Neonatal antigen composition for treating colorectal cancer liver metastasis as well as preparation method and application thereof

Abstract

The invention discloses a neoantigen composition for treating colorectal cancer liver metastasis as well as a preparation method and application thereof, and provides a novel combined immunotherapy scheme based on a tumor neoantigen vaccine by identifying and screening tumor neoantigen, synthesizing a neoantigen polypeptide vaccine and combining with a multi-target inhibitor. The problems that an existing immunotherapy with an immune checkpoint inhibitor has immune escape, the response to ICIs single medicine or combined targeting medicine is poor, and the objective remission rate is low are solved.

Description

Technical Field

[0001] The present invention relates to the field of tumor immunotherapy, and in particular to a neoantigen composition for treating colorectal cancer liver metastasis, a preparation method and an application thereof. Background Art

[0002] Colorectal cancer (CRC) has a high incidence and rapid progression. About 50% of CRC patients develop synchronous or metachronous colorectal cancer liver metastases (CRLM). Studies have shown that chronic HBV infection promotes the occurrence of CRLM. The tumor microenvironment of HBV-CRLM patients is highly immunosuppressive, resulting in poor prognosis. The median survival without treatment is only 6.9 months, and the 5-year survival rate is less than 5%. In recent years, immunotherapy represented by immune checkpoint inhibitors (ICIs) has developed rapidly in the field of advanced solid tumor treatment. Studies have shown that immunotherapy has the characteristics of high safety, few side effects, and definite efficacy, and is expected to make up for the shortcomings of the current traditional treatments for CRLM (surgical resection, chemoradiotherapy, radiofrequency ablation, targeted therapy, etc.). However, about 90%-95% of HBV-CRLM patients still have poor responses to ICIs alone or in combination with targeted drugs, with an objective response rate (ORR) of only 0-8.7%, due to low tumor TMB, lack of tumor-specific immune cell infiltration, and continuous chronic HBV inflammation stimulation leading to enrichment of immunosuppressive cells and immune checkpoint upregulation of PD-1, which are jointly mediated by cytotoxic T cell exhaustion, disorder of antigen-presenting cell subsets in the liver, and loss of antigen presentation ability mediated by major histocompatibility complex (MHC)-mediated immune escape. Therefore, exploring new immune strategies to induce tumor-specific immune cell infiltration, reverse the exhausted tumor microenvironment caused by persistent HBV infection, reactivate the immune system, and block the immune escape pathway of tumor cells is an important strategy to improve the efficacy of immunotherapy in HBV-CRLM patients.

[0003] Neoantigens are a type of tumor-specific antigens with strong immunogenicity. They exist specifically in tumor cells and are produced by frameshift mutations, splice variants, gene fusions, post-translational modifications, etc. They can be transcribed into mRNA and translated into mutated peptides with immunogenicity. Neoantigen peptides are presented to the cell surface after binding to the major histocompatibility complex (MHC) and are specifically recognized by the T cell surface receptor (TCR), thereby inducing the body to specifically kill tumor cells. Compared with traditional tumor-associated antigens, immunotherapy targeting neoantigens has strong specificity, no central tolerance and autoimmunity problems, and few side effects, thus achieving personalized precision immunotherapy. In recent years, the results of multiple basic research and clinical trials have shown that neoantigen vaccines are safe and effective in treating a variety of cancers, have broad application prospects, and can serve as a new breakthrough in cancer immunotherapy. However, due to the enrichment of immunosuppressive cells in the HBV-CRLM microenvironment, limited infiltration and functional loss of antigen-presenting cells and effector immune cells, and significantly weakened antigen presentation signaling pathways and T cell surface receptor (T cell receptor, TCR) immune response signaling pathways, although the vaccination of neoantigen vaccines can induce the body to produce specific immune responses, the effector lymphocytes infiltrating into tumor tissues are still limited, and most of them are in an exhausted state and cannot effectively kill tumors. Therefore, while inducing the body's immune system to respond specifically and accurately identify tumors, different drugs are used in a coordinated sequential treatment to reshape the immune microenvironment and solve the problem of HBV-CRLM immune escape from multiple dimensions, which can further improve the efficacy of immunotherapy.

[0004] Tyrosine kinase (TK) is a class of tyrosine-specific protein kinases. As an important mediator of signal cascades, it plays a key role in controlling various biological processes such as cell growth, differentiation, adhesion and apoptosis. Dysregulation of TK signaling can lead to the occurrence and development of various malignant tumors such as colorectal cancer (CRC). Current studies have shown that a variety of tyrosine kinases (TK) are significantly overexpressed in the microenvironment of HBV-CRLM. On the one hand, they jointly induce abnormal growth of blood vessels in the tumor site, hindering drug delivery and infiltration of effector immune cells. On the other hand, TK can inhibit the maturation and antigen presentation ability of dendritic cells (DC), inhibit the proliferation, differentiation and cytotoxicity of effector immune cells, and recruit immunosuppressive cells such as M2-like tumor-associated macrophages (M2-like TAMs), tumor-associated fibroblasts (CAFs), regulatory T cells (Tregs), and bone marrow-derived macrophages (MDSCs), and upregulate the immune checkpoint PD-L1, thereby forming a complex immunosuppressive microenvironment of HCC-CRLM in multiple dimensions and ultimately leading to its immune escape. Tyrosine kinase inhibitors (TKIs) can effectively block tyrosine kinase activity, inhibit cell signal transduction, and jointly induce tumor vascular normalization, promote the maturation and proliferation of DC cells and effector immune cells, and inhibit the enrichment and function of immunosuppressive cells, thereby achieving the effect of inhibiting tumor cell growth and proliferation.

[0005] Therefore, while inducing the body to produce tumor-specific immune responses through neoantigen vaccines, combining multi-target inhibitors to increase tumor-specific immune cell infiltration and reprogram the immunosuppressive microenvironment, thereby blocking the immune escape of HBV-CRLM in multiple dimensions, will be the future direction of precision immunotherapy for HBV-CRLM. Summary of the invention

[0006] The present invention provides a new combined immunization strategy based on neoantigen vaccines by identifying and screening CRLM tumor neoantigens, synthesizing neoantigen polypeptide vaccines, and combining the use of tyrosine kinase inhibitors TKIs, which solves the problems of poor tumor specificity, low immune cell infiltration and exhaustion in existing immunotherapy.

[0007] Neoantigens are a type of tumor-specific antigens that are specifically recognized by T cell surface receptors (TCR), thereby inducing the body to specifically kill tumor cells. Immunotherapy targeting neoantigens has the advantages of strong specificity, no central tolerance and autoimmunity problems, few side effects, and can achieve personalized precision immunotherapy. However, in the microenvironment of HBV-CRLM, multiple TKs are highly expressed, blood vessels grow abnormally in the tumor site, drug delivery and effector immune cell infiltration are hindered, and DC maturation and antigen presentation ability, effector immune cell proliferation and differentiation, and cytotoxicity are all affected. Immunosuppressive cells are enriched and immune checkpoints are upregulated, which together constitute a highly immunosuppressive microenvironment and mediate the immune escape of CRLM.

[0008] Therefore, in order to achieve the above objectives, the present invention combines tumor neoantigens with tyrosine kinase inhibitors TKIs to inhibit tyrosine kinase TK through multiple targets, reshape the immune microenvironment, and further enhance the efficacy of neoantigen vaccines against HBV-CRLM.

[0009] Preferably, the TKIs are selected from EGFR / VEGFR / PDGFR / FGFR / RET / KIT / TIE / DDR / BRAF

[0010] / CSF1R / HER inhibitors or one or more.

[0011] Preferably, the TKIs are selected from the following ingredients: one or more of regorafenib, lenvatinib, bevacizumab, and cetuximab.

[0012] Preferably, the TKI is regorafenib.

[0013] Advantageously, regorafenib is an oral multi-target TKI. Recent studies have shown that in addition to inhibiting related signal transduction, jointly inducing normalization of tumor blood vessels, promoting the maturation and proliferation of DCs and effector immune cells, and inhibiting the enrichment and function of immunosuppressive cells, regorafenib is the only TKI that can reduce the expression of PD-L1 in tumor cells by inhibiting pathways such as IFN-γ / STAT while upregulating MHC expression. Neoantigen vaccines combined with regorafenib can induce Rgs2+CD8+T cells with strong cytotoxicity, further enhancing the anti-tumor effect of neoantigen vaccine combined with immune strategy in CRLM.

[0014] Rgs2+CD8+T cells play an important role in tumor immunotherapy. G protein signaling regulator 2 (Rgs2) is a regulatory factor of G protein-coupled receptors. It can promote signal transduction by antagonizing G protein Gaq subunit coupled receptors. Through its GTPase activity, it reduces the level of intracellular adenylate diphosphatase (cAMP) during signal transduction, thereby regulating immune cell activation, migration and cytokine secretion. Tumor-infiltrating Rgs2+CD8+T cells have a high degree of activation and proliferation ability. They can bind to tumor antigens expressed on the surface of tumor cells through their surface TCR and kill tumor cells by secreting cytotoxins and chemokines. In addition, Rgs2+CD8+T cells regulate the tumor-targeted migration and full activation of immune cells such as B lymphocytes and macrophages by regulating factors such as cytoskeleton dynamics and calcium ion concentration, thereby further increasing the synergistic tumor killing ability.

[0015] Compared with the prior art, the present invention has the beneficial effect that after treatment with tumor neoantigen combined with inhibitor, mice can obtain the highest long-term complete tumor remission rate with high safety. In-depth research has found that combined treatment can induce downregulation of PD-L1 expression on the surface of tumor cells and upregulation of MHC expression, a large number of neoantigen-specific lymphocytes infiltrate into tumor tissues and remain activated, and B cells, effector CD8+T cells, DC cells, etc. in the microenvironment increase significantly, and immunosuppressive cells such as Treg, mast cells, and CAF decrease significantly. In addition, combined therapy can induce a class of effector immune cells Rgs2+CD8+T cells that have strong cytotoxicity and can regulate the recruitment of immune cells such as B cells to the tumor site to synergistically kill tumors, exerting a strong anti-tumor effect. At the same time, we found that the hepatitis B surface antigen (HbsAg) and hepatitis B DNA load (HBV-DNA) in the blood of HCC-CRLM patients / mice that underwent combined treatment dropped significantly to below the detection level, and the HBsAg expression level in liver biopsy tissue was significantly downregulated, indicating that this regimen can exert a strong and lasting anti-HBV virus ability while fighting CRLM, and may even be expected to achieve clinical cure standards, thereby preventing and treating the occurrence and development of colorectal metastasis by improving the highly inhibitory liver microenvironment caused by chronic HBV inflammatory stimulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The process of identifying neoantigen epitopes in the mouse colorectal cancer cell line MC38;

[0017] Figure 2 ELISPOT verifies the immunogenicity of potential neoantigen peptides;

[0018] Figure 3 Direct cross-immunogenicity analysis of 7 identified neoantigen peptides and their wild-type counterparts;

[0019] Figure 4 To evaluate the efficacy of RegoNeo, an immune strategy combining neoantigen peptide vaccine and regorafenib, based on the HBV-CRLM mouse model; (A) Schematic diagram of the process of RegoNeo immune strategy for treating HBV-CRLM; (B) Small animal in vivo imaging system monitors tumor activity in mice receiving different therapies;

[0020] Figure 5 Biosafety assessment of RegoNeo immune strategy for treating MSS-CRLM mice; (AM) Peripheral serum biochemical detection of biochemical indicators (ALB, TBil, ALT, AST, γ-GGT, AKP, CK, Urea, SCr, Glu, TG, TCHO) of MSS-CRLM mice receiving different immunotherapies. (N) Pathological evaluation of the effect of RegoNeo immune strategy on the main organs of MSS-CRLM mice, scale 100μm (10×), 40μm (40×); (O) Body weight monitoring to evaluate the effect of different immunotherapies on the growth of MSS-CRLM;

[0021] Figure 6 Immunofluorescence analysis of the effects of different immunotherapies on CD4 + and CD8 + Effect of T cell infiltration, scale bar, 100 μm (10×), 40 μm (40×);

[0022] Figure 7 Flow cytometry was used to evaluate the effects of different immunotherapies on TILs activation (PD-1 and 4-1BB expression). Data were expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant. Different significance levels were divided into: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0023] Figure 8 Flow cytometry was used to evaluate the effects of different immunotherapies on the expression of MHCI on the surface of tumor tissues (CRLM cells / HBV-infected hepatocytes). Data were expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant. Different significance levels were divided into: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0024] Fig. 9 To evaluate the effects of different immunotherapies on the expression of PDL1 on the surface of tumor tissues (CRLM cells / HBV-infected hepatocytes) by flow cytometry;

[0025] Fig.10 Slc201a:H-2K b(Ptpn2) tetramer flow cytometry analysis was used to evaluate the induction of neoantigen-specific TILs by different immunotherapies; data were expressed as mean ± standard deviation, and p < 0.05 was considered statistically significant. Different significance levels were divided into: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0026] Fig.11 Single-cell transcriptome sequencing showed that combined therapy can induce the enrichment of Rgs2+CD8+T cells in tumor tissues;

[0027] Fig.12 Evaluation of the tumor killing ability of Rgs2+CD8+T cells in vivo and in vitro; (A) Schematic diagram of in vitro killing evaluation based on MC38 cell line; (BC) ELISA detection of IFN-γ and GZMB secretion by Rgs2+CD8+T cells after tumor cell stimulation; (D) Flow cytometry analysis of the ability of Rgs2+CD8+T cells to induce tumor cell apoptosis; (E) Schematic diagram of the evaluation of the killing ability of Rgs2+CD8+T cells in vivo; (F) Small animal in vivo imaging system to evaluate the anti-tumor ability of Rgs2+CD8+T cells in vivo;

[0028] Fig.13 The effect of different treatment regimens on peripheral blood HBsAg was evaluated by immunoenzyme-linked sorbent assay (ELISA);

[0029] Fig.14 To evaluate the effect of different treatment regimens on peripheral blood HBV DNA load by fluorescence quantitative PCR;

[0030] Fig.15 To detect the effect of different treatment regimens on the expression of HBsAg in liver tissue of HBV-CLRM by immunohistochemistry;

[0031] Fig.16 To investigate the regression of liver metastases in patients with HBV-CRLM who received neoantigen vaccine combined with regorafenib therapy;

[0032] Fig.17 To observe the changes in serum HBV-DNA / HbsAg during follow-up in patients with HBV-CRLM who received neoantigen vaccine combined with regorafenib therapy. DETAILED DESCRIPTION

[0033] The invention is further described below through examples in combination with the accompanying drawings. The examples are only used to further explain the technical solution of the present invention and cannot be considered as limiting the scope of protection of the present invention. Non-essential improvements or adjustments made by technicians in this field based on the contents of the above invention all fall within the scope of protection of the present invention.

[0034] HBV-C57BL / 6 is an experimental mouse model for studying human hepatitis B virus (HBV) infection. This mouse model was established by transfecting human hepatitis B virus (HBV) into C57BL / 6 mice.

[0035] MC38 cell line is a mouse colon cancer cell line.

[0036] Example 1 Identification, screening and validation of tumor neoantigen polypeptides of mouse colorectal cancer cell line MC38

[0037] 1. Identification and screening of mouse tumor neoantigens. The specific process is as follows: Figure 1 As shown, the following steps are included:

[0038] Step A1. Extract DNA and RNA from tail somatic cells of C57BL / 6 mice and colorectal cancer cell line MC38;

[0039] Step A2. Perform whole exome sequencing on DNA of C57BL / 6 mouse tail somatic cells and colorectal cancer cell line MC38 to obtain WES, and perform transcriptome sequencing on RNA of C57BL / 6 mouse tail somatic cells and colorectal cancer cell line MC38 to obtain RNA-seq;

[0040] Among them, the region of the gene that contains the "instructions" of the protein synthesis code is called "exon", and all the exons in the gene are collectively referred to as "exome". Whole exome sequencing refers to the use of sequence capture technology to capture and enrich the DNA in the exon region of the whole genome and then perform high-throughput sequencing; in this example, the SureSelect XT Mouse Whole Exon Kit was used for DNA library preparation and whole exon capture;

[0041] The transcriptome is a collection of all RNAs transcribed by specific tissues or cells at a certain developmental stage or functional state, mainly including mRNA and non-coding RNA (ncRNA). Transcriptome sequencing refers to the use of second-generation high-throughput sequencing technology for cDNA sequencing to comprehensively and quickly obtain almost all transcripts of a specific organ or tissue of a species in a certain state.

[0042] Step A3. The raw fastq data of DNA and RNA sequencing are quality controlled by FastQC software to obtain the data after quality control filtering, and the candidate neoantigens are identified by the following process:

[0043] (1) Using the GATK pipeline based on the WES obtained in step A2 and the C57BL / 6 mouse genome as a control, the somatic mutations of MC38 were identified to obtain a VCF file containing the mutations;

[0044] "https: / / www.docin.com / p-2446844586.html" provides the "Most Detailed Instructions on How to Use GATK for plob";

[0045] GATK is the abbreviation of Genome Analysis Toolkit, which is a set of software used to process high-throughput sequencing data. The use process of Gatk consists of 3 steps: raw data processing, variant detection, and filtering quality control;

[0046] Processing of raw data: Quality control of the data after being downloaded from the sequencer, removal of low-quality short sequences (reads), alignment of the filtered reads to the reference genome, and generation of a BAM format alignment file;

[0047] Mutation detection: The C57BL / 6 mouse genome was created as a panel of normal, and the mutations of the colorectal cancer cell line MC38 were screened using the Mutect2 algorithm of GATK. The basic principle of the mutect2 algorithm is to divide the active region according to the Normal panel, reassemble the sequences in the region, establish a similarity matrix, determine the genotype, and thus obtain mutation information;

[0048] (2) Call VarScan to detect the somatic mutations of MC38 obtained in step (1) and detect the variant allel frequency (VAF) based on WGS / WES and RNA-seq sequencing data, call the Kallisto tool to calculate the mutant gene expression value TPM, and only retain mutations with VAF>10% and sequencing depth>20×; when the sequencing depth is 20×, the coverage of the exon region by reads is close to saturation. The full name of VAF is Variant Allele Frequency or Variant Allele Fraction, which is the ratio of the read coverage depth supporting the alternate / mutant allele at a certain site in the genome to the total read coverage depth of this site. VAF>10% is used to filter out background noise.

[0049] (3) using wANNOVAR software to annotate the mutations obtained in step (2) and screen out mutations that change the amino acid sequence, i.e., non-synonymous mutations;

[0050] (4) In order to obtain high-quality mutations for identifying new antigens, mutations with less than 20 variant allele reads in step (3) were first excluded, and then mutations with VAF>60% were discarded;

[0051] (5) The bam2R algorithm in the R package DeepSNV (v1.24.0) was used to further verify all mutations retained in step (4), and mutations with sequencing depth ≥ 20× and VAF ≥ 10% in the RNA-seq data and TPM> 1 were retained to obtain the amino acid sequence of the mutant polypeptide after mutation;

[0052] (6) Use NetMHCpan to evaluate the immunogenicity of all mutations retained after validation in step (5), and select those that produce H2-K b 20 peptides were synthesized as candidate neoantigens using mutant peptides of 9 amino acids with allele affinity IC50 < 500 nm. Table 1 shows the information of candidate tumor neoantigens obtained in step (6).

[0053] Table 1

[0054]

[0055] 2. Immunogenicity assessment of neoantigen peptides in C57BL / 6 mice, including the following steps:

[0056] Step B1. Each candidate neoantigen polypeptide synthesized above was dissolved in DMSO (100 μg / 200 ml), and the neoantigen polypeptide was inoculated into normal C57BL / 6 mice by subcutaneous injection.

[0057] Step B2. On day 14, the mice were killed and spleen single cell suspension was extracted according to the following process:

[0058] (1) Isolate the spleen, draw PBS with a syringe, and puncture the spleen parenchyma to obtain spleen flushing fluid;

[0059] (2) Collect the washing fluid, centrifuge at 800g for 5 minutes, discard the supernatant, and resuspend in PBS;

[0060] (3) Slowly add Ficoll separation solution to the suspension, place it in a horizontal centrifuge for density gradient centrifugation, and collect the middle cloud layer cells;

[0061] Among them, Ficoll separation fluid is lymphocyte separation fluid, which is a commonly used reagent for separation and purification of cells based on cell density differences and the gravitational acceleration generated by centrifugation. The volume, morphology and density of mononuclear cells and monocytes in peripheral blood are different from other cells. Lymphocyte separation fluid is a solution with a density between 1.075 and 1.090 and is approximately isotonic. It is used for density gradient centrifugation to make cells of a certain density distributed according to the corresponding density gradient, thereby separating various blood cells.

[0062] (4) Add the cloud layer cells to a centrifuge tube containing 5 volumes of PBS and centrifuge at 800G for 5 minutes;

[0063] (5) Discard the supernatant, resuspend in red blood cell lysis buffer (Gibco), lyse the red blood cells for 2-4 minutes, and centrifuge at 800G for 5 minutes;

[0064] Red blood cell lysis buffer (Gibco) is the simplest and most convenient method to remove red blood cells, that is, using lysis buffer to lyse red blood cells. It does not damage nucleated cells and can fully remove red blood cells. The tissue cells obtained by lysis with red blood cell lysis buffer do not contain red blood cells and can be further used for primary culture, cell fusion, flow cytometry analysis, separation and extraction of nucleic acids and proteins, etc.

[0065] (6) Discard the supernatant and resuspend in PBS.

[0066] Step B3. Use the mouse interferon-γ ELISPOT assay kit (Mouse INF-γ ELISPOT plus kit, 3321-4APW-10) of Mabtech, Sweden, according to the following process. Figure 2 The potential new antigen peptides shown were evaluated for immunogenicity of new antigen peptides to obtain Figure 3 The identification results shown are:

[0067] (1) Dendritic cells (DCs) loaded with peptides

[0068] 1) Pre-treat the culture plate: wash with PBS 4 times, 200 μL each time. Then add 200 μL / well of culture medium containing 10% FBS into the wells of the culture plate and incubate at 37°C for 2 hours;

[0069] 2) The DCs concentration was adjusted to 3 × 10 using a medium containing 10% FBS and GM-CSF and IL-4. 5 / mL, added into the wells of the culture plate;

[0070] 3) Loading DCs with polypeptides: Dissolve each synthesized polypeptide in DMSO and adjust the concentration to 4 μg / well. Add one polypeptide to each well containing DCs in each culture plate, and add each polypeptide to two wells;

[0071] 4) Place the culture plate at 37°C, 5% CO 2 Incubate in an incubator for 12-48 hours. Do not move the culture plate during this period and avoid evaporation;

[0072] (2) DC-T cell co-incubation: The spleen T cells collected in step B2 were counted and the cell density was adjusted to 3×10 using a culture medium containing 10% FBS. 6 / mL, add 100 μL / well into the corresponding wells in the culture plate, and co-culture with DCs for 48 h, so that the DCs loaded with polypeptides stimulate memory T cells;

[0073] (3) ELISPOT assay: Prepare and filter: 0.5% FBS (PBS), ddH 2 O.

[0074] 1) Aspirate the cells in the wells and wash them 3-5 times with PBS at 200 μL / well;

[0075] 2) Dilute R4-6A2-biotin antibody in 0.5% FBS to a concentration of 1 μg / mL, add 100 μL / well, and incubate at room temperature for 2 hours;

[0076] 3) Aspirate the liquid and then wash 3-5 times with PBS at 200 μL / well;

[0077] 4) Dilute serum alkaline phosphatase (ALP) in 0.5% FBS to a concentration of 1 μL / mL, add 100 μL / well, and incubate at room temperature in the dark for 1 hour;

[0078] 5) Aspirate the liquid and then wash 3-5 times with PBS at 200 μL / well;

[0079] 6) Add 100 μL / well of ready-to-use colorimetric solution, observe the color development, and then rinse with filtered ddH 2 O stops color development; the ready-to-use color development solution is provided in the mouse interferon-γ ELISpot assay kit.

[0080] 7) Enzyme-linked immunosorbent spot image analyzer (AT-Spot-2200) was used to acquire and analyze spot images, and seven neoantigen peptides with the strongest immunogenicity and no obvious cross-reaction with their wild-type peptides were screened out, such as Figure 3 shown.

[0081] The principle of Elispot assay is that cells produce cytokines locally after being stimulated by antigens, and these cytokines are captured by specific monoclonal antibodies. After cell decomposition, the captured cytokines bind to the biotin-labeled secondary antibody, and then to the alkaline phosphatase-labeled avidin. After incubation with BCIP / NBT substrate, "purple" spots appear on the PVDF plate, indicating that the cells produce cytokines. The results are obtained by analyzing the spots with the ELISPOT enzyme-linked spot analysis system.

[0082] Example 2 Evaluation of the efficacy of the immunization strategy RegoNeo of neoantigen peptide vaccine combined with regorafenib based on the HBV-CRLM mouse model

[0083] Construction of HBV-CRLM mouse model: 6-8 week old HBV-C57BL / 6 mice were selected and intraperitoneally anesthetized with 1% sodium pentobarbital (80 mg / kg) before surgery and then tied. The mouse's abdominal hair was removed, and a 2.5-3 cm long longitudinal incision was made from the xiphoid process of the mouse along the linea alba to enter the abdominal cavity. The superior mesenteric vein was fully exposed with a moist cotton swab, and 5×10 cells of the colorectal cancer cell line MC38 (Luc-MC38) with a luciferase reporter gene (luciferase) were slowly injected into the mouse with an insulin syringe. 5 / 40μL was injected into the superior mesenteric vein. After injection, the needle was quickly withdrawn and pressed with a dry cotton swab for 5 minutes to avoid cell reflux. After the injection, the incision was sutured layer by layer and disinfected. The HBV-CRLM mouse model was obtained. Tumor activity was regularly monitored using a small animal in vivo imaging system. When the tumor activity reached 2-4×10 6 p / s / cm 2 / sr was recorded as day 0.

[0084] Efficacy evaluation: On day 0, the HBV-CRLM mouse model was randomly divided into 4 groups. The PBS group and the neoantigen vaccine group (NeoVAC) were subcutaneously injected with PBS / neoantigen vaccine (100 μg / strip, 7 peptides totaling 700 μg, dissolved in 200 μL DMSO, respectively, Tdg_H145Y, Slc20a1_T425I, Napg_P257L, Kat6a_S1773) on days 0, 4, and 8, respectively. F, Farp2_Q101L, Flrt2_N240T, Lap3_L334F) (both sides of the axilla), the regorafenib group (Rego) was treated with regorafenib (10 mg / kg) (oral) on days 0, 2, 4, 6, 8, 10, and 12, and the combined treatment group was treated with neoantigen vaccine + regorafenib (NeoVAC + Rego) according to the above method (neoantigen vaccine subcutaneous injection, regorafenib oral), the above process is as follows Figure 4 As shown in A. Small animal in vivo imaging is used to monitor tumor activity to evaluate therapeutic efficacy, such as Figure 4 As shown in B. All mice in the PBS group died, the survival rate of mice in the neoantigen vaccine group (NeoVAC) was 2 / 5, the survival rate of mice in the regorafenib group (Rego) was 2 / 5, and all mice in the combined treatment group (NeoVAC+Rego) survived.

[0085] Example 3 Biosafety Assessment of RegoNeo Immunization Strategy for the Treatment of MSS-CRLM Mice

[0086] Safety assessment: The mice were weighed every 2 days during the monitoring period. The heart, liver, spleen, lung, kidney and other solid organs of HBV-CRLM mice treated with different regimens were embedded and sectioned, and then HE staining was performed to evaluate tissue damage. The mouse serum was collected for biochemical testing to evaluate the safety of the neoantigen vaccine combined with regorafenib immunization strategy. The results are as follows: Figure 5 As shown, the results of tissue HE staining and serum biochemical tests confirmed that the RegoNeo immunization strategy would not cause acute organ damage and significant changes in biochemical indicators, indicating that the immunization strategy has good safety.

[0087] Example 4 Immunofluorescence evaluation of different immunotherapies on tumor tissue CD4 + and CD8 + The effect of T cell infiltration was investigated using the Maixin Bio ready-to-use immunofluorescence EliVisionTM plus kit. The experimental steps and methods are as follows:

[0088] (1) Baking: Place the tumor tissue slices in a 60°C oven and bake for 2 hours;

[0089] (2) Dewaxing: Use xylene I, xylene II, and xylene III for dewaxing, each for 10 min;

[0090] (3) Dewaxing to water: soak in gradient alcohol 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol and 55% ethanol for 10 minutes;

[0091] (4) Antigen repair: Select the antigen repair method (sodium citrate repair or EDTA alkali repair) according to the antibody instructions. Soak the tumor tissue sections in the repair solution and place them in the high pressure sterilizer. Add until the pressure cooker sprays air. Time the repair for 3-5 minutes. After the end, turn off the heat and leave the heat source. Cool naturally to room temperature. Wash twice with distilled water, then wash three times with PBS, each for 3 minutes.

[0092] (5) Endogenous peroxidase blocking: Prepare fresh 3% H 2 O 2 After discarding the PBS on the slide, add 200 μL of 3% HO 2 O 2 solution, incubated at room temperature for 10 min, and then washed three times with PBS for 3 min each;

[0093] (6) Blocking: remove the PBS solution, add 5% BSA solution (prepared in PBS), block at room temperature for 20 minutes, and then wash with PBS three times, 3 minutes each time;

[0094] (7) Primary antibody: remove PBS, add CD4 / CD8 primary antibody solution prepared with 5% BSA, incubate overnight in a 4°C refrigerator, and wash with PBS three times, 3 minutes each time;

[0095] (8) Enhancer: remove PBS, add enhancer, incubate at room temperature for 20 min, then wash with PBS three times, 3 min each time;

[0096] (9) Secondary antibody: remove PBS, add goat anti-rabbit or anti-mouse fluorescent IgG labeled with horseradish peroxidase, incubate at room temperature for 20 minutes, and then wash with PBS three times, 3 minutes each time;

[0097] (10) Color development: Remove the PBS solution and add DAB color development solution (850 μL of ddHO 2 O, AC color development solution and substrate each add 50μL), color development for 2-10min, observe under a microscope until the tissue turns brownish yellow without obvious background coloring, remove the color development solution, ddH 2 O flushing terminates the reaction;

[0098] (11) Sealing: Seal the slides with anti-fluorescence fading sealing medium and then take photos.

[0099] like Figure 6 As shown, the neoantigen vaccine combined with regorafenib immunotherapy has the best effect on CD4+ and CD8+ T cell infiltration in tumor tissue.

[0100] Example 5 Flow cytometry evaluation of the effects of different immunotherapies on TILs activation (PD-1 and 4-1BB expression)

[0101] 1) Tumor tissue single cell suspension was centrifuged at 800 g for 5 minutes, the supernatant was discarded, and the suspension was resuspended in 5% BSA and incubated for 10 minutes;

[0102] 2) Centrifuge at 800 g for 5 minutes, discard the supernatant, and resuspend in 0.5% BSA;

[0103] 3) Add appropriate amount of anti-CD3-FITC (eBioscience TM , 17-0032-82 / 11-0037-42), anti-CD8-PE (eBioscience TM , 12-0081-82 / 11-0081-82), anti-CD137-APC (eBioscience TM , 17-1371-82), anti-CD279-PE-Cyanine7 (eBioscience TM, 25-9985-82) antibody staining at room temperature in the dark for 20 minutes; (the antibodies required for flow cytometry analysis are antibodies for selecting TIL (CD3 / CD8), antibodies for marking PD-1 and 4-1BB (137 and 279))

[0104] 4) Wash twice with PBS and resuspend in PBS;

[0105] 5) FACSverse flow cytometer analysis.

[0106] like Figure 7 As shown, the neoantigen vaccine combined with regorafenib immunotherapy is optimal for TILs activation, in which PD-1 and 4-1BB are highly expressed.

[0107] Example 6 Flow cytometry evaluation of the effects of different immunotherapies on the expression of PDL1 / MHCI on the surface of tumor tissues (CRLM cells / HBV-infected hepatocytes)

[0108] 1) Tumor tissue single cell suspension was centrifuged at 800 g for 5 minutes, the supernatant was discarded, and the suspension was resuspended in 5% BSA and incubated for 10 minutes;

[0109] 2) Centrifuge at 800 g for 5 minutes, discard the supernatant, and resuspend in 0.5% BSA;

[0110] 3) Add appropriate amount of anti-CD274-FITC (eBioscience TM , 15-0084-82 / 11-0045-73), anti-MHCI-PE (eBioscience TM , 12-0078-12 / 11-0042-54) antibody staining at room temperature in the dark for 20 minutes;

[0111] 4) Wash twice with PBS and resuspend in PBS;

[0112] 5) FACSverse flow cytometer analysis.

[0113] like Figure 8 and Fig. 9As shown, the neoantigen vaccine combined with regorafenib immunotherapy induced a decrease in the expression of PDL1 on the surface of tumor tissues (CRLM cells / HBV-infected hepatocytes) and an increase in the expression of MHCI. (Although NeoVAC can induce antigen-specific TIL, due to the stimulation of specific immunity, tumor cells will escape immunity by highly expressing PD-L1 and reducing MHCI. Rego will reduce PDL1 and increase MHCI. The combined use of NeoVAC and rego will increase TIL while reducing PD-L1 and increasing MHCI. The combined use of NeoVAC and rego is better than the PBS group and NeoVAC alone, and is close to the Rego alone group. There is no statistically significant difference between the Rego group and the combined use of NeoVAC and rego group.)

[0114] Example 7 Tetramer flow cytometry analysis to evaluate the induction of neoantigen-specific TILs by different immunotherapies was performed using the T-Select MHC Tetramer kit from Beijing Bomeier Biotechnology Co., Ltd. The experimental steps are as follows:

[0115] (1) Prepare a single cell suspension of tumor tissue according to the method of Example 6 above, and adjust the concentration to 1×10 6-7 / mL;

[0116] (2) Add 10 μL of Clear Back to 50 μL of cell suspension and react at room temperature for 5 minutes;

[0117] (3) Add 10 μL of T-Select MHC Tetramer-PE;

[0118] (4) Incubate at room temperature for 15-30 minutes or at 4°C for 30-60 minutes;

[0119] (5) Add CD8 and other antibodies and incubate at 4°C for 20 minutes;

[0120] (6) Add an appropriate amount of FCM buffer and centrifuge at 400 g for 5 minutes;

[0121] (7) Carefully remove the supernatant;

[0122] (8) The cells were resuspended in 500 μL of FCM buffer or 0.5% paraformaldehyde;

[0123] (9) FACSverse analysis.

[0124] like Fig.10 As shown, the neoantigen vaccine combined with regorafenib immunotherapy is optimal for inducing the production of neoantigen-specific TILs.

[0125] Example 8 Single-cell transcriptome sequencing shows that combined therapy can induce the enrichment of Rgs2+CD8+T cells in tumor tissues

[0126] Single cell libraries were prepared according to the instructions of (10×Genomics Chromium single cell 5'V3 reagent kit); Cell Ranger was used for comparison with default parameters; and Seurat was used to filter the mapped data to obtain qualified cells and UMIs. The filtering criteria are as follows: genes expressed by <3 cells; cells expressing <500 or >5000 genes; cells with mitochondrial genes >5% UMI; cells with the highest number of expressed genes exceeding 20% ​​UMI. After normalizing the gene expression levels, t-distributed stochastic neighbor embedding (t-SNE) or unified manipulative approximation and projection (UMAP) dimensionality reduction was performed, and cell clustering was performed using the Seurat function. Cell clusters were annotated using the Single R software package. Findmarker was used to identify differentially expressed genes (DEGs) between different samples or continuous clusters, and key cell subpopulations induced by the RegoNeo immune strategy were explored. GO functional enrichment analysis was performed based on clusterProfiler to find key functions or pathways, such as Fig.11 As shown. ( Fig.11 The vertical axis is the ratio without unit, 1.0 is equivalent to 100%)

[0127] Example 9 Evaluation of the tumor killing ability of Rgs2+CD8+T cells in vivo and in vitro

[0128] (1) In vitro cytotoxicity assay of Rgs2+CD8+T cells

[0129] like Fig.12As shown in A, the HBV-CRLM mouse model was treated with the NeoVAC+Rego immune strategy. The mice were killed on the 14th day and the tumor tissue was isolated to prepare a single cell suspension. The single cell suspension was centrifuged at 800g for 5 minutes, the supernatant was discarded, and the suspension was resuspended in 5% BSA. After incubation for 10 minutes, the suspension was centrifuged at 800g for 5 minutes, the supernatant was discarded, and the suspension was resuspended in 0.5% BSA. Antibodies such as anti-Rgs2, CD8, and CD3 were added, incubated in the dark for 20 minutes, washed twice with PBS, and resuspended in PBS. Rgs2+CD8+T cells and Rgs2-CD8+T cells were collected by flow cytometry sorter FACSAria and co-cultured with MC38 cell line for 48 hours (efficacy-target ratio was 10:1). Cells and supernatants were collected, and the cells were stained with anti-mouse CD3-APC antibody, Annexin V-FITC and PI. MC38 apoptosis was analyzed by flow cytometry, and the supernatant was used to detect the secretion of cytokines such as GZMB / IFN-γ by ELISA. GZMB (granzyme B) is a cytoplasmic serine protease expressed in cytotoxic T lymphocytes (CTL) and natural killer cells (NK), which can lyse target cells and induce target cell apoptosis. IFN-γ (interferon-γ) is a type II interferon produced by NK cells, NKT cells, CD8+T cells and CD4+T cells in natural and acquired immune responses. IFN-γ has antiviral, antitumor and immunomodulatory effects, and has certain therapeutic effects on certain viral infections, melanoma, breast cancer, etc. The results are as follows Fig.12 B in Fig.12 C in Fig.12 As shown in D.

[0130] (2) Evaluation of the in vivo tumor killing ability of Rgs2+CD8+T cells

[0131] like Fig.12 As shown in A, the HBV-CRLM mouse model was treated with the NeoVAC+Rego immune strategy. The mice were killed on the 14th day and the tumor tissues were isolated to prepare single cell suspensions. The single cell suspension was centrifuged at 800g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 5% BSA. After incubation for 10 minutes, the cells were centrifuged at 800g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 0.5% BSA. Antibodies such as anti-Rgs2, CD8, and CD3 were added, incubated in the dark for 20 minutes, washed twice with PBS, and resuspended in PBS. Rgs2+CD8+T cells and Rgs2-CD8+T cells were collected by flow cytometry sorter FACSAria. The HBV-CRLM mouse model was constructed according to the above steps and divided into 3 groups, and PBS (200μL / mouse), Rgs2+CD8+T cells induced by the NeoVAC+Rego immune strategy, and Rgs2-CD8+T cells (3*10 5 / 200μL / mouse), and IL-2 (3×10 3 IU). Tumor activity was monitored using a small animal in vivo imaging system. Fig.12 As shown in F. Rgs2+CD8+T cells can effectively kill tumors.

[0132] Example 10 Evaluation of the effects of four treatment regimens, PBS / NeoVAC / Rego / NeoVAC+Rego, on peripheral blood HBsAg by ELISA

[0133] The C57BL / 6 mice with HBV-CRLM were randomly divided into four groups and treated with PBS / NeoVAC / Rego / NeoVAC+Rego using the above treatment regimen. The tumor activity was evaluated by small animal in vivo imaging, and the peripheral serum of the mice was collected by tail vein bleeding.

[0134] (1) Preparation and storage of reagents

[0135] 1) Dilution and use of standard: Prepare within 2 hours before use. The kit provides 2 tubes of standard, 10 ng per tube, and use 1 tube each time. The standard is HbsAg recombinant protein, and the sample is the serum of treated mice or humans;

[0136] (a) Prepare 10,000 pg / mL standard: add 1 mL of sample diluent to the standard tube, cover and let stand for more than 10 minutes, then invert / rub repeatedly to help dissolution;

[0137] (b) Prepare 1,000 pg / mL standard: add 100 μL of 10,000 pg / mL standard to 0.9 M sample diluent in an EP tube, mix well, and mark.

[0138] (c) Prepare 500pg / mL-15.6pg / mL standard: prepare 6 EP tubes, add 0.3mL of sample diluent to each tube, and label them 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.3pg / mL, and 15.6pg / mL. Take 0.3mL of 1,000pg / mL standard and add it to the tube labeled 500pg / mL. After mixing, take out 0.3mL and add it to the next tube. Repeat this process until the last sample tube.

[0139] (d) Note: The diluted standard (10,000 pg / mL) should be used within 12 hours. If stored at -20°C, it can be used within 2 days, but it should not be repeatedly frozen and thawed.

[0140] 2) Biotinylated antibody working solution: Prepare within 2 hours before use.

[0141] (a) Calculate the total volume based on 100 μL per well (add 100-200 μL when actually preparing);

[0142] (b) Prepare the working solution at a ratio of 1 μL of biotinylated antibody to 99 μL of antibody diluent and mix gently;

[0143] 3) Preparation of avidin-peroxidase complex (ABC) working solution: Prepare within 1 hour of use.

[0144] (a) The total amount is calculated based on the requirement of 100 μL per well (100-200 μL more should be prepared in actual preparation);

[0145] (b) Prepare the working solution at a ratio of 1 μL ABC to 99 μL ABC diluent and mix gently;

[0146] (2) Operation process

[0147] The diluted ABC and TMB colorimetric solutions should be pre-equilibrated at 37°C for at least 30 minutes before adding to the ELISA plate. When diluting reagents or samples, do not forget to mix them. A standard curve should be made for each test. The amount of the factor to be measured in the sample must be estimated to determine the appropriate dilution factor.

[0148] 1) Determine the number of wells on the ELISA plate that have been coated with antibodies required for this test, and add one well as a TMB blank color development well. Total = number of samples + 9; × 2 for duplicate tests, and re-wrap the rest and put them in the refrigerator;

[0149] 2) Combine 1,000pg / mL, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL,

[0150] 100 μL of each standard of 31.3 pg / mL and 15.6 pg / mL were added to 7 wells in a row, and 1 well with only sample diluent was used as zero well. For mouse serum, plasma, cell culture supernatant, and cell lysate, 100 μL of the sample diluted with sample diluent was directly added;

[0151] 3) Add sealing film to the ELISA plate and react at 37°C for 90 minutes;

[0152] 4) After the reaction, use an automatic plate washer to wash away the liquid in the ELISA plate, or shake off the liquid in the ELISA plate and tap it against absorbent paper a few times without washing;

[0153] 5) Add 100 μL of the prepared biotin antibody working solution to each well (except the TMB blank color development well). Add a sealing plate to the ELISA plate and react at 37°C for 60 minutes;

[0154] 6) Wash three times with 1× washing buffer, soaking for about 1 minute each time (at least 300 μL of washing buffer per well);

[0155] 7) Add 100 μL of the prepared ABC working solution to each well (except the TMB blank color development well).

[0156] Add a sealing plate to the ELISA plate and react at 37°C for 30 minutes;

[0157] 8) Wash 5 times with 1× washing buffer, soaking for about 1-2 minutes each time (at least 300 μL of washing buffer per well);

[0158] 9) Add 90 μL of TMB colorimetric solution to each well and react at 37°C in the dark for 15-20 minutes. Note: The color development time may vary. At this time, the first 3-4 wells of the standard sample can be seen with the naked eye to have a clear gradient of blue, while the difference in the last 3-4 wells is not obvious;

[0159] 10) Add 100 μL of TMB stop solution to each well, and the blue color will turn yellow immediately;

[0160] 11) Measure the OD value at 450nm using a Spectra Max M5 microplate reader; there are two options for setting blank controls:

[0161] (a) Set the TMB blank well (add only TMB colorimetric solution and stop solution) as the control. After subtracting the absorbance of the TMB blank well from the absorbance of all standards and samples, draw a curve on the coordinate paper with the absorbance as the ordinate and the concentration as the abscissa;

[0162] (b) Set the zero well as the control. After subtracting the absorbance value of the zero well from the absorbance values ​​of all standards and samples, the obtained data can be directly plotted on the coordinate paper.

[0163] 12) Find the corresponding concentration on the coordinates based on the absorbance of the sample. Remember that since the sample is diluted N times, its actual concentration should be ×N.

[0164] like Fig.13 As shown in the results, the neoantigen vaccine combined with regorafenib immunotherapy has a significant effect on reducing peripheral blood HBsAg.

[0165] Example 11 Fluorescence quantitative PCR method to evaluate the effects of different treatment regimens on peripheral blood HBV DNA load

[0166] The C57BL / 6 mice with HBV-CRLM were randomly divided into four groups and treated with PBS / NeoVAC / Rego / NeoVAC+Rego respectively using the treatment regimen of Example 2. The tumor activity was evaluated by small animal in vivo imaging, and the peripheral serum of the mice was collected by tail vein bleeding.

[0167] 1. Sample processing (sample processing area)

[0168] Take 100μl of the serum or plasma sample to be tested and the positive and negative reference substances in the kit, respectively, and add them to 0.5ml centrifuge tubes; add 100μl DNA extraction solution 1, shake and mix, centrifuge at 13,000rpm for 10 minutes, and discard the supernatant [pay attention to fix the direction of the centrifuge tube during centrifugation, and discard the supernatant as much as possible without touching the precipitate]; then add 25μl DNA extraction solution 2, shake for 10 seconds [the precipitate does not need to be broken up or mixed], dry bath or boiling water bath at 100℃ for 10 minutes, centrifuge at 13,000rpm for 10 minutes, and retain the supernatant. If the sample lysate is not used on the same day, it should be stored at -20℃.

[0169] 2. Preparation of amplification reagents (PCR preparation area)

[0170] Take out each tube of reagent from the kit and centrifuge briefly. Assume that the number of tubes required is n (n = number of samples + 2 tubes of negative control + 1 tube of strong positive control + 1 tube of critical positive control + 4 tubes of positive working standard [qualitative detection does not require positive working standard])

[0171] Calculate the amount of each reagent, add them to a centrifuge tube of appropriate volume, mix thoroughly, centrifuge at 2,000 rpm for 10 seconds, add the corresponding volume of reaction solution to the set n PCR reaction tubes (add 18μl to system I and 38μl to system II), and transfer them to the sample processing area.

[0172] 3. Sample addition (sample processing area)

[0173] If the sample lysate is stored at -20°C, thaw it at room temperature before use and centrifuge it at 12000rpm for 5 minutes. Add 2μl of the sample, negative control, strong positive control and critical positive control supernatant processed in step 1, and positive working standard [qualitative detection does not require positive working standard] to the set n reaction tubes, cover the PCR reaction tube cap tightly [when using Roche capillary, place the capillary together with the Roche special metal sleeve in the centrifuge and centrifuge at 4000rpm for 5 seconds], transfer to the detection area, and place it on the quantitative PCR instrument.

[0174] 4. PCR amplification (detection area)

[0175] 4.1. Loop condition settings

[0176] When using the RotorGene Q fluorescent PCR detector, the cycle program settings are as follows:

[0177] 37℃: 5min; 94℃: 1min;

[0178] 95℃: 5sec, 60℃: 30sec, 40 cycles. The reaction system is 20μl.

[0179] Use ABI 7000 / 7300 / 7500, LC480 (96well), Opticon,

[0180] The cycle program settings for LineGene fluorescent PCR detector are as follows:

[0181] 37℃: 5min; 94℃: 1min;

[0182] 95℃: 5sec, 60℃: 30sec, 40 cycles. The reaction system is 40μl.

[0183] When using the iCycler fluorescent PCR detector, the cycling program settings are as follows:

[0184] 37℃: 5min; 94℃: 1min;

[0185] 95℃: 5sec, 60℃: 30sec, 42 cycles. The reaction system is 40μl.

[0186] When using the LC1.2 / 2.0 fluorescent PCR detector, the cycle program settings are as follows:

[0187] 37℃: 3min; 92℃: 1min;

[0188] 92℃: 5sec, 60℃: 30sec, 40 cycles. The reaction system is 20μl.

[0189] 4.2. Instrument detection channel selection

[0190] The multi-fluorescence detector was set to Fam fluorescein for fluorescence signal collection. Fluorescence signal collection was set at 60°C.

[0191] LC1.2 / 2.0 fluorescence PCR detector, instrument channel select F1 channel. Fluorescence signal collection is set at 60℃, and collection mode is set to SINGLE.

[0192] For the specific cycle program setting methods of other fluorescent PCR detectors, please refer to the above setting methods and the instruction manuals of various instruments and make appropriate adjustments according to actual conditions.

[0193] 4.3. Sample settings:

[0194] When setting the conditions before amplification, the HBV positive working standard is set to "Standard"

[0195] And enter the given concentration value in the kit; the sample to be tested and the reference substance are set to "Unknown" or "Sample".

[0196] like Fig.14 As shown in the data, the neoantigen vaccine combined with regorafenib immunotherapy has a significant effect on reducing the HBV DNA load in peripheral blood.

[0197] Example 12 Immunohistochemical detection of the effects of different treatment regimens on the expression of HBsAg in liver tissue of HBV-CLRM

[0198] Using the Maixin Bio ready-to-use immunofluorescence EliVisionTM plus kit, the experimental steps and methods are as follows:

[0199] (1) Baking: Place the liver tissue slices in a 60°C oven and bake for 2 hours;

[0200] (2) Dewaxing: Use xylene I, xylene II, and xylene III for dewaxing, each for 10 min;

[0201] (3) Dewaxing to water: soak in gradient alcohol 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol and 55% ethanol for 10 minutes;

[0202] (4) Antigen repair: Select the antigen repair method (sodium citrate repair or EDTA alkali repair) according to the antibody instructions. Soak the liver tissue sections in the repair solution and place them in a high pressure sterilizer. Add until the pressure cooker sprays air. Time the repair for 3-5 minutes. After the repair is completed, turn off the heat and leave the heat source. Cool naturally to room temperature. Wash twice with distilled water, then wash three times with PBS, each for 3 minutes.

[0203] (5) Endogenous peroxidase blocking: Prepare fresh 3% H 2 O 2 After discarding the PBS on the slide, add 200 μL of 3% HO 2 O 2 solution, incubated at room temperature for 10 min, and then washed three times with PBS for 3 min each;

[0204] (6) Blocking: remove the PBS solution, add 5% BSA solution (prepared in PBS), block at room temperature for 20 minutes, and then wash with PBS three times, 3 minutes each time;

[0205] (7) Primary antibody: remove PBS, add 5% BSA-containing HBsAg primary antibody solution, incubate overnight in a 4°C refrigerator, and wash three times with PBS for 3 minutes each time;

[0206] (8) Enhancer: remove PBS, add enhancer, incubate at room temperature for 20 min, then wash with PBS three times, 3 min each time;

[0207] (9) Secondary antibody: remove PBS, add goat anti-rabbit or anti-mouse fluorescent IgG labeled with horseradish peroxidase, incubate at room temperature for 20 minutes, and then wash with PBS three times, 3 minutes each time;

[0208] (10) Color development: Remove the PBS solution and add DAB color development solution (850 μL of ddHO 2 O, AC color development solution and substrate each add 50μL), color development for 2-10min, observe under a microscope until the tissue turns brownish yellow without obvious background coloring, remove the color development solution, ddH 2 O flushing terminates the reaction;

[0209] (11) Counterstaining: Use hematoxylin solution for 30 seconds in ddH 2 After O rinsing, reverse blue for 30 seconds;

[0210] (12) Dehydration in graded alcohol (55% ethanol to 100% ethanol), 3 minutes each time;

[0211] (13) Sealing: After air drying, use neutral gum to seal the slides and observe under a microscope.

[0212] like Fig.15 As shown in the results, the neoantigen vaccine combined with regorafenib immunotherapy significantly reduced the expression of HBsAg in the liver tissue of HBV-CLRM.

[0213] Embodiment 13

[0214] Identification and screening of neoantigens in colorectal cancer patients

[0215] Step X1. extracting DNA and RNA from tumor tissue and somatic cell tissue (such as paracancerous tissue) of a colorectal cancer patient obtained by puncture;

[0216] Step X2. Perform whole exome and transcriptome sequencing on DNA and RNA of tumor tissue and adjacent paracancerous tissue of colorectal cancer patients, respectively;

[0217] Step X3. Use SureSelect XT to construct a DNA library of colorectal cancer patient paracancerous tissue and perform whole genome sequencing;

[0218] (1) using the GATK process to identify somatic mutations in the tumor tissues of colorectal cancer patients based on whole exome sequencing data of tumor tissues of colorectal cancer patients, with the genome of adjacent tissues of colorectal cancer patients as a control; calling VarScan software to detect somatic mutations in the tumor tissues of colorectal cancer patients obtained in step (1), and based on WGS / WES and RNA-seq sequencing data, detecting variant allel frequency (VAF), calling Kallisto tool to calculate mutant gene expression value TPM, and only retaining mutations with variant allele frequency (VAF)>10% and sequencing depth>20×;

[0219] (2) wANNOVAR was used to annotate mutations and screen out mutations that changed the amino acid sequence, i.e., non-synonymous mutations;

[0220] (3) To obtain high-quality mutations for identifying neoantigens, mutations with less than 20 variant allele reads were excluded, and then mutations with VAF>60% were discarded;

[0221] (4) The bam2R algorithm in the R package DeepSNV (v1.24.0) was used to further verify all mutations retained in step (3), and mutations with sequencing depth ≥20× and VAF ≥10% in the RNAseq data and TPM>1 were retained to obtain the amino acid sequence of the mutant polypeptide after mutation;

[0222] (5) Using NetMHCpan to evaluate the immunogenicity of all mutations retained after verification in step (4), a mutant polypeptide with a length of 9 amino acids and an HLA affinity IC50 < 150 nm was selected as a candidate neoantigen to synthesize a candidate neoantigen polypeptide.

[0223] 2. Construction of vectors expressing neoantigens:

[0224] (1) Prepare 50 μl of enzyme digestion system (containing 13 μL PresentER-Cassette mCherry plasmid, 1 μL Sfil enzyme, 5 μL Q.Cut Buffer (10X) and 31 μL double distilled water) and shake to mix, then digest in a 37°C dry incubator for 1 hour. After the digestion, add calf intestinal alkaline phosphatase (CIP) to the mixture, and finally perform electrophoresis to recover the PresentER-Cassette mCherry plasmid.

[0225] (2) Artificially synthesizing a nucleic acid sequence encoding one or more polypeptides, and using the nucleotide sequence as a template to perform PCR amplification using upstream / downstream primers.

[0226] The PCR amplification reaction system was: 10 ng of synthetic sequence, 2 μL of upstream primer, 2 μL of downstream primer and 25 μL of Takara PrimerSTAR Max DNA Polymerase (PCR reagent), and finally the system was supplemented to 50 μL with double distilled water.

[0227] (3) Then, the target nucleotide sequence and the empty plasmid were connected: 96 μL of ethidium bromide (EB) was added to 2 μL of the nucleotide sequence and its complementary chain, mixed well and placed in hot water, and then the temperature was gradually reduced to room temperature. The target DNA fragment was digested with 50 μL of the system (5 μL of the target DNA fragment, 1 μL of Sfil enzyme, 5 μL of Q.Cut Buffer (10X) and 39 μL of double distilled water) and inactivated at 70°C for 10 minutes. Then, 10 μL of the system (3 μL PresentER-Cassette mCherry plasmid, 2 μL of the target DNA fragment, 0.5 μL T4 ligase, 1 μL T4 ligase Buffer (10X) and 8.5 μL of double distilled water) was used for connection. The connection was carried out at room temperature for 1 hour. (4) Transform the ligation product: Place the competent E. coli DH5α on ice. After melting, add 30 μL of the competent E. coli to the ligation product in step (2) and mix gently. Incubate on ice for 30 minutes. Then heat shock the incubated mixture in a 42°C water bath for 60 seconds, and then incubate on ice for 5 minutes. Then add the mixture to 600 μL of non-resistant LB medium and culture it at 37°C for 2 hours on a constant temperature shaker at 220 rpm / min. Centrifuge the 1000 g bacterial solution at room temperature for 2 minutes. After removing 400 μL of supernatant, reselect the remaining liquid and evenly drop it on the LB solid plate preheated at 37°C. Use a heated coating rod to gently spread it evenly. Place it until there is no obvious liquid on the surface of the plate. Invert and culture it in a 37°C bacterial incubator for 14 hours.

[0228] (5) Selecting monoclonal colonies and performing small shaking verification: Add 8 mL of LB liquid culture medium (containing ampicillin) to a 15 mL centrifuge tube, pick a monoclonal colony from the LB plate cultured in step (3) in a clean bench, place it in the above LB liquid culture medium, and culture it at 37°C on a constant temperature shaker at 220 rpm / min for 13 h until the bacterial solution becomes turbid, then extract the target plasmid.

[0229] (6) Prepare 20 μL of enzyme digestion system (5 μL target plasmid (500 ng), 0.5 μL Q.Cut EcoRⅠ, 0.5 μL Q.CutHindⅢ, 2 μL Q.Cut Buffer (10X) and 12 μL double distilled water) and shake to mix, then digest in a 37°C dry incubator for half an hour. After digestion, verify the plasmid structure by running gel and sequencing.

[0230] The nucleotide sequence expressing the neoantigen was inserted into the PresentER-Cassette mCherry empty plasmid to obtain the DNA-encapsulated plasmid pDNA. The plasmid structure was determined by gene sequencing, and its nucleotide sequence is shown in SEQ ID.NO.1.

[0231] Neoantigen peptide vaccine combined with regorafenib in the treatment of colorectal cancer patients

[0232] 1. The candidate neoantigen peptide synthesized above was dissolved in DMSO and injected subcutaneously into the axillary and inguinal lymph nodes of colorectal cancer patients on days 1, 4, 8, 15, and 22 respectively;

[0233] And give ICIs treatment (including but not limited to regorafenib) 200 mg q3w, this is not exhaustive, as long as the framework of the method of the present invention is adopted, it is within the scope of protection of the present invention)

[0234] 2. CT or MRI monitoring of tumor size changes and peripheral blood HBV DNA load.

[0235] like Fig.16 Magnetic resonance imaging showed that the tumor size of colorectal cancer patients decreased after neoantigen vaccine combined with ICIs treatment. Fig.17 It shows that the neoantigen vaccine combined with ICIs treatment has a significant effect on reducing the HBV DNA load in the peripheral blood of patients with colorectal cancer.

Claims

1. A neoantigen composition for treating colorectal cancer liver metastasis, characterized in that: It includes an immune composition, a pharmaceutically acceptable carrier, and a carrier; the immune composition includes: a plasmid as an active ingredient, the plasmid is inserted with a nucleic acid sequence encoding one or more polypeptides, each polypeptide is a liver cancer tumor-specific antigen, and is a mutated new antigen expressed by the cancer cells of the liver cancer patient; the immune composition also includes a multi-target inhibitor or a functional fragment thereof.

2. The neoantigen composition according to claim 1, characterized in that The multi-target inhibitor is a tyrosine kinase inhibitor TKIs.

3. The neoantigen composition according to claim 2, characterized in that: The tyrosine kinase inhibitors TKIs are selected from one or more of EGFR / VEGFR / PDGFR / FGFR / RET / KIT / TIE / DDR / BRAF / CSF1R / HER inhibitors.

4. The neoantigen composition according to claim 3, characterized in that The components of the tyrosine kinase inhibitors TKIs include one or more of regorafenib, lenvatinib, bevacizumab, and cetuximab.

5. The neoantigen composition according to claim 4, characterized in that The tyrosine kinase inhibitor TKIs is regorafenib.

6. The neoantigen composition according to any one of claim 1, characterized in that: The polypeptide is H2-K b A sequence of 9 consecutive amino acids with an allelic affinity IC50 of no greater than 500 nM.

7. The neoantigen composition according to claim 6, characterized in that: The polypeptide is screened and synthesized by the following steps: S1. Obtain DNA and RNA from tumor tissues and somatic cells, construct sequencing libraries and perform whole genome sequencing of somatic cells, and simultaneously obtain WGS / WES and RNA-seq sequencing data of tumor tissues and somatic cells; S2. Using the somatic genome of tumor patients as a control, the DNA and RNA of tumor tissues are compared respectively, somatic mutations of tumor tissues are detected, the characteristic values ​​of somatic mutations of tumor tissues are detected, tumor tissue somatic cells are filtered out according to the characteristic values, non-synonymous mutations are filtered out from the somatic cells of tumor tissues and annotated; S3. Filter out tumor tissue somatic mutations again based on the characteristic values ​​and verify them, extract the MHC binding neoantigen sequences corresponding to the tumor tissue somatic mutations, calculate the polypeptide characteristic values, filter out candidate neoantigen sequences based on the polypeptide characteristic values, and synthesize candidate neoantigen polypeptides; The characteristic values ​​of the tumor tissue somatic mutations include gene mutation frequency VAF, mutant gene expression value TPM, and sequencing depth.

8. Use of the neoantigen composition as described in claims 1 to 7 in the preparation of a drug for treating hepatitis B.

Images