mouse was measured with a digital vernier caliper every other day, and the tumor volume was calculated according to the following formula, and the weight of each mouse was recorded at the same time. On day 30, the mice were dissected, and the tumor volume was measured and calculated. The results are shown in the figure. Figure 11 shown.

[0114] 15. Immune cell analysis

[0115] After treatment, the lymph nodes, spleens, and tumors of the mice were removed, filtered through a grinding mesh, washed with PBS, centrifuged, and red blood cells were removed with red blood cell lysis buffer. The cells were then incubated with the corresponding flow cytometry antibodies in a 1% BSA solution. Dendritic cells were labeled with CD11c, CD80, and CD86, and T cells were labeled with CD3, CD4, CD8, CD44, and CD62L. Five samples were tested on a flow cytometer in each group. The results are shown below. Figure 13-15 As shown. Figure 15 It can be seen that pGPC3 saRNA6@Lipi (i.e., having six repeating units) has significantly better effects than pGPC3saRNA1@Lipi (i.e., having only one repeating unit).

[0116] 16. Preliminary evaluation of saRNA vaccines in preventing liver cancer

[0117] On day -7, the tumor-bearing mice were randomly divided into 3 groups, with 12 mice in each group for the experiment (n=12). Normal saline was used as the negative control group. On day -4, the tumor was implanted, and normal saline, pGPC3 polypeptide (i.e., P4 sequence in Table 3), and pGPC3 saRNA lipid were intradermally injected into the ipsilateral groin. Each injection contained 25 μg of antigen polypeptide (pGPC3=25 μg) or 10 μg of pGPC3 saRNA (pGPC3saRNA=10 μg). On day 0, Hepa1-6 cells were inoculated into the right buttocks of the mice. Starting from day 0, the tumor size of each mouse was measured every other day with a digital vernier caliper, and the tumor volume was calculated according to the following formula. At the same time, the weight of each mouse was recorded. On day 30, the mice were dissected, and the tumor volume was measured and calculated. The results are shown in the figure below. Figure 16 shown.

[0118] 17. Preliminary evaluation of the efficacy of saRNA vaccines in preventing liver cancer recurrence after surgery

[0119] On day -12, the tumor-bearing mice were randomly divided into 3 groups, with 12 mice in each group for the experiment (n=12), and Hepa1-6 cells were inoculated into the right buttocks of the mice. On day -5, subcutaneous liver cancer tissue of about 300 mm3 in size was surgically removed, and normal saline was used as the negative control group. On day 0, normal saline, pGPC3 polypeptide (i.e., P4 sequence in Table 3), and pGPC3saRNA lipid were intradermally injected into the ipsilateral groin. Each injection contained 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 with a digital vernier caliper, and the tumor volume was calculated according to the following formula. At the same time, the weight of each mouse was recorded. On day 30, the mice were dissected, and the tumor volume was measured and calculated. The results are as follows. Figure 17 shown.

[0120] 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 the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A GPC3-derived antigen peptide vaccine, characterized in that: The amino acid sequence of the antigen peptide vaccine is the amino acid sequence shown in SEQ ID NO: 4; the amino acid sequence of the antigen peptide vaccine is used to prepare a drug for treating hepatocellular carcinoma.

2. A GPC3-derived antigen peptide saRNA vaccine, characterized in that: The self-amplifying virus of the GPC3-derived antigen peptide saRNA vaccine is VEEV virus. The nucleotide sequence of the CDS region of the GPC3-derived antigen peptide saRNA vaccine is shown in SEQ ID NO:

9. The 5' end of the saRNA vaccine encodes the nucleotide coding region of the four non-structural proteins of the VEEV virus.

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