A screening method for tumor neoantigens for gastric cancer treatment and a preparation method and application of a novel nano vaccine

Abstract

The application discloses a tumor neoantigen screening method for stomach cancer treatment and a preparation method and application of a novel nano vaccine. The application uses single-cell sequencing technology to perform whole-exon (genome) sequencing on stomach cancer tissues and large analysis combining single-cell multi-omics (transcriptome), uses bioinformatics algorithm tools to analyze whole-exon and single-cell multi-omics sequencing data of stomach cancer tissues and stomach cancer paracancer tissues, obtains a group of potential neoantigen candidate sequences for stomach cancer treatment, and uses a nano adjuvant system of an active immune targeting activation technology platform as a delivery system to modify the neoantigen, so as to obtain a novel nano therapeutic vaccine ACTI of the active immune targeting activation technology. The vaccine can efficiently target immune activators and tumor neoantigens to professional antigen presenting cells pDCs cells, accurately activates specific immune responses of the body, and starts humoral immunity and cellular immunity to perform stomach cancer treatment.

Description

Technical Field

[0001] This invention relates to a method for screening tumor neoantigens for the treatment of gastric cancer and a method for preparing and applying novel nanovaccines, belonging to the field of biotechnology. Background Technology

[0002] Tumor immunotherapy has been increasingly applied to the treatment of gastric cancer in recent years. This therapy utilizes the principles and methods of immunology to activate the body's immune system, enhance the immunogenicity of tumor cells and their sensitivity to effector cell killing, stimulate and strengthen the body's anti-tumor immune response, and infuse immune cells and effector molecules into the host to synergistically kill tumors and inhibit tumor growth. Tumor therapeutic vaccines are currently one of the most popular tumor immunotherapies. These vaccines are peptides or recombinant protein fragments that can efficiently and specifically activate the immune system, breaking immune tolerance in chronically infected individuals and rebuilding or enhancing the immune response, thereby achieving a therapeutic effect on tumors. Their basic principle is to target tumor-associated or tumor-specific antigenic peptides, enhancing the immune system's ability to recognize and kill cancer cells containing these antigenic peptides. Tumor therapeutic vaccines can activate the immune system while effectively avoiding damage to healthy cells, showing great application and development potential.

[0003] The discovery and identification of specific tumor antigens is crucial for the successful development of therapeutic tumor vaccines. Neoantigens are gene mutations that occur during the development and progression of cancer cells. These mutations can activate the immune system (can be recognized by immune cells) and are abnormal proteins (abnormal / specific antigens) produced by cancer cell gene mutations. The discovery of neoantigens provides a new direction for the development of therapeutic tumor vaccines. Tumor immunomics refers to the comprehensive study of tumor immune status (TME) using multi-omics data reflecting tumor immune status, such as immunogenomics, immunoproteomics, and immunobioinformatics. It relies on the rapid development of next-generation sequencing technology. High-throughput genomic and transcriptomic data can be used to calculate the abundance of immune cells and predict tumor antigens; however, because batch sequencing represents the average characteristics of heterogeneous cell populations, it cannot distinguish different cell subtypes.

[0004] Furthermore, selecting appropriate adjuvants is crucial in the development of therapeutic vaccines. Immunoadjuvants are preparations that, when applied simultaneously with or before an antigen, enhance the body's immune response to that antigen or alter the type of immune response. As immunomodulators, adjuvants can enhance the immunogenicity of antigens and improve immunization efficacy. The mechanism of action of adjuvants is to alter the physical properties of antigens, delaying their degradation and excretion, thereby prolonging their retention time in the body and promoting antigen-specific humoral and cellular immune responses. This involves aspects such as antigen uptake by antigen-presenting cells, antigen processing, and antigen presentation. Selecting appropriate immunoadjuvants for effective delivery of neoantigens is one of the key technologies for the successful development of therapeutic cancer vaccines. Adjuvants can be classified according to their origin (natural, synthetic, or endogenous), mechanism of action, and physical or chemical properties. In its "Regulatory Considerations in the Safety Assessment of Adjuvants and Adjuvanted Preventive Vaccines," the US FDA classifies adjuvants into three categories: ① those that enhance the delivery of antigens to antigen-presenting cells and / or lymph nodes, thereby improving the immune response, such as aluminum salts and water-in-oil emulsions, like Novartis' MF59, GlaxoSmithKline's AS03 system, and other liposomes; ② immunostimulants or immune enhancers that primarily regulate the quality of the immune response through receptor-mediated signaling pathways, such as monophospholipids (MPL), QS21, CpG, and cytokines; and ③ combinations of ① and ②, known as adjuvant systems, such as GlaxoSmithKline's AS04 and AS01. Currently, the most studied adjuvants include oligonucleotides (CpG), adjuvant systems, live viral vectors, and emulsions. Although various novel adjuvants have been successively applied clinically, their mechanisms of action on the immune system remain not fully understood. The rise of nanomedicine has brought new opportunities for the development of tumor vaccines. Nanoadjuvant systems play an important role in the targeted delivery and controlled sustained release of antigens; they also provide co-stimulatory signals for immune activation, exerting a synergistic activation effect. Precise delivery of antigens and adjuvants to specific immune cells via nanomedicine delivery systems has become possible. Developing effective, safe, and easily manufactured nanocarrier vaccines by combining cutting-edge achievements in nanomedicine, structural biology, and immunology is one of the most promising directions for future vaccine development. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a tumor neoantigen screening method for the treatment of gastric cancer, which solves the problem that existing batch sequencing represents the average characteristics of heterogeneous cell populations and cannot distinguish different cell subtypes.

[0006] The technical problem to be solved by the present invention is achieved by the following technical solution: a method for screening tumor neoantigens for the treatment of gastric cancer, comprising: collection of tumor tissue samples from gastric cancer patients, whole exome sequencing (WES), RNA sequencing, prediction of polypeptide sequence epitopes, neoantigen prediction, in vitro assessment of neoantigen immunogenicity, detection of cytokines by flow cytometry, and IFN-γELISPOT assay.

[0007] As a preferred example, the in vitro assessment of neoantigen immunogenicity includes thawing frozen human PBMCs and culturing them in AIM-V medium supplemented with 10% FCS, adding 25 ng / mL of the medium. -1 IL-7 (PeproTech) was added at 20 U / ml on day 2. -1 IL-2 (PeproTech), replace half of the cytokine-containing culture medium every 3 days, using 2×10 5 One PBMC was inoculated with a single peptide (25 μg / mL) -1 Cells were cultured in 96-well plates and incubated overnight. Peptide-free medium (PBS) and stimulating phytohemagglutinin (PHA) were used as negative and positive controls, respectively. For in vitro stimulation of antigen-specific T cells, cells were collected one day after stimulation for further immunoassay to detect changes in cytokines.

[0008] As a preferred example, the steps for detecting cytokines using flow cytometry include: determining the IFN-γ concentration in the culture supernatant of the in vitro stimulation study according to the flow cytometry multifactorial assay (CBA) product instructions (BD Biosciences); using peptide-free culture medium (PBS) and stimulating phytohemagglutinin (PHA) as blank (negative) and positive controls, respectively; detecting and analyzing the data using a CytoFLEX LX flow cytometer (Beckman Coulter); mixing the synthesized mutant peptides together to stimulate PBMCs; and detecting and analyzing changes in CD8+ and IFN-γ in the PBMC samples.

[0009] As a preferred example, in the IFN-γ ELISPOT assay, for pre-stimulated PBMCs in vitro, after overnight stimulation with irradiated autologous PBMCs carrying the corresponding peptide, the secretion of IFN-γ released by T cells is assessed using the IFN-γ ELISPOT kit (Dakow), including pre-stimulated PBMCs (10 per well). 5 (each) and loaded with the corresponding peptide (1-20 μg mL) -1Irradiated autologous PBMCs were added to the wells in triplicate and incubated in AIM-V medium for 18-20 hours. After washing, diluted detection antibody was added and incubated at 37°C for 1 hour. Then, a mixture of streptavidin-HRP (1:100 dilution) and 3-amino-9-ethylcarbazole solution was added to each well sequentially. The plate was kept in the dark at room temperature for about 20 minutes, and then deionized water was added to terminate the reaction. The plate was scanned under an ELISPOT reader (Cellular Technology Inc.) and the data were analyzed using ELISPOT software (AID).

[0010] The beneficial effects of this invention are as follows: This invention utilizes single-cell sequencing technology and next-generation sequencing technology (NGS) to perform sequencing analysis combining whole-exome sequencing and tumor single-cell immunomics of gastric cancer tissue. Using our self-developed bioinformatics algorithm tools, we perform big data analysis and comparison on the whole-exome sequencing data and tumor single-cell immunomics data of gastric cancer tissue and adjacent normal tissue, and obtain a set of candidate sequences of neoantigens with potential for the treatment of gastric cancer.

[0011] In addition, a method for preparing a novel nanovaccine for the treatment of gastric cancer is provided. As a nanoadjuvant with active immune-targeting activation, it achieves efficient delivery of the screened neoantigen vaccine to immune-presenting cells, while also effectively activating the immune system.

[0012] The technical problem to be solved by the present invention is achieved by the following technical solution: a method for preparing a novel nanovaccine for the treatment of gastric cancer, characterized in that it includes: selection of linking peptides, preparation of PLGA nanospheres, preparation of proteins loaded on nanospheres, synthesis of SYNZIP1-neoantigen-specific polypeptides, preparation of the novel ACTI nanovaccine, and activity verification of the novel ACTI nanovaccine.

[0013] As a preferred example, the linker peptide is a SYNZIP1 / SYNZIP2 combination, the SYNZIP1 nucleic acid sequence is shown in SEQ ID NO: 1, the SYNZIP1 amino acid sequence is shown in SEQ ID NO: 2, the SYNZIP2 nucleic acid sequence is shown in SEQ ID NO: 3, and the SYNZIP2 amino acid sequence is shown in SEQ ID NO: 4.

[0014] As a preferred example, the PLGA nanosphere preparation steps include a 20 mg SYNZIP2 (sulfo-SMCC) cross-linking activation reaction, a 10 mg CpG (CpG ODN1018) nucleic acid molecule cross-linking reaction with sulfo-SMCC, and coupling the CpG nucleic acid molecule and the SYNZIP2 linker to the PLGA nanospheres.

[0015] As a preferred example, the preparation steps of the protein loaded on the nanospheres include the selection of the loaded protein SYNZIP1-Fc and IL-21-SYNZIP1 expression, SYNZIP1-Fc and IL-21-SYNZIP1 gene cloning, and transfection and protein expression of IL-21-SYNZIP1 and SYNZIP1-Fc.

[0016] As a preferred example, the activity validation steps of the novel ACTI nanovaccine include detecting the activation activity of ACTI using the U937 fluorescein reporter gene assay, changes in cytokines after co-culturing ACTI with PBMC cells, and an immunotherapy trial of the novel ACTI nanovaccine in a mouse gastric cancer tumor model.

[0017] The beneficial effects of this invention are as follows: This invention employs a combination of single-cell sequencing and whole-exome sequencing, along with neoantigen prediction and experimental verification, to establish a screening method for gastric cancer tumor neoantigens. Furthermore, it utilizes a nano-adjuvant system based on active immune-targeted activation technology as a delivery system to modify the screened neoantigens, resulting in a novel active checkpoint-targeted activated immunotherapy (ACTI) nano-therapeutic vaccine. This novel ACTI nanosphere vaccine can efficiently deliver immune activators and tumor neoantigens to specialized antigen-presenting cells (pDCs), precisely activating the body's specific immune response for the treatment of gastric cancer. Attached Figure Description

[0018] Figure 1 This is a flowchart of a neoantigen screening technology for a tumor neoantigen screening method for gastric cancer treatment according to the present invention.

[0019] Figure 2 This is a diagram showing the HLA typing frequencies and corresponding gene names of neoantigens screened out by a tumor neoantigen screening method for gastric cancer treatment according to the present invention.

[0020] Figure 3 This is a diagram showing the in vitro stimulation of PBMCs with predicted neoantigens selected by the tumor neoantigen screening method for gastric cancer treatment according to the present invention to produce cytokines.

[0021] Figure 4 This is a flow cytometry diagram showing the expression of cytokines in a tumor neoantigen screening method for gastric cancer treatment according to the present invention.

[0022] Figure 5 This is a diagram illustrating the effect of in vitro stimulation of PBMCs to produce IFN-γELISPOT in a tumor neoantigen screening method for gastric cancer treatment according to the present invention.

[0023] Figure 6 This is a schematic diagram illustrating the binding and affinity of the SYNZIP1 / SYNZIP2 helical peptides in a method for preparing a novel nanovaccine for the treatment of gastric cancer according to the present invention.

[0024] Figure 7 This image shows the ACTI activity detection of the U937 reporter gene cell line in the preparation method of a novel nanovaccine for gastric cancer treatment according to the present invention.

[0025] Figure 8 This diagram illustrates the ACTI stimulation of PBMCs to produce cytokines using a method for preparing a novel nanovaccine for gastric cancer treatment according to the present invention.

[0026] Figure 9 The figure shows the results of an ACTI novel nanovaccine tumor-killing experiment, which is a method for preparing a novel nanovaccine for the treatment of gastric cancer according to the present invention. Detailed Implementation

[0027] To facilitate a clear understanding of the technical means, creative features, objectives, and effects of this invention, the invention will be further described below in conjunction with specific illustrations.

[0028] Example 1: Prediction, screening and validation of gastric cancer neoantigens

[0029] 1. Collection of sample data

[0030] like Figure 1 As shown, cancer tissue samples were collected from 55 patients diagnosed with gastric adenocarcinoma, with a median survival of 50.3 months. Exome and RNA sequencing BAM files from 1099 gastric cancer patients were downloaded from ENA, TCGA, and CPTAC. Tumor-specific neoantigen data (SNAdb) from 441 TCGA gastric adenocarcinoma (STAD) samples were also obtained (http: / / biopharm.zju.edu.cn / tsnadb). A list of novel epitopes for gastric adenocarcinoma binding to MHC class I molecules was downloaded from the Immune Epitope Database (IEDB). 835 of these patients, with clinical variables including patient race, BRCA subtype, immune subtype, and nine tumor stages, were included in this study.

[0031] 2. Whole exome sequencing (WES)

[0032] like Figure 1 Blood and tumor tissue were collected from patients, and gDNA was extracted for library preparation. Tumor sections fixed in formalin and embedded in paraffin (FFPE) and corresponding blood samples were first processed. 50-50 ng of double-stranded DNA was fragmented to approximately 250 bp using sonication. The library was then constructed using the KAPA Hyper Prep Kit (KAPA Biosystems). Probes designed by a CRO covered over 20,000 human exon regions encoding genes, as well as introns with high pathogenicity fusion frequencies, to detect mutations. The captured library was sequenced on the Illumina NovaSeq 6000 platform (Illumina). The average coverage of the frozen section (FFPE) samples was 900-fold, and the average coverage of the matched blood samples was 300-fold.

[0033] 3. RNA sequencing.

[0034] RNA was extracted from unstained FFPE sections using the miRNeasy FFPE kit (catalog number 217504, Qiagen) according to the kit instructions. Qubit was used to extract RNA from the sections. TM The yield and quality of extracted RNA were assessed using a Thermo Fisher Scientific and LabChip GXTouch HT nucleic acid analyzer (Perkin Elmer). Ribosomal RNA was removed using the NEBNex rRNA Depletion Kit (cat#E6310L, New England Biolabs), followed by sequencing using M-MLV RTRNase (H-) (Ct#M3683, Promega) and the NEB Second Strand mRNA Synthesis Kit (at#E6111L, New England Biolabs). Sample libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) and sequenced on a NovaSeq 6000 platform at 2×151bp paired-end reads according to the manufacturer's instructions. The relative abundance of each annotated transcript was reported as parts per million (ppm) and log2 transformation prior to analysis. Sequencing and data analysis were outsourced to Zhiyi Medical.

[0035] 4. Epitope prediction

[0036] Mutations selected for expression were those with FPKM ≥ 30 and RNA variant allele frequency (VAF) ≥ 0.04. Then, NetMHCpan 2.8 and NetMHCpan 4.1 were used to examine 8-, 9-, and 10-mer epitopes containing mutated amino acids to predict IC50.50 Elution ligand (EL) gradations were determined to H-2Db and H-2Kb. The percentiles of MHCflurry (ver. 2.0.1) presentation were predicted using the 21-mer sequence with a mutated amino acid in the middle. The IC50 of NetMHCpan was selected. 50 Epitopes with ≤250 nM, EL rank ≤0.5 for NetMHCpan, and presentation percentile ≤0.5 for MHCflurry. As previously described, neoantigen peptides were synthesized using Syro I (Biotage, Uppsala, Sweden) via standard solid-phase synthesis.

[0037] 5. Neoantigen prediction

[0038] like Figure 1-2 As shown, human leukocyte antigen (HLA) genotyping was performed using WES data from blood samples or tumor-adjacent normal tissue in FASTQ format. OptiType (v1.3.5) was used to predict class I HLA genotypes with default settings. Raw RNA sequencing (RNA-seq) data from tumor tissue in FASTQ format was processed by Kallisto (v0.46.0) to obtain expression values ​​(TPM) with GRCh38 v78 coordinates. Tumor-specific somatic variant calls, RNA-seq expression values, and class I HLA genotypes were provided as inputs to the MuPeXI pipeline (v1.2) to predict neoantigen peptides. The binding affinity of mutant peptides to patient major histocompatibility complex class I (MHC-I) molecules was predicted using the NetMHCpan (v4.0) algorithm. Mutant peptides with an elution ligand percentile (EL% level) score ≤2% and an RNA expression level (TPM) >0.1 were defined as neoantigens. Neoantigens with a percentile score <0.5% are considered high-affinity neoantigens, as shown in the table below.

[0039]

[0040]

[0041] 6. In vitro assessment of the immunogenicity of the neoantigen.

[0042] Frozen human PBMCs were thawed and cultured in AIM-V medium supplemented with 10% FCS. 25 ng / mL of the medium was added. -1 IL-7 (PeproTech) was added at 20 U / ml on day 2. -1 IL-2 (PeproTech), replace half of the cytokine-containing culture medium every 3 days, using 2×10 5 One PBMC was inoculated with a single peptide (25 μg / mL) -1Cells were cultured in 96-well plates and incubated overnight. Peptide-free PBS and phytohemagglutinin (PHA) were used as negative and positive controls, respectively. For in vitro stimulation of antigen-specific T cells, cells were collected one day after stimulation for further immunoassay to detect changes in cytokines. Results are as follows: Figure 3 As shown.

[0043] 7. Flow cytometry detection of cytokines

[0044] like Figure 4 As shown, the concentration of IFN-γ in the culture supernatant for in vitro stimulation studies was determined according to the flow cytometry multifactorial assay (CBA) product instructions (BD Biosciences). Peptide-free medium (PBS) and stimulating phytohemagglutinin (PHA) were used as blank (negative) and positive controls, respectively. Samples were analyzed using a CytoFLEX LX flow cytometer (Beckman Coulter). Synthesized mutant peptides were mixed together to stimulate PBMCs, and changes in CD8+ and IFN-γ in the PBMC samples were detected and analyzed.

[0045] 8. IFN-γELISPOT assay

[0046] For pre-stimulated PBMCs in vitro, after overnight stimulation with irradiated autologous PBMCs loaded with the corresponding peptides, the secretion of IFN-γ released by T cells was assessed using the IFN-γ ELISPOT kit (Dakow). The procedure was as follows: Pre-stimulation of PBMCs (10 per well) 5 (each) and loaded with the corresponding peptide (1-20 μg mL) -1 Irradiated autologous PBMCs were added to each well in triplicate and incubated in AIM-V medium for 18–20 hours. After washing, diluted detection antibody was added and incubated at 37°C for 1 hour. Then, a mixture of streptavidin-HRP (1:100 dilution) and 3-amino-9-ethylcarbazole solution was added sequentially to each well. After incubating the plate in the dark at room temperature for approximately 20 minutes, deionized water was added to terminate the reaction. The plate was scanned using an ELISPOT reader (Cellular Technology Inc.), and the data were analyzed using ELISPOT software (AID). The reactivity of positive PBMCs was scored when the spots were more than twice the size of the negative controls, as shown in the results. Figure 5 As shown.

[0047] Example 2: Preparation of novel ACTI nanovaccines

[0048] 1. Selection of linker peptides

[0049] See J.AM.CHEM.SOC.2010,132,6025–6031, such as Figure 6 As shown, SYNZIP1 and SYNZIP2 peptides have a helical zipper structure. SYNZIP peptide molecules have a natural affinity for each other, forming a dimer structure through a coil-coil helix. By using these two peptides as linkers to fuse different protein fragments or nucleic acid molecules, a variety of different types of molecules can be obtained.

[0050] like Figure 1 and Figure 2 As shown, this invention uses the SYNZIP1 / SYNZIP2 combination as a linker for each molecule to complete the combination of multiple proteins. By fusing or coupling SYNZIP1 / SYNZIP2 with multiple proteins, the self-assembly of multiple proteins can be completed, and multiple different types of immunomodulatory drugs can be quickly and easily assembled.

[0051] SYNZIP1 nucleic acid sequence:

[0052] AACCTGGTTGCGCAGCTCGAAAACGAAGTTGCGTCTCTGGAAAATGAGAACGAAACCCTGAAGAAAAAGAACCTGCACAAAAAAGACCTGATCGCGTACCTGGAGAAAGAAATCGCGAATCTGCGTAAGAAAATCGAAGAATGA (SEQ ID NO: 1);

[0053] SYNZIP1 amino acid sequence:

[0054] NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEE

[0055] (SEQ ID NO: 2);

[0056] SYNZIP2 nucleic acid sequence:

[0057] GCGCGTAACGCGTATCTGCGTAAGAAAATCGCACGTCTGAAAAAAGACAACCTGCAGCTGGAACGTGATGAACAGAACCTGGAAAAAATCATCGCGAACCTGCGTGACGAAATCGCGCGTCTCGAAAACGAAGTTGCGTCTCACGAACAGTGA (SEQ ID NO: 3);

[0058] SYNZIP2 amino acid sequence:

[0059] ARNAYLRKKIARLKKDNLQLERDEQNLEKIIANLRDEIARLENEVAS HEQ (SEQ ID NO: 4);

[0060] 2. Preparation of PLGA nanospheres

[0061] (a) Cross-linking activation reaction of 20 mg SYNZIP2 (sulfo-SMCC): The SYNZIP2 polypeptide sequence was synthesized by GenScript. SYNZIP2 was activated by dissolving it in 10 ml PBS, adding TCEP to open the disulfide bonds, and dialysis to remove TCEP, yielding activated SYNZIP2. Sulfo-SMCC was then added, cross-linked, and excess sulfo-SMCC was removed by dialysis. After freeze-drying, 20 mg of SYNZIP2-sulfo-SMCC solid powder was obtained.

[0062] (b) Crosslinking reaction of 10 mg of nucleic acid molecule CpG (CpG ODN1018) with sulfo-SMCC: First, CpG was activated by dissolving it in ultrapure water and adding TCEP to open the disulfide bonds; then, TCEP was removed by desalting to obtain activated CpG. Sulfo-SMCC was then added, and after crosslinking, it was precipitated with alcohol and centrifuged to obtain solid CpG-sulfo-SMCC.

[0063] The sequence of CpG ODN 1018 is (5'-dT-dG-dA-dC-dT-dG-dT-dG-dA-dA-dC-dG-dT-dT-dC-dG-dA-dG-dA-dT-dG-dA-3') (SEQ ID NO: 5).

[0064] (c) 15 mg of polylactic acid-glycolic acid copolymer (PLGA) was dissolved in 15 mL of dichloromethane to obtain solution 1. 20 mg of SYNZIP2-sulfo-SMCC and 10 mg of CpG-sulfo-SMCC were added to complete the coupling of SYNZIP2-CpG and PLGA, which served as the oil phase. 15 mL of solution 1 was taken as the aqueous phase. Under ultrasonic power of 220 W, the oil phase was added dropwise to the aqueous phase to form a primary emulsion. The obtained primary emulsion was added dropwise to 20 mL of ultrapure water while stirring at 400 rpm. Stirring was continued for 4 h to allow the dichloromethane to completely evaporate. The resulting solution was first centrifuged at 1000 rpm for 5 minutes, and the supernatant was retained to remove large particles. Then, it was centrifuged at 12000 rpm for 10 minutes to retain the precipitate. The precipitate was washed three times with ultrapure water and freeze-dried to obtain SYNZIP2-CpG-PLGA nanospheres, which were stored for later use. The nanoparticle size was measured to be 200.3 ± 1.5 nm using a laser particle size analyzer, and the particle size after lyophilization was 223.2 ± 1.9 nm. The obtained nanoparticles were dissolved in PBS. This yielded PLGA nanospheres containing CpG nucleic acid molecules and SYNZIP2 linkers.

[0065] 3. Preparation of proteins loaded on nanospheres

[0066] (a) Selection of load protein

[0067] The Fc protein is the constant region of an antibody. It exerts biological activities such as antibody-dependent cell-mediated cytotoxicity (ADCC) through binding to FcγR, or complement-dependent cytotoxicity (CDC) through binding to complement C1q. The binding of Fc / FcγR can be used to target dendritic cells (DCs), thereby achieving targeted delivery to antigen-presenting cells. IL-21 belongs to the common cytokine receptor γ chain family and is a type I cytokine with a four-alpha helix bundle. IL-21 is mainly secreted by activated CD4+ T cells, NK cells, TFH cells, and Th17 cells. Stimulating immune cell activation with IL-2 is more effective than the commonly used IL-2.

[0068] (b) Expression of SYNZIP1-Fc and IL-21-SYNZIP1

[0069] The complete genome sequences of SYNZIP1-Fc and IL-21-SYNZIP1 were obtained through gene synthesis. The specific sequences are as follows:

[0070] SYNZIP1-Fc nucleic acid sequence (EcoRI+KOZAK+signal peptide+SYNZIP1+linker+Fc+NotI):

[0071] gaattcgccgccaccATGGAGTTCGGACTCAGTTGGCTGTTCCTGGTGGCCATCCTGAAGGGTGTGCAGTGTAACCTGGTTGCGCAGCTCGAAAACGAAGTTGCGTCTCTGGAAAATGAGAACGAAACCCTGAAGAAAAAGAACCTGCACAAAAAAGACCTGATCGCGTACCTGGAGAAAGAAATCGCGAATCTGCGTAAGAAAATCGAAGAAGAGATCAAAGGAGGAGGAGGATCAGGAGGAGGAGGATCAGGAGGAGGAGGATCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACGCCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGCCGCAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAgcggccgc(SEQ ID NO:6);

[0072] SYNZIP1-linker-Fc amino acid sequence (30KD):

[0073] MEFGLSWLFLVAILKGVQCNLVAQLENEVASLENENETLKKK NLHKKDLIAYLEKEIANLRKKIEE(GGGGS)3PELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 7);

[0074] IL-21-SYNZIP1 (EcoRI + KOZAK + signal peptide + IL-21 + linker + SYNZIP1 + Not I):

[0075] gaattcgccgccaccATGGAGTTCGGACTCAGTTGGCTGTTCCTGGTGGCCATCCTGAAGGGTGTGCAGTGTATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCGAGATCAAAGGAGGAGGAGGATCAGGAGGAGGAGGATCAGGAGGAGGAGGATCAAACCTGGTTGCGCAGCTCGAAAACGAAGTTGCGTCTCTGGAAAATGAGAACGAAACCCTGAAGAAAAAGAACCTGCACAAAAAAGACCTGATCGCGTACCTGGAGAAAGAAATCGCGAATCTGCGTAAGAAAATCGAAGAACATCATCATCATCATCATCATTGAgcggccgc(SEQ ID NO:8);

[0076] The amino acid sequence corresponding to IL-21-linker-SYNZIP1 28KD:

[0077] MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(GGGGS)3NLVAQLENEV ASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEHHHHHH (SEQ ID NO: 9).

[0078] (c) Cloning of SYNZIP1-Fc and IL-21-SYNZIP1 genes

[0079] EcoRI and NotI restriction sites were designed at both ends of SYNZIP1-Fc and IL-21-SYNZIP1. These sites were synthesized by Nanjing GenScript Biotech Co., Ltd. using a whole-genome synthesis method to obtain PCDNA3.1-IL-21-SYNZIP1 and PCDNA3.1-SYNZIP1-Fc cloning plasmids. Positive clones of PCDNA3.1-IL-21-SYNZIP1 and PCDNA3.1-SYNZIP1-Fc were screened, positive colonies were amplified, and sequencing confirmed the correctness of the cloning sequences. The PCDNA3.1-IL-21-SYNZIP1 and PCDNA3.1-SYNZIP1-Fc plasmids were then extracted using a plasmid endotoxin-free extraction kit for later use.

[0080] (d) Transfection and protein expression of IL-21-SYNZIP1 and SYNZIP1-Fc

[0081] Transient transfection and purification of cells: 293 cells were transfected according to Lipofectamine. TM Following the 2000 operating instructions, taking one well of a 6-well plate as an example, the steps are as follows: Inoculate the well with 4 × 10⁻⁶ ppm. 5 Cells were cultured at 37°C and 5% CO2 for 12 hours, with the medium changed once. Transfection began 12 hours later. The ideal confluence of adherent cells at transfection was 90-95%. 4 μg of plasmid DNA and 10 μl of Lipofectamine were added. TM2000 was diluted to 250 μl with antibiotic-free and serum-free culture medium, gently mixed, and incubated at room temperature for 15 min to form liposome complexes. After three days of continuous culture, the supernatant was collected, and the expression of the target protein was detected by indirect ELISA. The obtained cell supernatant was purified using a nickel affinity chromatography column to obtain PCDNA3.1-IL-21-SYNZIP1 and PCDNA3.1-SYNZIP1-Fc proteins. The protein concentrations were determined by BCA at 80 mg / ml and 100 mg / ml, respectively.

[0082] 4. Synthesis of SYNZIP1-neoantigen-specific peptides

[0083] The neoantigen peptide validated in Example 1 was ligated with SYNZIP1 as a linker, and the full-length peptide was synthesized by Nanjing Genscript Biotech Co., Ltd. The ligation sequence was as follows: (SYNZIP1-linker-neoantigen), resulting in the following sequence.

[0084]

[0085]

[0086] The synthesized SYNZIP1-linker-neo1-21 peptide lyophilized powder was dissolved in phosphate buffer and mixed, then converted to molar concentration for use.

[0087] 5. Preparation of novel ACTI nano-vaccines

[0088] The neoantigen peptides SYNZIP1-Fc, IL-21-SYNZIP1, and SYNZIP1-linker-neo1-21 (SEQ ID NO: 10-30) were mixed in equimolar concentrations to form a solution labeled Mix-neo. Mix-neo was slowly added to a PLGA nanosphere solution containing CpG nucleic acid molecules and SYNZIP2 linkers and mixed with magnetic stirring at a molar ratio of 1:1. Through the pairing and binding of SYNZIP1 and SYNZIP2, a novel ACTI nanovaccine was obtained. The concentration of the obtained nanospheres was quantitatively determined using the BCA method.

[0089] 6. Activity validation of the novel ACTI nano-vaccine

[0090] (a) Detection of ACTI activation activity using the U937 fluorescein reporter gene assay

[0091] First, U937 cells containing a reporter gene (luciferase) and overexpressing FcR molecules and TLR9 (CpG receptor) were used to identify ACTI activity. 5*10 5 U937 cells were cultured in 6-well plates with a V-bottom. After 12 hours, ACTI concentrations ranging from 1 μg / mL to 100 μg / mL were added, and the cells were cultured at 37°C with 5% CO2 for another 24 hours. Then, the substrate fluorescein was added, and the cells were shaken at 200 rpm for 3 minutes, followed by incubation for 10 minutes. Finally, the fluorescence value was detected using a multi-functional microplate reader. The experimental results showed that different concentrations of ACTI stimulated U937 cells to produce fluorescence of varying intensities with increasing concentration. The results are shown below. Figure 7 As shown.

[0092] (b) Changes in cytokines after co-culturing ACTI and PBMC cells

[0093] Using Miltenyi's MicroBead kit, following the instructions, PBMCs and pDCs were isolated from human peripheral blood. The isolated PBMCs were cultured for 12 hours in X-VIVO 15, 2% human serum albumin, and 10 ng / ml IL-2 medium. Then, 1x10⁻⁶ pDCs were... 5 PBMC cells were seeded into 6-well plates, and ACTI molecules were added at concentrations ranging from 1 μg / ml to 10 μg / ml. After stimulation for 48 hours, the culture supernatant was collected, and changes in IFN-γ and TNF-alpha cytokines were detected using a cytokine ELISA kit. Figure 8 As shown, the results indicate that different concentrations of ACTI can stimulate PBMC cells to produce different concentrations of cytokines.

[0094] (c) Immunotherapy experiment of a mouse tumor model using a novel ACTI nanovaccine

[0095] 2×10 6 MFC cells were subcutaneously injected into mice to establish a mouse model of gastric cancer. When the tumor reached approximately 100 mm... 3 When the tumor reached a certain volume, it was removed. Mice were then randomly divided into 6 groups: subcutaneous PBS group, PLGA group, free vaccine group, and ACTI nanovaccine group. Each group received 150 μg of the vaccine, administered once on day 0 and day 14. Tumor size was measured every two days in a non-blind manner using calipers, and the result was calculated using equation (a...). 2 Calculate their volume using (×b) / 2 (a, width; b, length). Figure 9 As shown, the experimental results indicate that the ACTI group had a better tumor suppression effect compared with other control groups.

[0096] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and various changes and modifications can be made without departing from the spirit and scope of the invention, all of which fall within the scope of the claims. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a novel nanovaccine for the treatment of gastric cancer, characterized in that, Includes the following steps: The selection of linker peptides, preparation of PLGA nanospheres, preparation of proteins loaded on nanospheres, synthesis of specific peptides by linking SYNZIP1 with a neoantigen, preparation of novel ACTI nanovaccines, and activity verification of novel ACTI nanovaccines, wherein the specific peptide is a SYNZIP1-linker-neoantigen peptide. The linker peptide is a SYNZIP1 / SYNZIP2 combination, and the amino acid sequence of SYNZIP1 is as follows: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEE; The amino acid sequence of SYNZIP2 is as follows: ARNAYLRKKIARLKKDNLQLERDEQNLEKIIANLRDEIARLENEVASHEQ; The preparation of the novel ACTI nanovaccine includes: (1) Mix SYNZIP1-Fc, IL-21-SYNZIP1 and SYNZIP1-linker-neo antigen peptides in equal molar concentrations to form a solution for use, and label it as Mix-neo; (2) The Mix-neo solution is slowly added to the PLGA nanosphere solution containing CpG nucleic acid molecules and SYNZIP2 linkers, and the mixture is stirred with magnetic force. The mixture is mixed at a molar ratio of 1:

1. Through the pairing and binding of SYNZIP1 and SYNZIP2, the novel ACTI nanovaccine is obtained. The SYNZIP1-linker-neo1-21 polypeptide mixture is composed of the following 21 SYNZIP1-linker-neo1-21 polypeptides, whose amino acid sequences are as follows: SYNZIP1-linker-Neo1: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSEERMVYVAFSEFVFDSAMESYFRAG SYNZIP1-linker-Neo2: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSAFFRNHPATWKNTIRHNLSLHKCFV SYNZIP1-linker-Neo3: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSKFIDAAKISNADEAGSRP SYNZIP1-linker-Neo4: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSRGRVLKKFKSDSRLAQRRFIRGWGL SYNZIP1-linker-Neo5: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSMTEYKLVVVGADGVGKSALTIQLIQ SYNZIP1-left-Neo6: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSFMKLRTDAVLPLTVAEVQKLLGPHV SYNZIP1-left-Neo7: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSDLKDQGGELLSLCYDLTVPFARYLA SYNZIP1-left-Neo8: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSSCGTPALGSLLFLLFSLGWVQPSRT SYNZIP1-left-Neo9: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSAASEDARQPAKPRYSYIALITMAIL SYNZIP1-left-Neo10: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSGQWDLGQEVLDDMIYRAQLELFSQP SYNZIP1-left-Neo11: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSTEGLLAPVGACESDYGGPLACFTHN SYNZIP1-left-Neo12: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSADNLPGINGLLQCKVAELEEEQSQG SYNZIP1-left-Neo13: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSEEKDSESHLAEDHHAVSTEAEDRSY SYNZIP1-left-Neo14: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSMCAWNPRDLPLMVLPPCHALCQFYV SYNZIP1-linker-Neo15: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSEALEYFMKQMNDARH SYNZIP1-linker-Neo16: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSQWFSPSNGRKRSYFS SYNZIP1-linker-Neo17: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSTPNSTGEEVPVQR SYNZIP1-linker-Neo18: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSQGDGEQSAGGGPGR SYNZIP1-linker-Neo19: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSPGSNGNPGPPPAGNTGAPGS SYNZIP1-linker-Neo20: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSQAGECLTVLPDGAACR SYNZIP1-linker-Neo21: NLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSSVEMGSVNEAYR; The amino acid sequence of the said SYNZIP1-Fc is: MEFGLSWLFLVAILKGVQCNLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEGGGGSGGGGSGGGGSPELLGGPSVFLFPPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; the amino acid sequence of IL-21-SYNZIP1 is: MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRR QKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSGGGGSNLVAQLENEVASLENENETLKKKNLHKKDLIAYLEKEIANLRKKIEEHHHHHH.

2. The method for preparing a novel nanovaccine for gastric cancer treatment according to claim 1, characterized in that, The preparation steps of the PLGA nanospheres include: a cross-linking activation reaction of 20 mg SYNZIP2 with sulfo-SMCC, a cross-linking reaction of 10 mg CpG nucleic acid molecules with sulfo-SMCC, and coupling the CpG nucleic acid molecules and SYNZIP2 linkers to the PLGA nanospheres; wherein the sequence of the CpG nucleic acid molecules is: 5′-dT-dG-dA-dC-dT-dG-dT-dG-dA-dA-dC-dG-dT-dT-dC-dG-dA-dG-dA-dT-dG-dA-3′.

3. The method for preparing a novel nanovaccine for gastric cancer treatment according to claim 1, characterized in that, The activity validation steps of the novel ACTI nanovaccine include validating the activation activity of ACTI as detected by the U937 fluorescein reporter gene assay, the changes in cytokines after co-culturing ACTI with PBMC cells, and the immunotherapy trial of the novel ACTI nanovaccine in a mouse gastric cancer tumor model.

4. A novel nano-vaccine for the treatment of gastric cancer, characterized in that, It is prepared by the preparation method described in any one of claims 1-3.

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