CN117883558A - Preparation method of personalized mRNA vaccine for targeting liver tumor - Google Patents
Preparation method of personalized mRNA vaccine for targeting liver tumor Download PDFInfo
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Abstract
The invention provides a preparation method of a targeted liver tumor personalized mRNA vaccine, belonging to the technical field of medical preparations; the preparation method comprises the steps of determining mutant polypeptide, preliminary screening mutant polypeptide, screening high-affinity peptide fragments, integrating to obtain new antigen polypeptide, obtaining new antigen polypeptide with strong immunogenicity, synthesizing mRNA, and preparing mRNA vaccine. The invention can obtain mutant polypeptide by sequencing tumor patients, screen to obtain high-affinity neoantigen polypeptide, further screen to obtain neoantigen polypeptide with strong immunogenicity, then connect neoantigen polypeptide by adopting a specific connection mode, and prepare personalized tumor mRNA vaccine; the mRNA vaccine can stimulate CD8+ T cells to enable IFN-gamma and TNF-alpha expression to be up-regulated, and the stimulated CD8+ T cells can kill tumor cells.
Description
Technical Field
The invention relates to a target liver tumor personalized mRNA vaccine and a preparation method thereof, belonging to the technical field of medical preparations.
Background
As a recent research hotspot, tumor vaccines are therapeutic anticancer vaccines tailored to tumor neoantigens specific to tumor cells of cancer patients, and tumor antigens are used to elicit a tumor immune response in vivo, which is mainly cellular immunity. Cancer vaccines mainly include cell vaccines, DNA vaccines, mRNA vaccines, polypeptide vaccines, and the like. The mRNA vaccine technology is the latest third-generation vaccine technology, and is translated in cytoplasm after being injected into organism without entering cell nucleus, thereby overcoming the difficult problem of DNA vaccine delivery system and avoiding the risk of integrative mutation. The technology not only can fully induce humoral immunity and cellular immunity response, but also can activate immune response adjuvant, has the advantages of rapid research and development, high yield, low cost and easier realization of multi-linked multivalent design, and becomes an important platform for cancer immunotherapy.
MRNA encoding neoantigens led to the development of personalized vaccines, which are "high-grade tailored" vaccines containing multiple sites for different mutation sites in each patient based on gene sequencing. The 4 basic steps in its preparation are the acquisition of tumor tissue and normal cells, the presumption of new tumor antigens, the determination of new epitopes and the preparation of vaccines. But none involved the optimization of the screening for tumor neoantigens, essentially all were directly deduced from the software.
For example, CN106645677a is directed to the detection of tumor neoantigen-specific T cells, and is not directed to the screening of neoantigen immunogenicity, while CN110706747a is directed to a method of detecting tumor antigen immunogenicity, but is also directed to tumor DC vaccine or mRNA vaccine after preparation, and is not directed to the screening of neoantigen immunogenicity. The detection of antigen immunogenicity against intact tumor DC vaccines, tumor mRNA vaccines or tumor neoantigen-specific T cells can result in high costs.
At present, the methods for determining the neoantigen of the mRNA vaccine in the prior art are various, but the immunogenicity screening of the neoantigen polypeptide is seldom concerned when the vaccine is prepared, and the sequence and the connection mode of the neoantigen polypeptide lead to the reduction of the immune effect of the prepared mRNA vaccine.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a preparation method of a target liver tumor personalized mRNA vaccine, which realizes the following aims: the expression quantity of IFN-gamma and TNF-alpha of the CD8+ T cells stimulated by the personalized mRNA vaccine is increased, and the killing efficiency of the CD8+ T cells stimulated by the personalized mRNA vaccine to tumor cells is improved.
In order to solve the technical problems, the invention adopts the following technical scheme:
The preparation method comprises the steps of determining mutant polypeptide, preliminary screening mutant polypeptide, screening high-affinity peptide fragments, integrating to obtain new antigen polypeptide, obtaining new antigen polypeptide with strong immunogenicity, synthesizing mRNA, and preparing mRNA vaccine.
The method for obtaining the neoantigen polypeptide with strong immunogenicity is to synthesize the neoantigen polypeptide, then test the immunogenicity by using a kit, and select the neoantigen polypeptide with more immune spots than the positive control, namely the neoantigen polypeptide with strong immunogenicity.
The method for synthesizing mRNA comprises the steps of connecting nascent antigen polypeptide with strong immunogenicity into mRNA according to the sequence of more immune spots;
His tag was added to the N-terminal of mRNA, and 5 '-capping and 3' -terminal Poly (A) tail structure were performed on the synthesized mRNA.
The method for determining the mutant polypeptide comprises the steps of sequencing and comparing tumor tissues and healthy blood of a tumor patient, and determining mutation sites, wherein mutation types comprise single nucleotide variation and insertion/deletion mutation; for the mutation of amino acid caused by single nucleotide variation, non-mutant amino acid is added before and after the mutation site to obtain mutant polypeptide; for the frameshift peptide generated by the insertion/deletion mutation, non-mutant amino acid is added at the N end of the whole frameshift peptide to obtain mutant polypeptide.
For the mutation of amino acid caused by single nucleotide variation, when the number of amino acids before and after a mutation site is more than or equal to 12, respectively adding 12 non-mutation amino acids before and after the mutation site to obtain mutant polypeptide;
When the number of amino acids before the mutation site is n and n is less than 12, and when the number of amino acids after the mutation site is more than or equal to 12, adding n non-mutation amino acids before the mutation site and adding 12 non-mutation amino acids after the mutation site to obtain mutant polypeptide;
When the number of amino acids before the mutation site is more than or equal to 12, the number of amino acids after the mutation site is m and m is less than 12, adding 12 non-mutation amino acids before the mutation site, and adding m non-mutation amino acids after the mutation site to obtain mutant polypeptide;
For a frameshift peptide (FSP) generated by insertion/deletion mutation, if the number of amino acids at the upstream of the frameshift peptide is more than or equal to 12, adding 12 non-mutant amino acids at the N-terminal of the whole fragment of the frameshift peptide to obtain mutant polypeptide; if the number of the amino acids upstream of the frame shift peptide is x and x is less than 12, adding x non-mutant amino acids at the N end of the whole frame shift peptide to obtain mutant polypeptide.
The primary screening method of the mutant polypeptide comprises the steps of carrying out MHC I and MHC II affinity analysis and prediction on the mutant polypeptide obtained by single nucleotide variation, and selecting sequences with high MHC I and MHC II affinities; and (3) for mutant polypeptides obtained by insertion/deletion mutation, all mutant sequences are incorporated, so that the mutant polypeptides after primary screening are obtained.
The MHC I and MHC II affinity analysis and prediction method is to adopt IEDB software to carry out MHC I and MHC II affinity analysis and prediction on mutant polypeptide obtained by single nucleotide variation, and select sequences with scores of 0.8-1.
The method for screening the high-affinity peptide fragment is to screen a 9-14mer polypeptide sequence with high affinity with MHC according to the mutation polypeptide sequence after initial screening and HLA typing results.
The method for integrating and obtaining the neoantigen polypeptide comprises the following steps: for the high-affinity peptide fragments of the 9-14mer screened from the same mutant polypeptide, if the high-affinity peptide fragments are not overlapped, each high-affinity peptide fragment is used as an independent neoantigen polypeptide; if there is an overlap between two high affinity peptide fragments, the overlap of 1 peptide fragment is deleted, and then the two peptide fragments are joined and integrated into one peptide fragment to form the neoantigen polypeptide.
The method for preparing the mRNA vaccine comprises the steps of purifying mRNA with a 5 '-capping structure and a 3' -tailing structure, loading the mRNA with a liposome mixture, synthesizing LNP nano particles of the mRNA, and obtaining the mRNA vaccine after dialysis, concentration and filtration.
The mRNA concentration in the mRNA vaccine is adjusted to 75-85 mug/mL.
Compared with the prior art, the invention has the following beneficial effects:
The invention can obtain mutant polypeptide by sequencing tumor patients, screen to obtain high-affinity neoantigen polypeptide, further screen to obtain neoantigen polypeptide with strong immunogenicity, then connect neoantigen polypeptide by adopting a specific connection mode, and prepare personalized tumor mRNA vaccine; the mRNA vaccine can stimulate CD8+ T cells to enable IFN-gamma and TNF-alpha expression to be up-regulated, and the stimulated CD8+ T cells can kill tumor cells.
Drawings
FIG. 1 is a flow chart of a screening for tumor neoantigens;
FIG. 2 is a sequence diagram showing the synthesis of 3 mRNAs;
FIG. 3 is a running gel validation graph of 3 mRNAs;
FIG. 4 is a flow chart of the expression rate of His protein in mRNA 1-loaded DC cells;
FIG. 5 is a bar graph of marker expression of tumor-specific CD8+ T cells;
wherein A is a histogram of positive cell occupancy of tumor-specific CD8+ T cells expressing IFN-gamma; b is a histogram of the positive cell occupancy of tumor-specific CD8+ T cells expressing TNF- ;
FIG. 6 is a histogram of killing efficiency of CD8+ T cells against liver cancer tumor MHCC-97H cells.
Detailed Description
Example 1 screening for tumor neoantigens
The flow of the screening for tumor neoantigens is shown in FIG. 1.
(1) Determination of mutant polypeptides
Firstly, obtaining tumor tissues and healthy blood of a tumor patient, carrying out exon sequencing, comparing sequencing results of the tumor tissues and the healthy blood, and determining different sites as mutation sites, wherein mutation types comprise Single Nucleotide Variation (SNV) and insertion/deletion mutation (also called frame shift mutation).
For the mutation of amino acid caused by single nucleotide variation, when the number of amino acids before and after a mutation site is more than or equal to 12, respectively adding 12 non-mutation amino acids before and after the mutation site to obtain mutant polypeptide;
when the number of amino acids before the mutation site is n and n is less than 12, the number of amino acids after the mutation site is more than or equal to 12, then
N non-mutant amino acids are added before the mutation site, and 12 non-mutant amino acids are added after the mutation site to obtain mutant polypeptide;
when the number of amino acids before the mutation site is more than or equal to 12 and the number of amino acids after the mutation site is m and m is less than 12, then
Adding 12 non-mutant amino acids before the mutation site, and adding m non-mutant amino acids after the mutation site to obtain mutant polypeptide;
For a frameshift peptide (FSP) generated by insertion/deletion mutation, if the number of amino acids at the upstream of the frameshift peptide is more than or equal to 12, adding 12 non-mutant amino acids at the N-terminal of the whole fragment of the frameshift peptide to obtain mutant polypeptide; if the number of the amino acids upstream of the frame shift peptide is x and x is less than 12, adding x non-mutant amino acids at the N end of the whole frame shift peptide to obtain mutant polypeptide.
(2) Primary screening of mutant polypeptides
Carrying out MHC I and MHC II affinity analysis and prediction on the obtained mutant polypeptide by using IEDB software, and selecting a sequence with the score of 0.8-1 for the mutant polypeptide obtained by SNV; and (3) for mutant polypeptides obtained by insertion/deletion mutation, all mutant sequences are incorporated, so that the mutant polypeptides after primary screening are obtained.
(3) Screening for high affinity peptide fragments
According to the mutation polypeptide sequence after primary screening and HLA typing result, using NETMHCPAN-4.0 software to screen the mutation site for the polypeptide sequence of 9-14mer with high affinity to MHC, and according to the matching result, selecting peptide segment with% Rank value less than 0.5 and BindLevel being SW.
(4) Integration to give a novel antigen polypeptide
In the same mutant polypeptide, the high-affinity peptide fragments of the 9-14mer are screened, and if the high-affinity peptide fragments are not overlapped, each high-affinity peptide fragment is used as an independent neoantigen polypeptide; if there is an overlap between two high affinity peptide fragments, the overlap of 1 peptide fragment is deleted, and then the two peptide fragments are joined and integrated into one peptide fragment to form the neoantigen polypeptide.
(5) Obtaining a nascent antigen polypeptide with strong immunogenicity
The new antigen polypeptide is synthesized, added into immune cells of a patient, the immunogenicity of the new antigen polypeptide is verified by using an IFN-gamma ELISPOT kit, the new antigen polypeptide with strong immunogenicity is obtained, and the screened new antigen polypeptide is connected in series according to the sequence from strong immunogenicity to weak immunogenicity, so as to synthesize mRNA.
Taking A liver cancer patient as an example, the HLA type measured by the liver cancer patient is A.times 30:01 A*02:01 B*50:01 B*13:02 C*06:02, 87 mutant polypeptides obtained according to the step (1) are obtained through screening in the step (2), 25 peptide fragments with high affinity of 9-14mer are obtained through analyzing and screening in the step (3), 68 neoantigen polypeptides are obtained through comprehensive analysis and integration in the step (4), the amino acid sequences of the neoantigen polypeptides are SEQ ID NO 2-SEQ ID NO 69, and the neoantigen polypeptides are respectively synthesized.
Screening of nascent antigen polypeptides was performed using human IFN-gamma Precoated ELISPOT Kit (strips) from Daidae, bioengineering, inc., the specific steps of which are shown in the kit instructions.
Based on the ELISPOT spot counts, neoantigen polypeptides with more spots than the positive control were finally selected, as shown in table 1.
The neoantigen polypeptides in Table 1 are connected and synthesized into mRNA1 according to the sequence of the number of spots from more to less, his tag is added at the N end of the mRNA1, and the nucleotide sequence is shown as SEQ ID NO. 1. mRNA2 is synthesized according to the number of spots from small to large, a His tag is added at the N end of the mRNA2, and the nucleotide sequence of the His tag is shown as SEQ ID NO. 70. mRNA3 is synthesized according to the sequence with more spots and less spots, his tag is added at the N end of the mRNA3, and the nucleotide sequence is shown as SEQ ID NO. 71.
The three mRNA sequencing sequences are shown in FIG. 2.
TABLE 1 ELISPOT Spot count results
Example 2 mRNA Synthesis and identification
For in vitro synthesized mRNA, 5 'capped mRNA was synthesized in vitro using cap MMESSAGE MMACHINE T kit (Ambion Corp.) and simultaneously 3' end-capped Poly (A) tail structure was added to mRNA to obtain 5 'capped and 3' tail structure mRNA, which was purified using NEB Monarch RNACleanup Kit (T2040) series kit, the specific steps are described in the specification.
The concentration of the purified mRNA is detected, electrophoresis is carried out in agarose gel, a gel imager photographs, and the size and the integrity of the mRNA are verified.
The concentration of mRNA1 purified in the present invention was 2.12. Mu.g/. Mu.L, the concentration of mRNA2 purified was 2.43. Mu.g/. Mu.L, and the concentration of mRNA3 purified was 2.31. Mu.g/. Mu.L; mRNA1, mRNA2 and mRNA3 obtained by agarose gel electrophoresis were matched with the actual nucleotide sizes (see FIG. 3).
Example 3 preparation of mRNA-LNP vaccine and characterization analysis thereof
The mRNA-LNP vaccine was prepared according to the conventional method for preparing LNP, and the specific method is as follows:
(1) Preparation of aqueous mRNA solutions
Dissolving the purified mRNA obtained in the example 2 in a citric acid buffer solution, adding PBS, and fully and uniformly mixing to obtain an mRNA aqueous solution;
wherein the volume ratio of the purified mRNA to the citric acid buffer solution and PBS is 1:1250:2500;
The concentration of the citrate buffer was 10mM and the pH was 4.
(2) Preparation of ethanol solution of Liposome mixture
Mixing the liposome mixture with ethanol to obtain an ethanol solution of the liposome mixture;
in the ethanol solution of the liposome mixture, the mass ratio of the liposome mixture to ethanol is 1:48.8;
The molar ratio of DLin-MC3-DMA, DSPC, cholesterol and PEG200-DMG in the liposome mixture was 50:10:38.5:1.5.
(3) Synthesis of LNP nanoparticles
LNP nanoparticles of mRNA were synthesized by mixing an ethanol solution of the liposome mixture with an aqueous mRNA solution in a ratio of 1:3 (volume ratio) using a nanoparticle synthesis system (Ignite).
(4) Preparation of mRNA-LNP vaccine
Dialyzing the obtained LNP nanoparticles in PBS (pH 7.4) for 24h times, concentrating with Amicon ultracentrifugation filter, filtering with 0.22 m filter membrane for 2 times, and adjusting mRNA concentration to 80 g/mL to obtain mRNA-LNP vaccine.
The purified mRNA1, mRNA2 and mRNA3 of example 2 were prepared as described above to obtain mRNA-LNP vaccines, respectively, and labeled as mRNA1-LNP vaccine, mRNA2-LNP vaccine and mRNA3-LNP vaccine.
And carrying out characterization analysis on the prepared mRNA-LNP vaccine, wherein the characterization analysis comprises measurement of the size, dispersion index and encapsulation efficiency of the nano particles.
The size and dispersion index (PDI) of the nanoparticles of the mRNA-LNP vaccine were analyzed by Dynamic Light Scattering (DLS) and the encapsulation efficiency of the nanoparticles was measured by quant-iT RiboGreen RNA REAGENT AND KIT (from Invitorgen).
The result shows that the size of the nano particles in the mRNA1-LNP vaccine is 86-112nm, the dispersion index PDI is 0.139, and the encapsulation rate of the nano particles is 86.7%; the size of the nano particles in the mRNA2-LNP vaccine is 89-113nm, the dispersion index PDI is 0.148, and the encapsulation rate of the nano particles is 85.4%; the size of the nano particles in the mRNA3-LNP vaccine is 87-116nm, the dispersion index is 0.154, and the encapsulation rate of the nano particles is 84.9%.
EXAMPLE 4 DC cell-loaded mRNA-LNP vaccine
MRNA1-LNP vaccine was loaded into mature DC cells according to conventional procedures by: after the DC cells were first cultured for 5 days, 25. Mu.g of mRNA1-LNP vaccine was co-cultured with 1X 10 6 DC cells for 1 day, and then the DC cells were induced to mature for 2 days, to obtain mRNA1-LNP vaccine-loaded DC cells.
Taking part of DC cells loaded with mRNA1-LNP vaccine, carrying out flow detection by using His tag flow antibody, and carrying out flow detection according to a conventional flow detection method, wherein the result is shown in figure 4, 65.6% of DC cells have His protein expression, which shows that the DC cells can phagocytize mRNA-LNP nano particles and translate into protein in the cells.
MRNA2-LNP vaccine and mRNA3-LNP vaccine are loaded into mature DC cells according to the method to obtain DC cells loaded with mRNA2-LNP vaccine and DC cells loaded with mRNA3-LNP vaccine.
EXAMPLE 5 expression of the CD8+ T cell marker IFN-gamma, TNF-alpha by mRNA-LNP vaccine loaded DC cells
(1) Collection of purified CD8+ T cells
Collecting separated lymphocytes, centrifuging, discarding supernatant, referring to the specification of STEMCELL CD8 ENRICHMENT KIT (Human CD8+T Cell) kit, adding 1mL Robosep buffer solution to every 510 7 cells, transferring the resuspended cells to a centrifuge tube, adding 30 l of Human CD8+T Cell enriched antibody mixture to every mL of Cell suspension, mixing uniformly, standing for 5min, adding 50 l magnetic beads, mixing uniformly, and incubating at room temperature for 1min; 1mL Robosep buffer was added and allowed to stand, and the supernatant was allowed to contain enriched CD8+ T cells.
(2) Co-stimulation culture
The control group is CD8+ T cells cultured alone;
experimental group 1 was co-stimulated with DC cells and cd8+ T cells loaded with mRNA1-LNP vaccine;
Experimental group 2 was co-stimulated with DC cells loaded with mRNA2-LNP vaccine and cd8+ T cells;
experimental group 3 was co-stimulated with DC cells loaded with mRNA3-LNP vaccine and cd8+ T cells;
the addition amount of DC cells loaded with mRNA-LNP vaccine in each hole of the experimental group is 1.53X10 6, and the addition amount of CD8+ T cells in each hole is 5 6;
The addition amount of CD8+ T cells in each hole of the control group is 510 6;
The culture conditions for the control and experimental groups were 3 days incubation of cells in a 5% CO 2 cell incubator at 37 .
Each group of cells was subjected to fixation treatment according to the instructions of the flow antibody kit (purchased from Thermo), and then purified IFN-gamma antibody and purified TNF-alpha antibody were added to the control group and the experimental group, respectively, and incubated at room temperature for 30min in the absence of light, and the expression rates of IFN-gamma and TNF-alpha in the cells were analyzed using a Backman flow cytometer.
The experimental results are shown in fig. 5 and table 2, and compared with the control group, the specific cd8+ T cell markers IFN- , TNF- , stimulated by the mRNA-LNP vaccine-loaded DC cells, were both up-regulated, demonstrating a greater potential to kill tumor cells. The IFN-gamma and TNF-alpha expression level in the specific CD8+ T cells stimulated by the DC cells loaded with the mRNA1-LNP vaccine is higher than that in the specific CD8+ T cells stimulated by the DC cells loaded with the mRNA2-LNP or mRNA3-LNP vaccine, which proves that the ordering of the neoantigens has a larger influence on the immunogenicity of the mRNA-LNP vaccine.
TABLE 2 positive cell fraction for specific CD8+ T cell markers IFN-. Gamma.and TNF-. Alpha.
Example 6 in vitro tumor cell killing by tumor neoantigen specific T cells validation
(1) Collection of purified CD8+ T cells
The isolated lymphocytes were collected and centrifuged to discard the supernatant, and 1mL of Robosep buffer was added per 510 7 cells, as described in STEMCELL CD 8: 8 ENRICHMENT KIT (Human cd8+tcell) kit. Transferring the resuspended cells to a centrifuge tube, adding 30 mu L of human CD8+ T cell enrichment antibody mixture into each milliliter of cell suspension, uniformly mixing, standing for 5min, adding 50 mu L of magnetic beads, uniformly mixing, and incubating for 1min at room temperature; 1mL Robosep buffer was added and allowed to stand, and the supernatant was allowed to contain enriched CD8+ T cells.
(2) Preparation of DC cell stimulated CD8+ T cells loaded with mRNA-LNP vaccine
The mRNA1-LNP vaccine loaded DC cells were co-cultured with CD8+ T cells. Wherein, 510 6 DC cells loaded with mRNA1-LNP vaccine and 1.5310 6 DC cells loaded with mRNA1-LNP vaccine are added into each hole, and cultured for 7 days at 37 under 5% CO 2 in a T cell culture medium, so as to obtain the DC cell stimulated CD8+ T cells loaded with mRNA1-LNP vaccine, which has the tumor neoantigen TCR specificity.
And preparing DC cells loaded with the mRNA2-LNP vaccine and DC cells loaded with the mRNA3-LNP vaccine respectively according to the method to obtain CD8+ T cells stimulated by the DC cells loaded with the mRNA2-LNP vaccine and CD8+ T cells stimulated by the DC cells loaded with the mRNA3-LNP vaccine.
(3) In vitro killing experiments
Liver cancer tumor MHCC-97H cells containing luciferase markers are selected as target cells.
Blank groups are MHCC-97H cells;
Control group 1 is a mixture containing MHCC-97H cells and mRNA1-LNP vaccine, control group 2 is a mixture containing MHCC-97H cells and mRNA2-LNP vaccine, and control group3 is a mixture containing MHCC-97H cells and mRNA3-LNP vaccine;
Experiment group 1 was cocultured with MHCC-97H cells and DC-stimulated CD8+ T cells loaded with mRNA1-LNP vaccine, experiment group 2 was cocultured with MHCC-97H cells and DC-stimulated CD8+ T cells loaded with mRNA2-LNP vaccine, and experiment group 3 was cocultured with MHCC-97H cells and DC-stimulated CD8+ T cells loaded with mRNA3-LNP vaccine.
Wherein the addition amount of MHCC-97H cells in the blank group is 1.0X10 5 per well;
The addition amount of MHCC-97H cells per well in the control group was 1.0X10 5, and the final concentration of mRNA-LNP vaccine per well was 10. Mu.g/mL;
The loading per well of MHCC-97H cells in the experimental group was 1.0X10 5, and the loading per well of DC-cell stimulated CD8+ T cells loaded with mRNA-LNP vaccine was 1.0X10 5.
Then incubating the cells of the blank group, the experimental group and the control group in a cell culture box with 5% CO 2 at 37 for 10 hours, measuring the fluorescein value by an enzyme-labeling instrument, and calculating the killing efficiency of the CD8+ T cells to the tumor cells, wherein the calculation formula is as follows: killing efficiency of experimental group= (1-experimental group fluorescein value/blank group fluorescein value) 100%; control killing efficiency= (1-control fluorescein value/blank fluorescein value) 100%.
Experimental results show that the control group has almost no killing power on liver cancer tumor MHCC-97H cells; the DC cell stimulated CD8+ T cell loaded with mRNA-LNP has obvious killing power to liver cancer tumor MHCC-97H cell. The killing efficiency (86.5%) of the DC cell-stimulated CD8+ T cells loaded with mRNA1-LNP vaccine against MHCC-97H cells was higher than that of the DC cell-stimulated CD8+ T cells loaded with mRNA2-LNP vaccine and the DC cell-stimulated CD8+ T cells loaded with mRNA3-LNP vaccine against MHCC-97H cells (55.6%, 64.2%), as shown in FIG. 6, demonstrating that the ordering of neoantigens has an effect on the immunogenicity of the mRNA vaccine.
Claims (8)
1. The preparation method of the target liver tumor personalized mRNA vaccine is characterized by comprising the following steps: the preparation method comprises determining mutant polypeptide, preliminary screening mutant polypeptide, screening high affinity peptide fragment, integrating to obtain new antigen polypeptide, obtaining new antigen polypeptide with strong immunogenicity, synthesizing mRNA, and preparing mRNA vaccine; the method for obtaining the neoantigen polypeptide with strong immunogenicity is to synthesize the neoantigen polypeptide, then test the immunogenicity by using a kit, and select the neoantigen polypeptide with more immune spots than the positive control, namely the neoantigen polypeptide with strong immunogenicity.
2. The method for preparing the personalized mRNA vaccine for targeting liver tumor according to claim 1, wherein: the method for synthesizing mRNA is to connect the nascent antigen polypeptide with strong immunogenicity into mRNA according to the sequence of more immune spots.
3. The method for preparing the personalized mRNA vaccine for targeting liver tumor according to claim 1, wherein: the method for determining the mutant polypeptide comprises the steps of sequencing and comparing tumor tissues and healthy blood of a tumor patient, and determining mutation sites, wherein mutation types comprise single nucleotide variation and insertion/deletion mutation; for the mutation of amino acid caused by single nucleotide variation, non-mutant amino acid is added before and after the mutation site to obtain mutant polypeptide; for the frameshift peptide generated by the insertion/deletion mutation, non-mutant amino acid is added at the N end of the whole frameshift peptide to obtain mutant polypeptide.
4. A method of preparing a personalized mRNA vaccine for targeting liver tumors according to claim 3, wherein: the primary screening method of the mutant polypeptide comprises the steps of carrying out MHC I and MHC II affinity analysis and prediction on the mutant polypeptide obtained by single nucleotide variation, and selecting sequences with high MHC I and MHC II affinities; and (3) for mutant polypeptides obtained by insertion/deletion mutation, all mutant sequences are incorporated, so that the mutant polypeptides after primary screening are obtained.
5. The method for preparing the personalized mRNA vaccine for targeting liver tumor according to claim 4, wherein the method comprises the following steps: the method for screening the high-affinity peptide fragment is to screen a 9-14mer polypeptide sequence with high affinity with MHC according to the mutation polypeptide sequence after initial screening and HLA typing results.
6. The method for preparing the personalized mRNA vaccine for targeting liver tumors according to claim 5, wherein: the method for integrating and obtaining the neoantigen polypeptide comprises the following steps: for the high-affinity peptide fragments of the 9-14mer screened from the same mutant polypeptide, if the high-affinity peptide fragments are not overlapped, each high-affinity peptide fragment is used as an independent neoantigen polypeptide; if there is an overlap between two high affinity peptide fragments, the overlap of 1 peptide fragment is deleted, and then the two peptide fragments are joined and integrated into one peptide fragment to form the neoantigen polypeptide.
7. The method for preparing the personalized mRNA vaccine for targeting liver tumor according to claim 2, wherein: his tag was added to the N-terminal of mRNA, and 5 '-capping and 3' -terminal Poly (A) tail structure were performed on the synthesized mRNA.
8. The method of preparing a personalized mRNA vaccine for targeting a liver tumor according to claim 7, wherein: the method for preparing the mRNA vaccine comprises the steps of purifying mRNA with a 5 '-capping structure and a 3' -tailing structure, loading the mRNA with a liposome mixture, synthesizing LNP nano particles of the mRNA, and obtaining the mRNA vaccine after dialysis, concentration and filtration.
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