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: 4, SEQ ID NO: 5 and SEQ ID NO: 6. According to the invention, an efficient pGPC3 model antigen peptide is screened on the basis of GPC protein, and pGPC3 mRNA and pGPC3 saRNA are designed according to a pGPC3 sequence example. The pGPC3saRNA (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-2I type molecules (H-2Kb) are adopted during pGPC3 polypeptide antigen screening, APC cells are facilitated to present antigens through an MHCI restrictive pathway, CD8 + T cells are activated, and GPC3 specific CTL is triggered to efficiently kill liver cancer cells. Finally, the pGPC3saRNA (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 present invention relates to the field of biotechnology, and in particular to a GPC3-derived antigen peptide saRNA vaccine design, preparation method and application. 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 antigen properties. It exists on the surface of cancer cells differentiated from endoderm cells. It is a structural protein of the cell membrane and also a broad-spectrum tumor marker. Glypican 3 (GPC3) is an oncofetal glycoprotein attached to the cell membrane through a glycophosphatidylinositol (GPI) anchor. It is composed of 580 amino acids and is 70 kDa in size. It is believed to play a crucial regulatory role in cell proliferation in embryonic mesoderm tissue. GPC3 is widely expressed in the placenta, as well as the liver, lungs, and kidneys of embryos, but is almost undetectable 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] At present, 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 melanoma cells modified with OVA gene (B16-OVA) on the market, which makes the body's immune cells accept the "model antigen" OVA. 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, and 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 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 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: Venezuelan encephalitis virus. Summary of the invention

[0007] In order 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 among 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 among SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ IDNO:6, or the nucleotide sequence of the CDS region of the mRNA vaccine encodes a repeating unit coding region formed by multiple 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 as shown in SEQ ID NO 7.

[0013] A further improvement is 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 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 as shown in SEQ ID 9, the 5' end of the saRNA vaccine encodes the nucleotide coding region of 4 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 repeating unit coding region.

[0015] A use of the above vaccine, wherein the vaccine is used as a drug for treating or preventing tumor diseases, wherein the tumor diseases include but are 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, and specifically comprises the following steps:

[0017] Step 1: Use IEDB software to predict antigenic epitopes of polypeptide sequences in the entire amino acid sequence of GPC3. The obtained software scores the immunogenicity of polypeptide sequences in the GPC3 protein sequence and arranges them from large to small according to the scores;

[0018] Step 2: dock the top n ranked peptide sequences with the H-2Kb molecule through the MHC-Ⅰ binding prediction tool on the IEDB website to predict the docking affinity, and use the peptide sequences with predicted docking affinity within the preset threshold range 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 with H-2Kb molecule by flow cytometry;

[0020] Step 4: Arrange the candidate polypeptides in descending order according to their actual affinity 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, and immunize them twice with an interval of seven days; the first injection is prepared by mixing 100 μg of the candidate polypeptide antigen with complete Freund's adjuvant, and the booster injection is prepared by mixing 50 μg of the candidate polypeptide antigen with incomplete Freund's adjuvant. On the seventh day after the last immunization, spleen lymphocytes are extracted and restimulated with 20 μg / mL of the corresponding polypeptide antigen, and incubated with Hepa1-6 liver cancer cells for 24 hours two days later. The killing efficiency of antigen peptide-specific cytotoxic T cells on Hepa1-6 liver cancer cells is detected by CCK-8, and 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 the GPC3-derived antigen peptide vaccine, use Escherichia coli to amplify the plasmid containing the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence, purify after in vitro transcription, and then verify after sequencing whether the extracted RNA is consistent with the designed pGPC3 mRNA or pGPC3 saRNA nucleotide sequence, if they are consistent, a 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 six: Load the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence onto liposomes to prepare GPC3-derived antigen peptide mRNA vaccine or 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 a plurality of tandem repeat unit coding regions, and each repeat unit coding region is 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 out and used for vaccine adjuvant and carrier research in the Hepa1-6 liver cancer subcutaneous transplant tumor model of C57BL / 6j mice.

[0029] (2) The use of nonapeptides and high-affinity binding to H-2 type I molecules (H-2Kb) during GPC3 peptide antigen screening is conducive to APC cells presenting antigens through the MHC I restricted pathway and activating CD8+ T cells, thereby inducing 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-cut-GPC3-derived antigen short peptide" nucleotide alternating repeating units, that is, one mRNA and saRNA can obtain 6-14 pGPC3 short peptides through enzyme cutting 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 accompanying drawings do not constitute any limitation to the present invention.

[0034] Figure 1 This is 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 to MHC-I molecules on dendritic cells. Data are expressed as mean ± standard deviation, n = 3. ***P < 0.001, ns: no significant difference.

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

[0038] Figure 3b The affinity diagram of candidate peptides to MHC-I molecules on spleen lymphocytes was quantified by flow cytometry. Data are expressed as mean ± standard deviation, n = 3. ***P < 0.001, ns: no significant difference.

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

[0040] Figure 5a To screen GPC3-specific epitope peptides for detecting 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 detect the IFN-γ content in the cell supernatant after CTLs stimulated with each candidate peptide were co-incubated with Hepa1-6 cells. Data are expressed as mean ± standard deviation, 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 figure is the curve of body weight change of mice in each group.

[0048] Fig.9c The average tumor weight of the tumors 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.

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

[0050] Fig.11 The comparison results of the designed sequence and sample sequence of pGPC3 mRNA show that the obtained sample sequence is completely consistent with the designed sequence.

[0051] Fig.12 The design sequence and sample sequence comparison results of pGPC3 saRNA show that the obtained sample sequence is completely consistent with the design sequence.

[0052] Fig.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] Fig.14 This is the morphological feature of the pGPC3 saRNA lipid vaccine under transmission electron microscopy, which is a multi-compartment liposome structure.

[0054] Fig.15 The pGPC3@Lipi vaccine treatment regimen and mouse tumor growth curve. Data are expressed as mean ± standard deviation, n = 8. *P < 0.05; **P < 0.01; ***P < 0.001.

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

[0056] Fig.17 Figure 2 shows the phenotyping of immune cells in the lymph nodes of treated mice analyzed by flow cytometry. Data are expressed as mean ± standard deviation, n = 5. *P < 0.05; **P < 0.01; ***P < 0.001.

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

[0058] Fig.19Figure 2 shows the phenotyping of immune cells in the spleen of treated mice analyzed by flow cytometry. Data are expressed as mean ± standard deviation, n = 5. *P < 0.05; **P < 0.01; ***P < 0.001.

[0059] Fig. 20 The pGPC3@Lipi vaccine treatment scheme for preventing liver cancer and the tumor growth curve of mice. Data are expressed as mean ± standard deviation, n = 12. *P < 0.05; **P < 0.01; ***P < 0.001.

[0060] Fig.21 The pGPC3@Lipi vaccine is used to prevent liver cancer recurrence after surgery and the tumor growth curve of mice. Data are expressed as mean ± standard deviation, 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 clearly understood, 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 with the assistance of the antigen screening website IEDB (http: / / tools.iedb.org / processing / ), that is, the full-length mouse GPC3 protein sequence was input, the screening method, mouse MHC molecule, MHC allele (H-2Kb, H-2Db) and amino acid length were selected, and 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 (http: / / tools.iedb.org / mhci / ) on the IEDB website, that is, the top ten peptide sequences with higher 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, and the prediction results were submitted. 50 A value between 0 and 300 indicates that the screened polypeptide has a strong affinity to the mouse MHC-Ⅰ (H-2Ⅰ) molecule and can be used as a candidate vaccine antigen, as shown in Table 2.

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

[0065]

[0066] Table 2 IC values ​​of peptides and MHC-Ⅰ 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, predicting the affinity of each candidate antigen peptide with H-2Kb, among which, Figure 1 The PC (positive control) sequence is FAPGNYPAL, which is derived from the Sendai virus nuclear protein SV9 and is a peptide that has been shown to have high affinity with H-2Kb. The NC (negative control) sequence is EYILSLEEL, which is a peptide derived from human GPC3 protein and has low affinity with H-2Kb molecules.

[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 taken out and placed in a 70 micron (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 cells were 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)) for culture.

[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 taken. 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 and passed through a 70 micron (200 mesh) cell sieve. The cells were centrifuged at 1000-1200 r / min for 5 min, and the supernatant after centrifugation was discarded. 1 mL of red blood cell lysis buffer was added, and the cells were lysed for 5 min. The cells were 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 stimulating 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. On the sixth day, the cells were immature BMDCs.

[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 with 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 then blocked with PBS containing 1% BSA for 30 minutes, and 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 polypeptide (the amount of antigen for the first immunization was 100 μg / mouse, mixed with complete Freund's adjuvant; the amount of antigen for the booster injection was 50 μg / mouse, mixed with incomplete Freund's adjuvant), and the mixture was completely emulsified by homogenizer and ultrasound, and then dropped into ice-cold PBS and left to stand for 5-10 minutes to observe the emulsification state. A qualified oil-in-water preparation could keep the complete droplets floating on the water surface, and a dispersible emulsion was an unqualified emulsion if it was dispersed into flakes. One day in advance, the groins of normal C57BL / 6j mice aged 6 to 8 weeks were depilated on both sides and the immunization injection was injected. A total of two injections were given, and spleen lymphocytes after immunization were extracted one week after the last treatment for subsequent experiments.

[0081] 7. CTL killing experiment in vitro

[0082] Splenic lymphocytes of immunized mice were extracted and stimulated with 20 μg / mL peptide for 48 h. Hepa1-6 and LO2 cells were plated in 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 and without peptides with Hepa1-6 cells in serum-free 1640 medium at effector-target ratios of 10:1, 20:1, and 40:1 for 24 hours, with a total volume of 100μL of culture medium per well. After the culture is completed, add 10μL CCK-8 to each well, incubate at 37℃ for 2h, and detect at a wavelength of 450nm using an enzyme reader. Calculate the CTL killing rate 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 with good growth status were collected, resuspended in PBS, and the cell number was adjusted to an appropriate concentration. 0.1 mL of cell suspension was subcutaneously inoculated on the right buttocks of 6-8 week old male C57BL / 6 animals, and the day of tumor inoculation was recorded as day -7.

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

[0088] On day 0, the tumor-bearing mice were randomly divided into 2 groups, with 5 mice in each group for tumor inhibition experiments (n=5). Physiological saline was used as the negative control group. On days 0, 7, and 14 of tumor inoculation, physiological saline and pGPC3 (i.e., P4 sequence in Table 3) were intradermally injected into the ipsilateral groin, respectively. Each injection of the vaccine 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. The weight of each mouse was also 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. Fig.10 shown.

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

[0093] The design of mRNA and saRNA includes 5'Cap, 5'UTR, 3'UTR, PolyA tail, wherein the CDS region of mRNA is 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 mRNA corresponds to the base U); the 5' end of saRNA encodes 4 non-structural proteins (NSPs) from Venezuelan encephalitis virus (VEEV) (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 according to 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 2000uL of the enzymatic reaction solution to a clean 15mL centrifuge tube, add 10ml of Buffer B3, and mix thoroughly. Pass the entire mixture through the adsorption column and centrifuge at 8,000xg for 30s. Pour out the liquid in the collection tube and place the adsorption column in the same collection tube. Use 750uL each time and apply the column multiple times. Add 500ul Wash Solution to the adsorption column and centrifuge at 9,000xg for 30s. Pour out 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 into a centrifuge and centrifuge at 9,000xg for 1min. Add 30uL of Elution Buffer preheated to 60℃ to the center of the adsorption membrane. Let stand at room temperature for 2min and centrifuge at 9.000xg for 1min. Store the resulting DNA solution at -20℃ 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 method: RNA length must be greater than 300 nt, and the concentration must not be less than 100 ng / uL. Add 30 μL RNase-free H20 and 30 μL 7.5 M lithium chloride to 20 μL of reaction mixture. After mixing evenly, place at -20°C for at least 30 min, centrifuge at maximum speed of 4°C for 15 min, and collect the precipitate. Add 500 μL ice-cold 70% ethanol to wash the RNA precipitate. Dissolve the RNA precipitate with 20 μL RNase-free H2O. Store the purified RNA solution at -20°C.

[0109] Magnetic bead purification method: VAHTS Clean Beads were taken out from 2-8℃ 30min in advance, equilibrated to room temperature, and inverted or vortexed to mix the beads thoroughly; 36μL RNA Clean Beads were added to 20μL sample, pipetted 10 times to mix thoroughly, and incubated at room temperature for 5min to allow RNA to bind to the magnetic beads; the sample was placed on the magnetic rack for 5min, and after the solution was clarified, the supernatant was carefully removed; the sample was kept in the magnetic rack, and 200μL 70% ethanol was added to rinse the magnetic beads, and be careful not to blow the magnetic beads apart, and repeat the previous step. Keeping the sample in the magnetic rack, 378μL of freshly prepared 70% ethanol (prepared with RNase free water) was added to rinse the magnetic beads, and be careful not to blow the magnetic beads apart; the sample was kept in the magnetic rack, and the lid was opened for 5min. After the magnetic beads were dry, the sample was removed from the magnetic rack, 210ul of RNase free water was added, and pipetted 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.

[0110] 13. Preparation of saRNA vaccines

[0111] The liposome formula includes: DSPE-PEG\cholesterol and egg yolk lecithin. The saRNA and liposomes are fully 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 vaccine 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 planted, 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 25μg of antigen polypeptide (pGPC3=25μg) or 10μg of pGPC3 saRNA (pGPC3saRNA=10μg). 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 body weight of each mouse was recorded. On the 30th day, the mice were dissected, and the tumor volume was calculated by measuring the mouse tumor. The results are as follows. Fig.11 shown.

[0114] 15. Immune cell analysis

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

[0116] 16. Preliminary evaluation of saRNA vaccine 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 planted, 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 25μg of antigen polypeptide (pGPC3=25μg) or 10μg of pGPC3 saRNA (pGPC3saRNA=10μg). On day 0, Hepa1-6 cells were inoculated in the right buttocks of the mice. 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. The body weight of each mouse was also recorded. On day 30, the mice were dissected, and the tumor volume was calculated by measuring the volume. The results are as follows. Fig.16 shown.

[0118] 17. Preliminary evaluation of the efficacy of saRNA vaccine 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 in the right buttocks of the mice. On day -5, subcutaneous liver cancer tissue of about 300 mm3 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, respectively. 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, and the weight of each mouse was recorded. On the 30th day, the mice were dissected, and the tumor volume was measured and calculated. The results are as follows Fig.17 shown.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution 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 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.

2. A GPC3-derived antigen peptide mRNA vaccine, characterized in that: The nucleotide sequence of the CDS region of the GPC3-derived antigen peptide mRNA vaccine encodes an antigen peptide vaccine among 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.

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

4. A GPC3-derived antigen peptide saRNA vaccine, characterized in that: 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, and 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 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 IDNO:6; the amino acid sequence of the P2A cleavage region is as shown in SEQ ID NO 7.

5. The GPC3-derived antigen peptide saRNA vaccine according to 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 repeating unit coding region formed by 6-14 P2A enzyme cleavage regions-GPC3 antigen peptide vaccine regions.

6. The GPC3-derived antigen peptide saRNA vaccine according to claim 5, 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 9. The 5' end of the saRNA vaccine encodes the nucleotide coding region of 4 non-structural proteins of the VEEV virus. Finally, the CDS region of the VEEV virus located behind the subgenomic promoter is replaced with the repeating unit coding region.

7. Use of the vaccine according to any one of claims 1 to 6, characterized in that: The vaccine is used as a drug for treating or preventing tumor diseases, including but not limited to liver cancer.

8. A method for designing a vaccine, characterized in that: 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, and specifically comprises the following steps: Step 1: Use IEDB software to predict antigenic epitopes of polypeptide sequences in the entire amino acid sequence of GPC3. The obtained software scores the immunogenicity of polypeptide sequences in the GPC3 protein sequence and arranges them from large to small according to the scores; Step 2: dock the top n ranked peptide sequences with the H-2Kb molecule through the MHC-Ⅰ binding prediction tool on the IEDB website to predict the docking affinity, and use the peptide sequences with predicted docking affinity within the preset threshold range as candidate peptides; 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; Step 4: Arrange the candidate polypeptides in descending order according to their actual affinity 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, and immunize them twice with an interval of seven days; the first injection is prepared by mixing 100 μg of the candidate polypeptide antigen with complete Freund's adjuvant, and the booster injection is prepared by mixing 50 μg of the candidate polypeptide antigen with incomplete Freund's adjuvant. On the seventh day after the last immunization, spleen lymphocytes are extracted and restimulated with 20 μg / mL of the corresponding polypeptide antigen, and incubated with Hepa1-6 liver cancer cells for 24 hours two days later. The killing efficiency of antigen peptide-specific cytotoxic T cells on Hepa1-6 liver cancer cells is detected by CCK-8, and the candidate polypeptide with a killing efficiency higher than that of the control group is used as the GPC3-derived antigen peptide vaccine; 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: Step 5, design at least one of the pGPC3 mRNA and pGPC3 saRNA nucleotide sequences for translation to generate the GPC3-derived antigen peptide vaccine, use Escherichia coli to amplify the plasmid containing the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence, purify after in vitro transcription, and then verify after sequencing whether the extracted RNA is consistent with the designed pGPC3 mRNA or pGPC3 saRNA nucleotide sequence, if they are consistent, a successfully transformed Escherichia coli is obtained, and the successfully transformed Escherichia coli is used to produce the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence; Step six: Load the pGPC3 mRNA or pGPC3 saRNA nucleotide sequence onto liposomes to prepare GPC3-derived antigen peptide mRNA vaccine or GPC3-derived antigen peptide saRNA vaccine.

9. The method for designing a vaccine according to claim 8, characterized in that: In the step 1, 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.

10. A vaccine, characterized in that The vaccine is a saRNA vaccine or an mRNA vaccine, and the CDS region of the vaccine comprises a plurality of tandem repeat unit coding regions, each repeat unit coding region being formed by a P2A enzyme cutting coding region + an antigen peptide vaccine coding region.

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