Preparation method and application of dendritic cell vaccine

Abstract

The invention discloses a preparation method and application of a dendritic cell vaccine. The preparation method of the dendritic cell vaccine comprises the following steps: designing a new antigen mRNA sequence; preparing virus-like particles containing annular mRNA according to a new antigen mRNA sequence; dC is transfected through virus-like particles containing annular mRNA, and the DC vaccine of the tumor new antigen is obtained. The DC vaccine of the annular mRNA can be individually designed and prepared according to an amino acid sequence or a nucleotide sequence of a tumor neoantigen, accurate application of different individuals is realized, the stability of the annular mRNA in cells is higher, the annular mRNA can be directly expressed in the cells, and antigen information is more efficiently ingested and presented by the DC.

Description

Technical Field

[0001] The present invention relates to the technical field of cell vaccine preparation, and particularly relates to a preparation method and application of a dendritic cell vaccine. Background Art

[0002] Dendritic cells (DCs) are professional antigen-presenting cells (APCs) of the body, which can efficiently uptake, process and present antigens.

[0003] There are three main sources of tumor antigens: tumor-associated antigens (TAAs), carcinogenic virus-derived antigens, and tumor-specific antigens (TSA, neoantigens). Mutations occurring in tumor cells can generate new self-antigen epitopes, called neoepitopes or neoantigens. Neoantigens are only expressed by tumor cells, so they can trigger a truly tumor-specific T cell response, thus preventing "off-target" damage to non-tumor tissues. Neoantigens are neoepitopes derived from somatic mutations, and they have the potential to bypass central tolerance of T cells to self-epitopes, thereby inducing an immune response against tumors. Therefore, DCs loaded with tumor neoantigens can be used to make personalized tumor vaccines, achieving tumor-specific immunity for different individuals.

[0004] Currently, there are two main aspects affecting the effectiveness of tumor vaccines. One is whether the targeting of antigens is precise, and the other is the efficiency of DCs presenting antigen information. Summary of the Invention

[0005] Based on the above problems, the purpose of the present invention is to provide a preparation method of a DC vaccine that can realize the amino acid sequence or nucleotide sequence of a personalized tumor neoantigen and enable the mRNA vaccine to be directly expressed in DC cells, thereby allowing DCs to uptake and present antigen information more efficiently.

[0006] The technical solution of the present invention is as follows:

[0007] A preparation method of a DC vaccine based on tumor neoantigens, comprising the following steps:

[0008] Design a neoantigen mRNA sequence;

[0009] After converting the neoantigen mRNA sequence into the corresponding neoantigen DNA sequence, prepare mRNA packaged in the outer shell of virus-like particles to obtain virus-like particles containing circular mRNA;

[0010] Transfect DCs with the virus-like particles containing circular mRNA to obtain a DC vaccine of tumor neoantigens.

[0011] In one embodiment, in the DC vaccine preparation method, when designing the neoantigen mRNA sequence, the technological steps are as follows:

[0012] Obtain tumor samples and normal tissue samples from tumor patients, extract DNA separately for exon sequencing, analyze and compare the sequencing results of the two groups of samples, identify DNA mutation sequences unique to tumors, and obtain the corresponding amino acid mutations for all DNA mutation sequences; at the same time, perform HLA typing determination on the DNA whole-exome sequencing data of the normal tissue samples to obtain the polypeptide sequences of MHC class I subtypes in HLA typing;

[0013] Obtain the corresponding mutant peptide chain sequences and their flanking sequences from the mutant amino acids, and respectively extract the polypeptide sequences containing the mutant amino acids accordingly, and the polypeptide sequences of each mutant amino acid have a length of 8-15 peptides;

[0014] Analyze the affinity of the extracted polypeptide sequences of the mutant amino acids with the polypeptide sequences of MHC class I subtypes in the HLA typing, and screen out 20-30 polypeptide sequences with excellent affinity according to the analysis results. According to the screened polypeptide sequences, use SnapGene software to convert to obtain the corresponding mRNA sequences;

[0015] Sort and splice all the mRNA sequences from high to low according to the affinity score values obtained by HLA analysis of the corresponding polypeptide sequences to obtain Neo-Seq mRNA sequences;

[0016] Use SnapGene software to sort the internal ribosome entry site sequence, start codon, Neo-Seq mRNA sequence, and stop codon in sequence to construct a complete neoantigen mRNA sequence.

[0017] In one embodiment, in the method for preparing the DC vaccine, for preparing the virus-like particles of the circular mRNA, the following steps are further included:

[0018] Use SnapGene software to convert the neoantigen mRNA sequence to obtain the corresponding neoantigen DNA sequence;

[0019] Adopt the Tornado circular mRNA expression system to prepare the virus-like particles containing circular mRNA from the neoantigen DNA sequence. In one embodiment, in the method for preparing the DC vaccine, when using the Tornado circular mRNA expression system to prepare the virus-like particles containing circular mRNA, the following steps are further included:

[0020] Synthesize the neoantigen DNA sequence and the Tornado component sequence into a gene block; then clone it into the NotI and SacII restriction sites of the pAV-U6+27-Tornado-Broccoli plasmid to obtain plasmid A;

[0021] Using a DNA transfection reagent, plasmid A, plasmid psPAX2-D64V-NC-MCP, and plasmid pMD2.G were added to HEK293T cells resuspended in Opti-MEM I medium and incubated for 24 h.

[0022] After the incubation, the cell lysate was collected and centrifuged at 300 g for 5 min to remove the supernatant. An equal volume of fresh serum-free HEK293T medium was added to the cell pellet and the mixture was cultured on a shaker for 48 h. After the culture, the culture medium was collected and centrifuged at 300 g for 5 min to collect the supernatant, which was the culture medium containing virus-like particles with circular mRNA.

[0023] The culture medium containing virus-like particles with circular mRNA was filtered through a 0.45 μm filter and then purified and concentrated to obtain a concentrated solution of virus-like particles with circular mRNA, which was stored frozen for later use.

[0024] In one embodiment, in the transfection and incubation step of the DC vaccine preparation method, the mass ratio of plasmid A, plasmid psPAX2-D64V-NC-MCP, and plasmid pMD2.G is 3:2:1.

[0025] In one embodiment, in the transfection and incubation step of the DC vaccine preparation method, the total mass ratio of plasmid A, plasmid psPAX2-D64V-NC-MCP, and plasmid pMD2.G to the DNA transfection reagent is 1:3, and in the transfection and incubation culture medium, the HEK293T cell density is maintained at 0.5 - 1×10 6 cells / mL.

[0026] In one embodiment, in the step of transfecting DC with virus-like particles containing circular mRNA in the DC vaccine preparation method, the following treatment is also included:

[0027] Mature DC was resuspended in RPMI-1640 medium and the cell density was controlled to be 3×10 6 cells / mL to obtain a cell suspension.

[0028] The filtrate of virus-like particles containing circular mRNA was added to the cell suspension, and 1 μg of virus-like particles containing circular mRNA was added per 10 6 DC cells, and the mixture was cultured in a shaker at 37 °C and 5% CO2 for 48 h.

[0029] After the culture, the culture medium was collected and centrifuged at 500 g for 6 min. The supernatant was removed and the cell pellet was washed multiple times with PBS to obtain the DC vaccine of tumor neoantigen, which was stored frozen for later use.

[0030] The DC vaccines prepared by any of the above preparation methods can be widely used in the preparation of anti-tumor drugs; for example, the preparation of drugs for treating liver cancer, lung cancer, colorectal cancer, gastric cancer, etc.

[0031] The DC vaccine preparation method provided by the present invention has the following technical advantages:

[0032] 1. The amino acid sequence or nucleotide sequence of the tumor neoantigen of the present invention can be personalized designed and prepared, which can provide the optimal prediction results for different individuals and achieve precise application for different individuals;

[0033] 2. Compared with linear mRNA, circular mRNA has stronger stability and longer action time in cells, and can play a greater role with a smaller amount of circular mRNA;

[0034] 3. Compared with traditional polypeptide vaccines, mRNA vaccines can be directly expressed in cells, and can more efficiently enable DC to uptake and present antigen information. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the structural diagram of circular mRNA sequence in Example 1;

[0036] Figure 2 It is the structural diagram of linear mRNA sequence in Example 2;

[0037] Figure 3 It is the curve graph of the tumor volume change in mice after tumor prevention by the DC vaccine in Example 6;

[0038] Figure 4 It is the curve graph of the tumor volume change in mice after tumor inhibition by the DC vaccine in Example 6;

[0039] Figure 5 It is the curve graph of the concentration change of TNF-α and IFN-γ in mice after tumor prevention by the DC vaccine in Example 6;

[0040] Figure 6 It is the curve graph of the concentration change of TNF-α and IFN-γ in mice after tumor inhibition by the DC vaccine in Example 6. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] The following further describes in detail the preferred embodiments of the present invention with reference to the accompanying drawings.

[0042] The DC vaccine preparation method based on tumor neoantigens provided by the present invention predicts the neoantigens of different tumors through deep learning of neural networks, provides more individualized and targeted antigen information, and combined with the high expression level of circular mRNA, further enhances the ability of DC to uptake and present antigens, providing better tools and means for the clinical application of tumors.

[0043] I. DC Culture

[0044] The DC used in the present invention is obtained by inducing and culturing mononuclear cells separated from peripheral blood. The acquisition steps are as follows:

[0045] (1) Preparation of dendritic cell culture medium

[0046] Two kinds of culture media are required during the DC culture process, namely DC culture medium and DC induction culture medium. Their preparations are as follows:

[0047] DC culture medium: Using RPMI - 1640 as the basal medium, which contains GM - CSF with a final concentration of 20 - 200 ng / mL, rhIL - 4 with a final concentration of 20 - 100 ng / mL, and AB serum with a final concentration of 5% v / v;

[0048] DC induction culture medium: Using RPMI - 1640 as the basal medium, which contains TNF - α with a final concentration of 5 - 40 ng / mL, rhIL - 6 with a final concentration of 50 - 200 ng / mL, prostaglandin E2 with a final concentration of 2 - 50 μg / mL, and AB serum with a final concentration of 1% v / v;

[0049] (2) Obtaining mononuclear cells

[0050] First, collect 100 mL of peripheral blood from a healthy person using a heparin sodium anticoagulant blood collection tube;

[0051] Secondly, transfer the blood in the blood collection tube to a 100 mL centrifuge tube containing lymphocyte separation solution, and centrifuge at 800 g for 20 min at room temperature;

[0052] Then, after centrifugation, aspirate the mononuclear cells in the middle buffy coat layer and transfer them to a 50 mL centrifuge tube, and use CD14 positive selection magnetic beads to separate mononuclear cells with CD14+≥95%;

[0053] Finally, wash the separated mononuclear cells with DPBS solution, and centrifuge the washed mononuclear cells at 600 g for 8 min at room temperature; repeat washing and centrifugation 2 - 3 times, and collect the cell precipitate to obtain mononuclear cells for standby.

[0054] (3) DC cell culture

[0055] On day 0, transfer the mononuclear cells sorted in step (2) of item (1) above into a TC75 culture flask containing 20 mL of the DC culture medium prepared in step (1) of item (1) above, and statically culture at 37°C and 5% CO2 for 5 days; during this period, change the medium every two days, and supplement 20 mL of DC culture medium each time when changing the medium;

[0056] On the 5th day, change the culture medium completely. Digest the cells in the culture flask with 5 mL of 0.05% trypsin digestion solution. After centrifuging the digestion solution and collecting the immature DCs, resuspend the cells with 20 mL of the DC induction culture medium prepared in step (1) of the first item above and inoculate them into another new TC75 culture flask, and continue static culture for 24 h;

[0057] On the 6th day, collect the culture medium, centrifuge at 500 g for 10 min, and discard the supernatant; wash the cell pellet with PBS 2 - 3 times to obtain mature DCs, and store them frozen for later use.

[0058] II. Prediction and design of tumor neoantigen polypeptide sequences

[0059] (1) Collect tumor samples and normal tissue samples from the same individual, extract DNA respectively for whole - exome sequencing; then use the Trimmomatic software to analyze and compare the sequencing results of the two groups of samples, identify the DNA mutation sequences unique to the tumor, and use the Annovar software to identify the amino acid mutations corresponding to all DNA mutation sequences; at the same time, perform HLA typing determination on the DNA whole - exome sequencing data of the normal tissue samples to obtain the polypeptide sequences of MHC class I subtypes in HLA typing;

[0060] (2) Use the Annovar software to identify the corresponding mutant peptide chain sequences and their flanking sequences from the mutant amino acids, and respectively extract the polypeptide sequences containing the mutant amino acids accordingly, and the length of the polypeptide sequences of each mutant amino acid is 8 - 15 peptides;

[0061] (3) Use the NetMHCpan software to perform affinity analysis on the polypeptide sequences of the mutant amino acids extracted in step (1) of the second item above and the polypeptide sequences of MHC class I subtypes in HLA typing obtained in step (2) of the second item respectively, and according to the analysis results, screen out 20 - 30 polypeptide sequences with high - quality affinity. According to the screened polypeptide sequences, use the SnapGene software to convert them into the corresponding mRNA sequences;

[0062] (4) Sort and splice all the mRNA sequences in descending order according to the affinity score values obtained from the HLA analysis of the corresponding polypeptide sequences to obtain the Neo - Seq mRNA sequence;

[0063] (5) Use the SnapGene software to sort the internal ribosome entry site (IRSE) sequence, start codon, Neo - Seq mRNA sequence, and stop codon in sequence to construct a complete neoantigen mRNA sequence.

[0064] III. Preparation of virus - like particles containing circular mRNA (i.e., mRNA - VLP)

[0065] (1) Using the SnapGene software, convert the neoantigen mRNA sequence obtained in step (5) of the second item above to obtain a neoantigen DNA sequence;

[0066] (2) Using the SnapGene software, splice the neoantigen DNA sequence and the Tornado component sequence, and prepare gene blocks by chemical synthesis; then clone the gene blocks into the two restriction sites of NotI and SacII of the pAV-U6+27-Tornado-Broccoli plasmid to obtain plasmid A;

[0067] (3) Mix DNA transfection reagents (such as FuGENE), plasmid A, plasmid psPAX2-D64V-NC-MCP, and plasmid pMD2.G, etc., and add them to HEK293T cells resuspended with Opti-MEM I medium. The HEK293T cells are obtained by suspension culture with HEK293T serum-free medium, and co-culture for 24 h;

[0068] Among them, plasmid A, plasmid psPAX2-D64V-NC-MCP, and plasmid pMD2.G are added according to a mass ratio of 3:2:1, and plasmid A is the target plasmid, while plasmid psPAX2-D64V-NC-MCP and pMD2.G are packaging plasmids; according to 3 μg of DNA transfection reagent and 1 μg of the total mixed plasmid (the sum of plasmid A, plasmid psPAX2-D64V-NC-MCP, and plasmid pMD2.G), 1 mL of Opti-MEM I medium needs to be added; after resuspending the cells with Opti-MEM I medium, the density of HEK293T cells is maintained at 0.5 - 1×10 6 cells / mL;

[0069] (4) After the co-culture is completed, collect the cell culture fluid and centrifuge at 300 g for 5 min to remove the supernatant; add fresh HEK293T serum-free medium to resuspend the HEK293T cell pellet, and the cell density is maintained at 0.5 - 1×10 6 cells / mL; continue to culture on a shaker for 48 h;

[0070] (5) Collect the culture medium, centrifuge at 300 g for 5 min, and collect the centrifuged supernatant to obtain the culture medium containing mRNA packaged in the virus-like particle (VLP) shell;

[0071] (6) Filter the culture medium containing mRNA packaged in the virus-like particle (VLP) shell through a 0.45 μm filter, and purify and concentrate the filtrate to obtain a concentrated solution of virus-like particles containing circular mRNA (i.e., mRNA-VLP);

[0072] (7) Sample the concentrated solution for virus RNA titer detection, calculate the concentration of circular mRNA containing the neoantigen in the concentrated solution, which serves as the basis for adding the volume of mRNA-VLP when transfecting DCs.

[0073] IV. Preparation of DC vaccine based on tumor neoantigen

[0074] (1) Resuspend the mature DCs obtained in the first item above with RPMI-1640 medium and control the cell density to be 3×10 6 cells / mL to obtain a cell suspension;

[0075] (2) Add the mRNA-VLP obtained in step (6) of the third item above to the cell suspension. According to the virus RNA titer measured in step (7) of the third item above, add 1 μg of mRNA-VLP for every 10 6 DC cells, and culture in a shaker at 37°C and 5% CO2 for 48 h;

[0076] (3) After the culture is completed, collect the culture medium and centrifuge at 500 g for 6 min to remove the supernatant; wash the cell pellet with PBS 2-3 times to obtain the DC vaccine of tumor neoantigen, and store it frozen for later use.

[0077] The DC vaccine of tumor neoantigen prepared by the present invention can be used in the preparation of drugs for preventing or treating solid tumors (such as liver cancer, lung cancer, colorectal cancer, gastric cancer, etc.), especially has good application in the preparation of drugs for treating liver cancer.

[0078] The quantity and frequency of administration of such vaccine drugs will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, and will be determined by the clinical protocol. When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition of the patient (subject). The optimal dosage and treatment regimen for a specific patient can be readily determined by those skilled in the medical art by monitoring the signs of the patient's disease and thus adjusting the treatment.

[0079] The administration of the DC vaccine of the present invention can be carried out in any convenient manner, including by injection, infusion, implantation or transplantation. The compositions described herein can be administered subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally to a patient. In one embodiment, the DC vaccine of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the DC vaccine of the present invention is preferably administered by i.v. injection, or the DC vaccine can be directly injected into the tumor, lymph node or infected site.

[0080] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding DC vaccines to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously, or after) with any number of relevant treatment modalities, including but not limited to treatment with the following reagents: such reagents as antiviral therapies, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or treatment with natalizumab for MS patients or efalizumab for psoriasis patients or other treatments for PML patients. In further embodiments, the DC vaccines of the present invention can be used in combination with: chemotherapy, radiation, immunosuppressive agents, such as, cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination (e.g., before, simultaneously, or after) with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In an additional embodiment, the expanded cells are administered before or after surgery. The doses of the above treatments administered to the patient will vary with the precise nature of the disorder being treated and the recipient of the treatment. Dosage ratios for human administration can be practiced according to accepted practice in the art. Generally, 1×10 6 -1×10 10 cells / mL can be administered to the patient, for example, by intravenous infusion.

[0081] Taking liver cancer as an example below, DCs are derived from the first item of the present invention to further describe the technical solution of the present invention in detail.

[0082] Example 1 (DC vaccine of circular mRNA-VLP)

[0083] 1.1. Predict and design neoantigen polypeptide sequences

[0084] 1.1.1. Obtain tumor samples and normal tissue samples from liver cancer patients, extract DNA respectively for whole exome sequencing; subsequently, use the Trimmomatic software to analyze and compare the sequencing results of the two groups of samples, identify the DNA mutation sequences unique to the tumor, and use the Annovar software to identify the amino acid mutations corresponding to all DNA mutation sequences; at the same time, perform HLA typing determination on the DNA whole exome sequencing data of the normal tissue samples to obtain the polypeptide sequences of MHC class I subtypes in HLA typing;

[0085] 1.1.2. Use the Annovar software to identify the corresponding mutant peptide chain sequences and their flanking sequences from the mutant amino acids, and respectively extract the polypeptide sequences containing the mutant amino acids accordingly. The length of the polypeptide sequences for each mutant amino acid is 8 - 15 peptides;

[0086] 1.1.3. Use the NetMHCpan software to perform affinity analysis on the polypeptide sequences of each mutant amino acid with the polypeptide sequences of MHC class I subtypes in the obtained HLA typing. According to the analysis results, screen out 25 high-quality affinity polypeptide sequences, and then use the SnapGene software to convert them to obtain the corresponding mRNA sequences;

[0087] 1.1.4. Sort and splice all the mRNA sequences in descending order according to the affinity score values obtained from the HLA analysis of the corresponding polypeptide sequences to obtain the Neo-Seq mRNA sequence;

[0088] 1.1.5. Use the SnapGene software to sequentially sort and construct a complete neoantigen mRNA sequence with the internal ribosome entry site (IRSE) sequence, start codon, Neo-Seq mRNA sequence, and stop codon.

[0089] 1.2. Prepare virus-like particles containing circular mRNA (i.e., mRNA-VLP)

[0090] 1.2.1. Use the SnapGene software to convert the neoantigen mRNA sequence obtained in step 1.1.5 of this example to obtain a neoantigen DNA sequence; this neoantigen mRNA sequence mainly contains an internal ribosome entry site (IRES) sequence, a neoantigen sequence, a start codon (AUG), and a stop codon, as Figure 1 shown;

[0091] 1.2.2. Adopt the Tornado (Twister-optimized RNA for durable overexpression) circular RNA expression system. Use the SnapGene software to ligate the neoantigen DNA sequence with the Tornado component sequence, and prepare a gene block through chemical synthesis; then clone the gene block into the NotI and SacII restriction sites of the pAV-U6+27-Tornado-Broccoli plasmid to obtain plasmid A;

[0092] 1.2.3. Add 54 μg of FuGENE, 9 μg of plasmid A, 6 μg of plasmid psPAX2-D64V-NC-MCP, and 3 μg of plasmid pMD2.G to HEK293T cells resuspended in Opti-MEM I medium, shake well, and incubate in a shaker at 37 °C and 5% CO2 for 24 h; among them, control the density of HEK293T cells to be maintained at 0.5 - 1×10 6 cells / mL, and the HEK293T cells are obtained by suspension culture in serum-free HEK293T medium;

[0093] 1.2.4. After the incubation is completed, collect the cell supernatant and centrifuge at 300 g for 5 min, discard the supernatant; add fresh serum-free HEK293T medium equal to the cell pellet and continue to culture in a shaker for 48 h;

[0094] 1.2.5. Collect the culture medium and centrifuge at 300 g for 5 min, and collect the centrifuged supernatant again to obtain the culture medium containing circular mRNA encapsulated in virus-like particle coats;

[0095] 1.2.6. Filter the culture medium containing circular mRNA encapsulated in virus-like particle coats through a 0.45 μm filter, and purify and concentrate the filtrate to obtain a concentrated solution of virus-like particles containing circular mRNA (i.e., mRNA-VLP);

[0096] 1.2.7. Take a sample of the concentrated solution for virus RNA titer detection, and calculate the concentration of circular mRNA containing the neoantigen in the concentrated solution according to the titer to determine the volume of mRNA-VLP to be added for subsequent transfection of DC.

[0097] 1.3. Preparation of DC vaccine of neoantigen

[0098] 1.3.1. Resuspend the mature DC obtained in the first item of the present invention with 10 mL of RPMI-1640 medium, and control the cell density to be 3×10 6 cells / mL to obtain a cell suspension;

[0099] 1.3.2. Add 1 μg of mRNA-VLP for every 10 6 DC cells, add 30 μg of circular mRNA-VLP to the cell suspension, and culture in a shaker at 37 °C and 5% CO2 for 48 h;

[0100] 1.3.3. After the culture is completed, collect the culture medium and centrifuge at 500 g for 6 min, discard the supernatant and wash the cell pellet with PBS 2 - 3 times to obtain the DC vaccine of tumor neoantigen, and store it frozen for later use.

[0101] Example 2 (Comparative Example 1, DC vaccine of linear mRNA-LNP)

[0102] 2.1. Prediction and Design of Neoantigen Polypeptide Sequences

[0103] 2.1.1. Collect tumor samples and normal tissue samples from liver cancer patients, extract DNA respectively for whole exome sequencing, then use the Trimmomatic software to analyze and compare the sequencing results of the two groups of samples, identify the DNA mutation sequences unique to the tumor, and use the Annovar software to identify the amino acid mutations corresponding to all DNA mutation sequences; at the same time, perform HLA typing determination on the DNA whole exome sequencing data of the normal tissue samples to obtain the polypeptide sequences of MHC class I subtypes in HLA typing;

[0104] 2.1.2. Use the Annovar software to identify the corresponding mutant peptide chain sequences and their flanking sequences from the mutant amino acids, and extract the polypeptide sequences containing the mutant amino acids respectively, and the polypeptide sequences of each mutant amino acid have a length of 8-15 peptides;

[0105] 2.1.3. Use the NetMHCpan software to perform affinity analysis on the polypeptide sequences of each mutant amino acid with the polypeptide sequences of MHC class I subtypes obtained in HLA typing respectively, and according to the analysis results, screen out 25 high-quality affinity polypeptide sequences, and according to the screened polypeptide sequences, use the SnapGene software to convert to obtain the corresponding mRNA sequences;

[0106] 2.1.4. Sort and splice all the mRNA sequences in descending order according to the affinity score values obtained from HLA analysis of the corresponding polypeptide sequences to obtain the Neo-Seq mRNA sequence;

[0107] 2.1.5. Use the SnapGene software to sort and construct the complete neoantigen mRNA sequence in turn with the 5' untranslated region (UTR) sequence, Neo-Seq mRNA sequence and 3' untranslated region (UTR) sequence.

[0108] 2.2. Preparation of Neoantigen Linear mRNA-LNP

[0109] 2.2.1. Perform IVT (i.e., in vitro synthesis of mRNA) on the neoantigen mRNA sequence, add a cap to the 5' end of the mRNA sequence by enzymatic method, and add a Poly(A) tail to the 3' end of the mRNA sequence by template coding to synthesize the initial product of linear mRNA, and then perform purification;

[0110] 2.2.2. When the purity of the linear mRNA detected by HPLC is ≥80%, dissolve the linear mRNA with a capping rate at the 5' end of the mRNA sequence and a tailing rate at the 3' end of the mRNA sequence both ≥90% in 1 mM sodium citrate solution to obtain purified linear mRNA, and make the volume 1 mg / mL;

[0111] 2.2.3. The linear mRNA after volume fixation is encapsulated with LNP to obtain a linear mRNA-LNP solution with a concentration of 0.1 mg / mL for standby.

[0112] 2.3. Transfect DC with the original linear mRNA-LNP

[0113] 2.3.1. Resuspend the mature DC obtained in the first item above of the present invention with 10 mL of RPMI-1640 medium and control the cell density to be 3×10 6 cells / mL to obtain a cell suspension;

[0114] 2.3.2. Add 1 μg of linear mRNA-LNP for every 10 6 DC cells, add 30 μg of linear mRNA-LNP to the cell suspension, and place it in a shaker at 37°C and 5% CO2 for transfection and culture for 48 h;

[0115] 2.3.3. After the transfection and culture are completed, collect the culture medium and centrifuge it at 500 g for 6 min, remove the supernatant, and wash the cell pellet with PBS 2-3 times to obtain a DC vaccine of tumor neoantigen of liposome-encapsulated linear mRNA (mRNA-LNP), which is frozen for standby.

[0116] Example 3 (Comparative Example 2, stimulating DC with neoantigen polypeptide)

[0117] 3.1. Predict and design the neoantigen polypeptide sequence

[0118] 3.1.1. Collect tumor samples and normal tissue samples from liver cancer patients, extract DNA respectively for whole exome sequencing, then use the Trimmomatic software to analyze and compare the sequencing results of the two groups of samples, identify the DNA mutation sequences unique to the tumor, and use the Annovar software to identify the amino acid mutations corresponding to all DNA mutation sequences; at the same time, perform HLA typing determination on the DNA whole exome sequencing data of the normal tissue samples to obtain the polypeptide sequences of MHC class I subtypes in HLA typing;

[0119] 3.1.2. Use the Annovar software to identify the corresponding mutant peptide chain sequences and their flanking sequences from the mutant amino acids, and respectively extract the polypeptide sequences containing the mutant amino acids accordingly, and the length of each polypeptide sequence of the mutant amino acids is 8-15 peptides;

[0120] 3.1.3. Use the NetMHCpan software to perform affinity analysis on the polypeptide sequences of each mutant amino acid respectively with the polypeptide sequences of MHC class I subtypes in the obtained HLA typing. According to the analysis results, screen out 25 high-quality affinity polypeptide sequences. According to the screened polypeptide sequences, use the SnapGene software to convert to obtain the corresponding mRNA sequences.

[0121] 3.2. Preparation of neoantigen polypeptides

[0122] 3.2.1. Score the 25 polypeptide sequences screened in step 3.1.3 of this example, splice them in the order from high to low, and synthesize polypeptides;

[0123] 3.2.2. Dissolve the synthesized polypeptides with PBS containing 10% DMSO and make the volume up to 1 mg / mL to obtain a polypeptide solution for standby.

[0124] 3.3. Stimulation of DC with neoantigen polypeptides

[0125] 3.3.1. Resuspend the mature DC obtained in the first item above of the present invention with 10 mL of RPMI-1640 medium and control the cell density to 2×10 6 cells / mL to obtain a cell suspension;

[0126] 3.3.2. Add 1 μg of polypeptide for every 10 6 DC cells. Add 20 μg of polypeptide to the cell suspension, and incubate in a shaker at 37°C and 5% CO2 for 48 h;

[0127] 3.3.3. After the incubation is completed, collect the culture medium and centrifuge at 500 g for 6 min. Discard the supernatant and wash the cell pellet with PBS multiple times, and store it frozen for standby.

[0128] Example 4 (Comparative Example 3, neoantigen polypeptide)

[0129] 4.1. Prediction and design of neoantigen polypeptide sequences

[0130] 4.1.1. Collect 0.3 - 1 cm of tumor samples from the same liver cancer patient as in Examples 1, 2, and 3 3 , cut off the surrounding non-tumor tissues, wash them repeatedly 3 - 5 times with physiological saline containing gentamicin, cut them into pieces, grind them with a 50-μm pore size steel mesh, and resuspend them with physiological saline to prepare a single-cell suspension;

[0131] 4.1.2. Repeatedly freeze-thaw the single-cell suspension 3 to 5 times, filter it through a 50-μm pore size sieve to obtain the tumor cell lysate, centrifuge at 16,000 g for 20 min, and take the supernatant, which is the neoantigen polypeptide solution. Sample and detect the protein concentration. The purpose of detecting the protein concentration is as follows: Tumor antigens are mainly proteins expressed by tumors, mainly on the membrane. Here, total protein is used as the antigen protein, and detecting the protein concentration is equivalent to detecting the antigen concentration.

[0132] 4.2. Stimulation of DC with neoantigen polypeptides

[0133] 4.2.1. Resuspend the mature DC obtained in the first item of the present invention with 10 mL of RPMI-1640 medium, and control the cell density to be 2×10 6 cells / mL to obtain a cell suspension;

[0134] 4.2.2. Add 5 μg of polypeptide per 10 6 DC cells. Add 100 μg of polypeptide to the cell suspension, and incubate in a shaker at 37°C and 5% CO2 for 48 h;

[0135] 4.2.3. After the incubation is completed, collect the culture medium and centrifuge at 500 g for 6 min. Discard the supernatant and wash the cell pellet with PBS multiple times, and store it frozen for later use.

[0136] Example 5 (Comparative Example 4, DC without loaded antigen)

[0137] 5.1 Preparation of DC cells without loaded antigen

[0138] 5.1.1. Resuspend the mature DC obtained in the first item of the present invention with 10 mL of RPMI-1640 medium, and control the cell density to be 2×10 6 cells / mL to obtain a cell suspension;

[0139] 5.1.2. Add 10 μL of PBS per 10 6 DC cells. Incubate in a shaker at 37°C and 5% CO2 for 48 h;

[0140] 5.1.3. After the culture is completed, collect the culture medium and centrifuge at 500 g for 6 min. Discard the supernatant and wash the cell pellet with PBS 2 to 3 times to obtain DC cells without loaded antigen, and store it frozen for later use.

[0141] Example 6

[0142] 6.1. Verification of the in vivo tumor prevention effect of DC vaccine

[0143] Experimental operation: On the 1st day and the 7th day, SCID mice (6 - 8 weeks old) were administered via the tail vein with normal saline (blank control), DCs prepared and collected in Examples 1 to 5 (i.e., Comparative Examples 1 to 4), and PBMCs isolated from the blood of the patients in Examples 1 to 5 above (3 - 5 mice per group, and each mouse was injected with 100,000 DCs + 2 million PBMCs). On the 10th day, the tumor cells of the patients in Examples 1 to 5 above (2×10^6 cells / mouse) were inoculated under the right lower abdomen of the mice, and then the tumor growth was observed. After 7 days of inoculating the tumor cells, the tumor volume was measured every 3 - 4 days, and the calculation formula was: length × width 2 / 2; After two administrations and then injecting the tumor, the intervention effect (tumor prevention effect) in the early stage of tumor occurrence was simulated, and the results are as Figure 3 shown.

[0144] It can be seen from Figure 3 the following: 1. After the SCID mice were administered twice and then inoculated with the DCs prepared in Example 1, the tumor volume basically did not increase, indicating that the DCs prepared in Example 1 could generate specific immune cells from the donor PBMCs in the mice, specifically kill the inoculated tumor cells, and achieve the prevention of tumor occurrence; 2. After the DC drugs corresponding to Comparative Examples 1 - 4 were injected into the SCID mice, the specific killing effects were as follows: in the order of the circular VLP - structured mRNA tumor neo - antigen DC vaccine, the linear LNP - structured mRNA tumor neo - antigen DC vaccine, the polypeptide tumor neo - antigen DC vaccine, the conventional - method tumor - antigen DC vaccine, and the antigen - unloaded DC, the effects decreased successively.

[0145] 6.2. Verification of the in - vivo tumor - inhibitory (therapeutic) effect of the DC vaccine

[0146] Experimental operation: On the 1st day, the tumor cells of the patients in the examples (2×10^6 cells / mouse) were inoculated under the right lower abdomen of SCID mice (6 - 8 weeks old). When the tumor volume was about 80 - 100 mm 3 , mice with similar tumor sizes were selected for grouping. Each group was administered via the tail vein with the DCs prepared and collected in Examples 1 to 5 (i.e., Comparative Examples 1 to 4) and the PBMCs isolated from the blood of the patients in Examples 1 to 5 above (100,000 DCs + 2 million PBMCs / mouse). After the first administration (on the 1st day after administration), the second administration was carried out after a 6 - day interval (on the 7th day after administration) (with the same dose, a total of two administrations). The tumor volume was measured every 3 - 4 days, and the calculation formula was: length × width × thickness / 2. And the secretion levels of TNF - α and IFN - γ in the mice and the proportion of specific T cells in T lymphocytes were detected 48 h after the first administration and 48 h after the second administration; After two administrations after tumor modeling, the inhibitory effect after in - vivo tumor occurrence (tumor therapeutic effect) was simulated; as Figure 4 shown.

[0147] It can be seen from Figure 4 that: 1) After the mice were inoculated with the DCs prepared in Examples 1 to 5 respectively after two administrations of the drug, the inoculated tumors increased after both administrations of the drug. However, the increase amplitude of Example 1 was the smallest, indicating that the DC prepared in Example 1 could generate specific immune cells against the donor PBMC in the mice, could specifically kill tumor cells, achieved the effect of inhibiting tumor growth, and realized the inhibitory effect on tumor growth; 2) After injecting the corresponding DC drugs into the mice in Comparative Examples 1 to 4, the specific killing effects were as follows: the specific killing effects decreased in turn according to the cyclic VLP structure mRNA tumor neoantigen DC vaccine, the linear LNP structure mRNA tumor neoantigen DC vaccine, the polypeptide tumor neoantigen DC vaccine, the conventional method tumor antigen DC vaccine, and the DC without loaded antigen. The DC without loaded antigen prepared in Example 5 had basically no inhibitory effect, which was similar to the results of the normal saline group.

[0148] 6.3 Detection of the Secretion Levels of Tumor Necrosis Factor α (TNF-α) and Interferon γ (IFN-γ) in the Mouse Model

[0149] Experimental operation: Respectively take the mice in the preventive effect experiment of Item 6.1 and the inhibitory effect experiment of Item 6.2 of this example. After 24 hours of the second administration, take blood samples, centrifuge to obtain plasma, and use an ELISA kit to detect the concentrations of TNF-α and IFN-γ in the plasma according to the manufacturer's instructions; the detection results are as Figure 5 and 6 shown.

[0150] It can be seen from Figure 5 and 6Results: ① After injecting the DC drugs of the experimental groups corresponding to Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 into mice, the concentration values of TNF-α and IFN-γ in the mouse blood were submitted in sequence, and the concentration values of TNF-α and IFN-γ in the experimental group corresponding to Comparative Example 4 were basically the same as those in the normal saline group; ② In the inhibition experiment (Test Item 6.1) of Example 1 and Comparative Examples 1-3, the numerical values of the two factors (TNF-α and IFN-γ concentrations) were higher than those in the prevention experiment (Test Item 6.2); ③ In the experimental group of Example 1, the increase amplitude of the numerical values of the two factors (TNF-α and IFN-γ concentrations) was also higher than that of other comparative example groups, that is, the increase amplitude of the numerical values of the two factors (TNF-α and IFN-γ concentrations) in Example 1 > 20%, the increase amplitude of the numerical values of the two factors (TNF-α and IFN-γ concentrations) in Comparative Example 1 was 15-20%, the increase amplitude of the numerical values of the two factors (TNF-α and IFN-γ concentrations) in Comparative Examples 2-3 was 5-10%, and the increase amplitude of the numerical values of the two factors (TNF-α and IFN-γ concentrations) in Comparative Example 4 was < 5% (similar to the normal saline group). This shows that tumor antigens can promote the level of tumor-killing factors secreted by immune cells, and the novel antigen DC vaccine provided by the present invention can further promote the increase of this level.

[0151] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed, and it should not be considered as a limitation to the protection scope of the invention patent of the present invention. The protection scope of the invention patent of the present invention shall be subject to the appended claims.

Claims

1. A method for preparing a DC vaccine based on tumor neoantigens, characterized in that, It includes the following steps: Design a novel antigen mRNA sequence; Convert the novel antigen mRNA sequence into the corresponding novel antigen DNA sequence, and then prepare mRNA packaged in the capsid of virus-like particles to obtain virus-like particles containing circular mRNA; Transfect the virus-like particles containing circular mRNA into DC to obtain a DC vaccine for tumor neoantigens.

2. The DC vaccine preparation method according to claim 1, characterized in that, Design the novel antigen mRNA sequence, and the technological steps are as follows: Obtain tumor samples and normal tissue samples of tumor patients, extract DNA respectively for exon sequencing, analyze and compare the sequencing results of the two groups of samples, identify DNA mutation sequences specific to tumors, and obtain amino acid mutations corresponding to all DNA mutation sequences; at the same time, perform HLA typing determination on the DNA whole-exome sequencing data of the normal tissue samples to obtain the polypeptide sequences of MHC class I subtypes in HLA typing; Obtain the corresponding mutant peptide chain sequences and their flanking sequences from the mutant amino acids, and respectively extract the polypeptide sequences containing the mutant amino acids accordingly, and the polypeptide sequences of each mutant amino acid have a length of 8-15 peptides; Perform affinity analysis on the extracted polypeptide sequences of the mutant amino acids and the polypeptide sequences of MHC class I subtypes in HLA typing respectively, and screen out 20-30 polypeptide sequences with high-quality affinity according to the analysis results, and convert the corresponding mRNA sequences according to the screened polypeptide sequences; Sort and splice all the mRNA sequences from high to low according to the affinity score values obtained from HLA analysis of the corresponding polypeptide sequences to obtain Neo-Seq mRNA sequences; Sort the internal ribosome entry site sequence, start codon, Neo-Seq mRNA sequence and stop codon in sequence to construct a complete novel antigen mRNA sequence.

3. The method for preparing a DC vaccine according to claim 1, wherein, To prepare the virus-like particles containing circular mRNA, the following steps are further included: Convert the novel antigen mRNA sequence into the corresponding novel antigen DNA sequence; Use the Tornado circular mRNA expression system to prepare virus-like particles containing circular mRNA from the novel antigen DNA sequence.

4. The DC vaccine preparation method according to claim 3, wherein In the preparation of virus-like particles containing circular mRNA using the Tornado circular mRNA expression system, the following steps are further included: Synthesize the novel antigen DNA sequence and the Tornado component sequence into a gene block; then clone it into the NotI and SacII restriction sites of the pAV-U6+27-Tornado-Broccoli plasmid to obtain plasmid A; Add a DNA transfection reagent, plasmid A, plasmid psPAX2-D64V-NC-MCP and plasmid pMD2.G to HEK293T cells resuspended in Opti-MEM I medium, and transfect and incubate for 24 h; After the incubation and culture, collect the cell fluid and centrifuge at 300 g for 5 min to remove the supernatant; resuspend the cell pellet with HEK293T serum-free medium and continue to culture for 48 h; After the culture, collect the culture medium and centrifuge at 300 g for 5 min, and collect the supernatant, which is the culture medium of virus-like particles containing circular mRNA; The culture solution of virus-like particles containing circular mRNA was filtered through a 0.45 μm filter and then purified and concentrated to obtain a concentrated solution of virus-like particles containing circular mRNA, which was stored frozen for later use.

5. The method for preparing a DC vaccine according to claim 4, wherein, In the transfection incubation culture step, the mass ratio of plasmid A, plasmid psPAX2-D64V-NC-MCP, and plasmid pMD2.G is 3:2:

1.

6. The method for preparing a DC vaccine according to claim 4, wherein In the transfection incubation culture step, the mass ratio of the total mass of plasmid A, plasmid psPAX2-D64V-NC-MCP, and plasmid pMD2.G to the mass of the DNA transfection reagent is 1:3, and in the culture medium for transfection incubation, the density of HEK293T cells is maintained at 0.5-1x10 6 cells / mL.

7. The method for preparing a DC vaccine according to claim 4, 5 or 6, characterized in that, In the transfection incubation culture step, the DNA transfection reagent is FuGENE.

8. The method for preparing a DC vaccine according to claim 1, wherein In the step of transfecting DC with the virus-like particles containing circular mRNA, the following treatment is further included: Resuspend the mature DCs with RPMI-1640 medium to obtain a cell suspension, and control the cell density in the suspension to be 3×10 6 cells / mL; Add the virus-like particles containing circular mRNA to the cell suspension, and add 1 μg of the virus-like particles containing circular mRNA per 10 6 DC cells, and culture in a shaker at 37 °C and 5% CO2 for 48 h; After the culture is completed, the culture solution is collected and centrifuged at 500 g for 6 min, and the supernatant is removed; the cell precipitate is washed multiple times with PBS to obtain the DC vaccine of tumor neoantigen, which is stored frozen for later use.

9. A DC vaccine prepared by the preparation method according to any one of claims 1 to 8.

10. Use of a DC vaccine prepared by the preparation method according to any one of claims 1 to 8 in the preparation of a medicament for treating tumors, preferably in the preparation of a medicament for treating liver cancer.

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