An oral frameshift peptide neoantigen vaccine and a preparation method and application thereof

Abstract

This invention discloses an oral frameshift peptide neoantigen vaccine, its preparation method, and its application, relating to the field of biomedical technology. The preparation method includes: 1) synthesizing a sea urchin-like metal-organic framework (MOF) via a hydrothermal method using zinc ions and 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid; 2) loading the sea urchin-like MOF with a frameshift peptide and a Toll-like receptor 9 agonist via electrostatic interactions and / or van der Waals forces to prepare the oral frameshift peptide neoantigen vaccine. This vaccine not only promotes antigen endocytosis and transcytosis via intestinal microfold cells by activating cyclin 42, but also alleviates immune tolerance mediated by the goblet cell-regulatory T cell immunosuppressive axis and activates specific CD8+. + T cells help prevent the development of Lynch syndrome-related colorectal cancer.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to an oral frameshift peptide neoantigen vaccine, its preparation method, and its application. Background Technology

[0002] Lynch syndrome (LS) is an autosomal dominant genetic disorder caused by pathogenic germline mutations in mismatch repair (MMR) genes (MLH1 / MSH2 / MSH6 / PMS2). MMR-deficient somatic cells continuously accumulate insertion / deletion mutations, significantly increasing the risk of developing various malignancies. LS most commonly develops into colorectal cancer (LS-CRC, approximately 70%). Currently, LS is the most common inherited cancer syndrome, posing a serious threat to human health. Given the high overall lifetime risk of CRC in LS patients, preventative interventions targeting LS-CRC are urgently needed.

[0003] Prevention methods for LS-CRC include prophylactic surgical resection, chemoprevention, and frameshift peptide (FSP) neoantigen vaccines. Prophylactic total colectomy can reduce the risk of LS-CRC to some extent, but it cannot significantly prolong the survival of LS patients, and lifelong side effects such as diarrhea and abdominal pain greatly affect the quality of life of patients. Chemoprophylaxis (such as aspirin) can effectively inhibit the occurrence and development of LS-CRC, but long-term high-dose use increases the risk of cardiovascular disease in patients. Summary of the Invention

[0004] This application provides an oral frameshift peptide neoantigen vaccine, its preparation method, and its application, in order to improve the problem of low oral delivery efficiency of existing frameshift peptide (FSP) neoantigen vaccines.

[0005] In a first aspect, this application provides an oral frameshift peptide neoantigen vaccine having a sea urchin-shaped metal-organic framework (SU-MOF) delivery carrier with a sea urchin spine structure on its surface.

[0006] The oral frameshift peptide neoantigen vaccine of this application, based on SU-MOF, can not only enhance antigen uptake by activating cyclin 42 (CDC42) to promote endocytosis and transcytosis of antigens via microfolded cells (M cells), but also alleviate goblet cell-CD103... + The "dendritic cell (DC)-regulatory T cell (Treg)" immunosuppressive axis-mediated immune tolerance can protect its loaded vaccine from degradation in simulated gastrointestinal fluid, exhibiting good biocompatibility and high antigen loading rate. It can enhance intestinal retention and intestinal epithelial penetration, and induce robust intestinal mucosal immune activation in vivo, contributing to the activation of antigen-specific CD8. + T-cell responses can prevent the development of Lynch syndrome-associated colorectal cancer (LS-CRC).

[0007] In some embodiments, the oral frameshift peptide neoantigen vaccine comprises two frameshift peptides, one of which has the amino acid sequence shown in SEQ ID NO: 1, and the other has the amino acid sequence shown in SEQ ID NO: 2.

[0008] SEQ ID NO: 1: AEGRWPCWLLRAH.

[0009] SEQ ID NO: 2: LSSPKRVMYRLSVGCLRPTL.

[0010] In some embodiments, the oral frameshift peptide neoantigen vaccine promotes the endocytosis and transcytosis of antigens via microfolded cells (M cells) by activating cell division cycle protein 42 (CDC42).

[0011] In some embodiments, the oral frameshift peptide neoantigen vaccine can alleviate goblet cell-CD103... + DC-Treg cell immunosuppressive axis-mediated immune tolerance, thereby activating antigen-specific CD8+. + T cell response.

[0012] Secondly, this application provides a method for preparing an oral frameshift peptide neoantigen vaccine, comprising the following steps: A sea urchin-shaped metal-organic framework was mixed with a frameshift peptide antigen in ultrapure water to obtain an oral frameshift peptide neoantigen vaccine.

[0013] In some embodiments, the preparation method of the sea urchin-like metal-organic framework includes the following steps: Zn(NO3)2·6H2O, 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid, trifluoroacetic acid, anhydrous ethanol and N , N - Dimethylformamide (DEF) was ultrasonicated, incubated, and cleaned to obtain a metal-organic framework.

[0014] In some embodiments, the mass ratio of Zn(NO3)2·6H2O to 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid is 1:(0.3~0.4).

[0015] In some embodiments, the method of mixing sea urchin-shaped metal-organic frameworks with frameshift peptide neoantigens in ultrapure water to obtain an oral frameshift peptide neoantigen vaccine includes: The sea urchin-like metal-organic framework was mixed with a first frameshift peptide, a second frameshift peptide, a Toll-like receptor-9 agonist and a solvent, and then freeze-dried to obtain an oral frameshift peptide neoantigen vaccine. The amino acid sequence of the first frameshift peptide is shown in SEQ ID NO: 1; The amino acid sequence of the second frameshift peptide is shown in SEQ ID NO: 2; The sequence of the Toll-like receptor-9 agonist is shown in SEQ ID NO: 3.

[0016] SEQ ID NO: 1: AEGRWPCWLLRAH.

[0017] SEQ ID NO: 2: LSSPKRVMYRLSVGCLRPTL.

[0018] SEQ ID NO: 3: 5'-TCCATGACGTTCCTGACGTT-3'.

[0019] In some embodiments, the mass ratio of the urchin-like metal-organic framework, the first frameshift peptide, and the second frameshift peptide is 20:(0.9~1.1):(0.9~1.1).

[0020] Thirdly, this application provides the use of the oral frameshift peptide neoantigen vaccine described in the first aspect, or the oral frameshift peptide neoantigen vaccine prepared by the method described in the second aspect, in the preparation of a drug for treating Lynch syndrome-related colorectal cancer. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The images show the material characterization diagrams of MOF Vac / SU-MOF Vac in Examples 1 and 2 of this application.

[0023] Figure 2 This is a diagram showing the intestinal retention and intestinal epithelial permeability of SU-MOF Vac in Example 3 of this application.

[0024] Figure 3 This is a diagram illustrating the immune mechanism of SU-MOF Vac in Example 4 of this application.

[0025] Figure 4 This is an image showing the immunization results of SU-MOF Vac in Example 5 of this application.

[0026] Figure 5This is a graph showing the mouse experimental results of SU-MOF Vac in Example 6 of this application.

[0027] Figure 6 This is a safety evaluation diagram of the SU-MOF Vac in Embodiment 7 of this application. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0029] Lynch syndrome (LS) is an autosomal dominant genetic disorder caused by pathogenic germline mutations in mismatch repair (MMR) genes (MLH1 / MSH2 / MSH6 / PMS2). MMR-deficient somatic cells continuously accumulate insertion / deletion mutations, significantly increasing the risk of developing various malignancies. LS most commonly develops into colorectal cancer (LS-CRC, approximately 70%). Currently, LS is the most common inherited cancer syndrome, posing a serious threat to human health. Given the high lifetime overall risk of CRC in LS patients, preventative interventions targeting LS-CRC are urgently needed.

[0030] Prevention methods for LS-CRC include prophylactic surgical resection, chemoprevention, and frameshift peptide (FSP) neoantigen vaccines. Prophylactic total colectomy can reduce the risk of LS-CRC to some extent, but it cannot significantly prolong the survival of LS patients, and lifelong side effects such as diarrhea and abdominal pain greatly affect their quality of life. Chemoprophylaxis (such as aspirin) can effectively inhibit the development of LS-CRC, but long-term high-dose use increases the risk of cardiovascular disease. Compared to the above prevention methods, tumor vaccines have better safety, compliance, and targeting. Compared to mismatch repair intact CRC (pMMR CRC), LS-CRC has a higher response rate to immunotherapy. Furthermore, MMR inactivation leads to the accumulation of insertion or deletion gene mutations at microsatellite sites in the coding region, producing frameshift sequences, which, after translation, ultimately form the FSP neoantigen unique to LS-CRC. FSP vaccines can effectively inhibit the development of LS-CRC and prolong median survival time in mouse models. Clinical phase I / IIa and Ib / 2 trials have shown that the FSP vaccine is safe and non-toxic, and can induce significant cellular and humoral immune responses in patients with advanced LS. Therefore, tumor vaccines are considered the most promising method for the prevention of LS-CRC.

[0031] Oral vaccine delivery systems offer a promising approach to immunization in LS-CRC due to their low cost, minimal operator skill requirements, and ability to directly activate the intestinal immune system. Various oral vaccine delivery systems have been developed to date, including liposome nanoparticles, polymer carriers, inorganic nanoparticles, plant-derived nanovesicles, and bacterial outer membrane vesicles. However, existing oral vaccine delivery systems are inefficient and fail to meet the required standards.

[0032] In view of this, this application provides an oral frameshift peptide neoantigen vaccine, its preparation method and application, to improve the problem of low oral delivery efficiency of existing FSP neoantigen vaccines.

[0033] In a first aspect, this application provides an oral frameshift peptide neoantigen vaccine having a delivery carrier with a sea urchin-like metal-organic framework and a sea urchin-like spine structure on its surface.

[0034] The oral frameshift peptide neoantigen vaccine of this application, based on a sea urchin-shaped metal-organic framework (SU-MOF), not only enhances antigen uptake by activating cyclin 42 (CDC42) to promote endocytosis and transcytosis of antigens via microfolded cells (M cells), but also alleviates goblet cell-CD103... + DC-Treg cell immunosuppressive axis-mediated immune tolerance can protect its loaded vaccine from degradation in simulated gastrointestinal fluid, exhibiting good biocompatibility and high antigen loading rate. It can enhance intestinal retention and intestinal epithelial penetration, and can induce robust intestinal mucosal immune activation in vivo, contributing to the activation of antigen-specific CD8. + T cell response helps prevent the occurrence of LS-CRC.

[0035] It should be noted that, compared to delivery systems with smooth surfaces, the urchin-like metal-organic framework (MOF)-inspired "spiky" delivery system possesses a richer array of interfacial sites, enabling higher load-bearing capacity and superior surface functionalization. Its external nanospikes can also generate stronger mechanical interactions with the cell membrane, thereby enhancing intestinal retention, intestinal mucosal penetration, and intracellular uptake. From a mechano-biological perspective, the nanospikes can apply mechanical stress to the cell membrane and modulate cell function through mechanosensitive ion channels.

[0036] In conjunction with the first aspect, in some embodiments provided in this application, the oral frameshift peptide neoantigen vaccine comprises two frameshift peptides, wherein the amino acid sequence of one frameshift peptide is shown in SEQ ID NO: 1, and the amino acid sequence of the other frameshift peptide is shown in SEQ ID NO: 2.

[0037] SEQ ID NO: 1: AEGRWPCWLLRAH.

[0038] SEQ ID NO: 2: LSSPKRVMYRLSVGCLRPTL.

[0039] In conjunction with the first aspect, in some embodiments provided in this application, the oral frameshift peptide neoantigen vaccine promotes the endocytosis and transcytosis of antigens via microfolded cells (M cells) by activating CDC42.

[0040] In conjunction with the first aspect, in some embodiments provided in this application, the oral frameshift peptide neoantigen vaccine can alleviate goblet cell-CD103... + DC-Treg cell immunosuppressive axis-mediated immune tolerance, thereby activating antigen-specific CD8+. + T cell response.

[0041] Secondly, this application provides a method for preparing an oral frameshift peptide neoantigen vaccine, comprising the following steps: A sea urchin-shaped metal-organic framework was mixed with a frameshift peptide in ultrapure water to obtain an oral frameshift peptide neoantigen vaccine.

[0042] In conjunction with the second aspect, in some embodiments provided in this application, the method for preparing the sea urchin-like metal-organic framework includes the following steps: Zn(NO3)2·6H2O, 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid, trifluoroacetic acid, anhydrous ethanol and N , N - Dimethylformamide (DEF) was ultrasonicated, incubated, and cleaned to obtain a metal-organic framework.

[0043] In conjunction with the second aspect, in some embodiments provided in this application, the mass ratio of Zn(NO3)2·6H2O to 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid is 1:(0.3~0.4).

[0044] In conjunction with the second aspect, in some embodiments provided in this application, the method of mixing sea urchin-like metal-organic frameworks with frameshift peptide antigens in ultrapure water to obtain an oral frameshift peptide neoantigen vaccine includes: The sea urchin-like metal-organic framework was mixed with the first frameshift peptide antigen, the second frameshift peptide antigen, the Toll-like receptor-9 agonist (CpG1826), and a solvent, and then freeze-dried to obtain an oral frameshift peptide neoantigen vaccine. The amino acid sequence of the first frameshift peptide is shown in SEQ ID NO: 1; The amino acid sequence of the second frameshift peptide is shown in SEQ ID NO: 2; The sequence of the Toll-like receptor-9 agonist (CpG1826) is shown in SEQ ID NO: 3.

[0045] SEQ ID NO: 1: AEGRWPCWLLRAH.

[0046] SEQ ID NO: 2: LSSPKRVMYRLSVGCLRPTL.

[0047] SEQ ID NO: 3: 5'-TCCATGACGTTCCTGACGTT-3'.

[0048] In conjunction with the second aspect, in some embodiments provided in this application, the mass ratio of the sea urchin-like metal-organic framework, the first frameshift peptide, and the second frameshift peptide is 20:(0.9~1.1):(0.9~1.1).

[0049] Specifically, the preparation of sea urchin-shaped metal-organic frameworks (SU-MOFs) includes the following steps: Combine 40 mg Zn(NO3)2·6H2O, 15 mg 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid, 2 μL trifluoroacetic acid, 100 μL anhydrous ethanol, and 2 mL of... N , N After sonicating with dimethylformamide (DEF) for 5 minutes, the mixture was incubated at 100 °C for 12 hours. The resulting products were then processed using... N , N SU-MOF was prepared by washing with dimethylformamide (DMF) and anhydrous ethanol.

[0050] The preparation of a frameshift peptide neoantigen vaccine based on a sea urchin-like metal-organic framework (SU-MOF Vac) includes the following steps: 1 mg SU-MOF was mixed with 100 μg of FSP neoantigen (FSP1 50 μg + FSP2 50 μg) and 100 μg of Toll-like receptor-9 agonist (CpG1826) in ultrapure water and gently shaken at room temperature for 1 hour, and then lyophilized to obtain SU-MOF Vac.

[0051] Thirdly, this application provides the use of the oral frameshift peptide neoantigen vaccine described in the first aspect, or the oral frameshift peptide neoantigen vaccine prepared by the method described in the second aspect, in the preparation of a drug for treating Lynch syndrome-related colorectal cancer.

[0052] The technical solution provided in this application will be described in detail below with reference to embodiments. In the accompanying drawings of the experimental results, "*, **, ***" are typically used to represent the significance level of a statistical test, corresponding to different significance levels. P Value range, where "*" represents P<0.05 ,"**"represent P<0.01 ,"***"represent P<0.001 .

[0053] Immunoprophylaxis against LS-CRC was achieved by regulating microfolded cells (M cells) and goblet cells through mechanomechanical-biological effects. A sea urchin-like metal-organic framework (SU-MOF) was synthesized by reacting zinc ions with 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid via a hydrothermal method. A relatively smooth metal-organic framework (MOF) was also synthesized as a control. Then, MOF Vac / SU-MOF Vac was prepared by loading FSPs with a Toll-like receptor 9 agonist (CpG1826) through electrostatic interactions and / or van der Waals forces.

[0054] in, Figure 1 A is a scanning electron microscope image of MOF / SU-MOF. Figure 1 B is the statistical diagram of hydrated particle size of MOF / SU-MOF. Figure 1 C represents the X-ray diffraction analysis of MOF / SU-MOF. Figure 1 D represents the X-ray photoelectron spectroscopy analysis of MOF / SU-MOF. Figure 1 E represents the specific surface area analysis of MOF / SU-MOF. Figure 1 F is the elemental analysis diagram obtained by transmission electron microscopy. Figure 1 G is a statistical plot of the zeta potential of MOF / MOF Vac / SU-MOF / SU-MOF Vac. Figure 1 H represents the particle size distribution of MOF Vac / SU-MOF Vac under simulated gastric fluid conditions. Figure 1 I represents the particle size distribution of MOF Vac / SU-MOF Vac under simulated intestinal fluid conditions. Figure 1 J represents the integrity map of vaccines (OVA and CpG1826) analyzed under simulated gastric fluid conditions using agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 K represents the integrity map of vaccines (OVA and CpG1826) under simulated intestinal fluid conditions, obtained by agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0055] Figure 2A shows the in vitro fluorescence imaging of CY5-labeled OVA in gastrointestinal tissue after oral vaccination and the corresponding average fluorescence intensity statistics. Figure 2 B shows the immunofluorescence image of Pascal's knot and the corresponding value per mm. 2 The number of OVA-positive cells; the white circle represents Pareto's knot, and the yellow circle represents the corresponding magnified immunofluorescence image. Figure 2 C is a schematic diagram of a human intestinal M-cell-like model. Figure 2 D is the mean fluorescence intensity (MFI) plot of the lower chamber culture supernatant. Figure 2 E is the MFI diagram of RAJI cells in the lower chamber.

[0056] Figure 3 A represents the H&E analysis of enriched M-cell mouse small intestinal organoids and the presence of Gp2 in the organoids. + Immunofluorescence analysis of cells. Figure 3 B is a dot plot of marker genes for each cell subtype. Figure 3 C is the UMAP analysis diagram of the overall cell subtypes. Figure 3 D is a graph showing the overall percentage of cell subtypes. Figure 3 E represents the percentage of goblet cells. Figure 3 F is a graph showing the enrichment of Reactome in M ​​cells. Figure 3 G represents the RNA expression map of CDC42 in M ​​cells. Figure 3 H is Gp2 + Flow cytometry analysis of activated CDC42 expression in cells. Figure 3 I represents Gp2 enriched in the small intestinal organoids of M-cell-type mice. + Flow cytometry histograms of OVA in cells and corresponding mean MFI plots Figure 3 J is the MFI image of RAJI cells in the lower chamber of Transwell. Figure 3 K is the MFI diagram of the lower chamber supernatant of the Transwell. Figure 3 L shows the PAS staining analysis of the small intestine near the cecum and the number of goblet cells in each villus. Figure 3 M is an immunofluorescence image of dextran-FITC / Muc2-CY3 and a map showing the number of GAPs for each villus; white arrows indicate GAPs. Figure 3 N represents the CD103 concentration in mesenteric lymph nodes (mLN). + Statistical analysis diagram of dendritic cells (DCs) Figure 3 O is CD3 in mLN + CD4 + FoxP3 + Flow cytometry plots and quantitative analysis graphs of cells.

[0057] Figure 4 A represents CD11c in mLN. + CD80+ CD86 + Flow cytometry plots and quantitative analysis diagrams of cells. Figure 4 B represents CD11c in mLN. + SIINFEKL-H2K b+ Flow cytometry plots and quantitative analysis diagrams of cells. Figure 4 C represents CD8 after SIINFEKL restimulation. + CD8 in T cells + Gran-B + Perf + Flow cytometry plots and quantitative analysis graphs of cells. Figure 4 D is related to CD8 + JC-1 staining images and corresponding J-monomer / J-aggregate ratios of B16-OVA cells co-cultured with T cells. Figure 4 E is a graph showing B16-OVA cell-specific lysis as determined by the CCK-8 assay. Figure 4 F is an analysis graph of interferon-γ (IFN-γ) by enzyme-linked immunosorbent assay (ELISA).

[0058] Figure 5 A is a schematic diagram of the LS-CRC mouse model construction. Figure 5 B shows the agarose gel electrophoresis analysis of Lgr5-CreERT2 and Msh2. Figure 5 C is an immunohistochemical staining analysis diagram of Msh2 in colon tissue. Figure 5 D is a timeline diagram illustrating the efficacy of SU-MOF Vac in the prevention of LS-CRC. Figure 5 E shows colonoscopy images at different time points, with black arrows indicating polyps. Figure 5 F is a gross view of the colon; the red arrows indicate polyps. Figure 5 G is a statistical analysis chart of the number of tumor nodules in the colon. Figure 5 H is a statistical analysis graph showing the size distribution of tumors in the colon. Figure 5 Image I shows the H&E staining analysis of the colon, where the black arrows indicate adenocarcinoma lesions and the black boxes represent magnified areas.

[0059] Figure 6 A shows H&E staining images of major organs in mice from different treatment groups. Figure 6 B shows the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (Urea), and creatinine (Crea) in mice from different treatment groups. Figure 6 C is a line graph showing the weight of mice in different treatment groups.

[0060] Example 1: Preparation of MOF Vac / SU-MOF Vac Preparation of MOF / SU-MOF 40 mg Zn(NO3)2·6H2O, 15 mg 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid, 100 μL ethanol, and 2 mL of [unclear text - likely a typo, should be removed] N , N After sonicating with dimethylformamide (DMF) for 5 minutes, the mixture was incubated at 100°C for 3 hours. The precipitate was then collected, washed with DMF for 2 days, and then washed with ethanol for 2 days to obtain MOF with a thornless surface topology.

[0061] 40 mg Zn(NO3)2·6H2O, 15 mg 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid, 2 μL trifluoroacetic acid, 100 μL ethanol, and 2 mL N , N SU-MOF can be prepared by sonicating dimethylformamide (DEF) for 5 minutes and incubating it at 100°C for 12 hours. The product is then washed with DMF and ethanol.

[0062] Preparation of MOF Vac / SU-MOF Vac 1 mg MOF / SU-MOF was mixed with 100 μg of FSP neoantigen (FSP1 50 μg + FSP2 50 μg) and 100 μg of Toll-like receptor-9 agonist (CpG oligodeoxynucleotide (CpG1826)) in ultrapure water at room temperature with gentle shaking for 1 hour, and then lyophilized to obtain MOF Vac / SU-MOF Vac.

[0063] The amino acid sequence of FSP1 is AEGRWPCWLLRAH; The amino acid sequence of FSP2 is LSSPKRVMYRLSVGCLRPTL; The CpG1826 sequence is: 5'-TCCATGACGTTCCTGACGTT-3'.

[0064] Example 2 Characterization of MOF Vac / SU-MOF Vac microstructure The hydrated particle size and surface potential of MOF / SU-MOF were detected using dynamic light scattering (DLS, Malvern Instruments Nano-ZS90). The surface morphology of MOF / SU-MOF was characterized using scanning electron microscopy (SEM, FEI Sirion 200). X-ray diffraction (XRD, Rigaku SmartLab SE) and X-ray photoelectron spectroscopy (XPS, ThermoScientific ESCALAB Xi) were used to determine the hydration size and surface potential. + The crystal structure and valence state of MOF / SU-MOF were analyzed. The specific surface area of ​​MOF / SU-MOF was characterized using the Brunauer–Emmett–Teller (BET, Micromeritics ASAP 2460) method. The major elements of SU-MOF Vac were analyzed using transmission electron microscopy (TEM, FEI Talos F200X G2).

[0065] Experimental results are as follows Figure 1 As shown in the figure, SEM analysis revealed that SU-MOF exhibits a more significant and sharper nanospike-like surface topology compared to MOF. Figure 1 A), which is expected to generate stronger interfacial mechanical stimulation. The particle sizes of MOF and SU-MOF are 28.3±4.4 μm and 48.6±10.3 μm, respectively. Figure 1 B). XRD results show that the diffraction peaks of both MOF and SU-MOF match those of the simulated MOF-74-Ⅲ. Figure 1 C). XPS showed clear Zn 2p in both MOFs. 1 / 2 With Zn 2p 3 / 2 peak( Figure 1 (D) indicates the presence of Zn-O bonds. The specific surface areas of MOF and SU-MOF, as determined by the BET method, were 420.0 m². 2 / g and 321.4 m 2 / g ( Figure 1 E). After loading FSP and CpG onto SU-MOF, TEM elemental analysis detected the presence of Zn, P, and S elements in the SU-MOF Vac. Figure 1 F). DLS results showed that the zeta potential of both MOFs decreased by approximately 30% (F). Figure 1 G).

[0066] The above results indicate that both MOF Vac and SU-MOF Vac have been successfully prepared.

[0067] SU-MOF Vac in vitro biostability assessment MOF / SU-MOF Vac (1 mg MOF / SU-MOF, 100 μg OVA, and 100 μg CpG) were incubated in simulated gastric and intestinal fluids, respectively. Particle size was measured and photographed using an inverted microscope (Nikon ECLIPSE Ti2-U). The protective efficacy against CpG and OVA was evaluated using agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively.

[0068] Experimental results are as follows Figure 1 As shown in HK, MOF Vac and SU-MOF Vac almost completely maintained their original particle size during 6 hours of incubation in simulated gastric fluid. Figure 1 H), and compared with MOF Vac, SU-MOF Vac showed stronger vaccine protection (H). Figure 1 J). Both MOF Vac and SU-MOF Vac showed varying degrees of disintegration within 72 hours in simulated intestinal fluid. MOF Vac completely disintegrated into small rod-like structures, while SU-MOF Vac retained its sea urchin-like morphology but was smaller in size. Figure 1 I). Furthermore, MOFVac and SU-MOF Vac almost completely maintained the integrity of the vaccine structure in simulated intestinal fluid. Figure 1 (K), indicating that the difference in the degree of disintegration of the MOF crystal structure does not affect its protective ability against vaccines.

[0069] These results indicate that SU-MOF Vac possesses excellent biostability and provides outstanding vaccine protection under gastrointestinal stimulation. Therefore, SU-MOF Vac demonstrates great potential in enhancing intestinal retention and mechanodynamically mediated immune activation.

[0070] Example 3: SU-MOF Vac enhances intestinal retention and intestinal epithelial permeability SU-MOF Vac enhances intestinal retention Mice were first orally administered castor oil (400 μL per mouse) and fasted for 12 hours, followed by oral administration of different vaccines (0.2 mg MOF / SU-MOF, 20 μg OVA-CY5, and 20 μg CpG). Gastrointestinal tissues were harvested at different time points and fluorescence imaging was performed using a small animal multimodal imaging system (BLT AniView100).

[0071] Experimental results are as follows Figure 2As shown in Figure A, the fluorescence of OVA-CY5 in the Vac group was almost undetectable within 36 hours; while both MOFVac and SU-MOF Vac maintained a continuous fluorescence signal until 72 hours. Furthermore, compared to MOF Vac, SU-MOF Vac exhibited significantly stronger intestinal retention. P <0.05).

[0072] The above results indicate that SU-MOF Vac can significantly enhance intestinal retention.

[0073] SU-MOF Vac enhances intestinal epithelial permeability In the mucosal permeability assessment, a 4 cm segment of the intestine containing Peyer's patches was ligated and injected with the appropriate vaccine (0.2 mg MOF / SU-MOF, 20 μg OVA-CY3, and 20 μg CpG). After incubation in the dark for 30 minutes, the tissue was fixed overnight for CD11c immunofluorescence staining.

[0074] Experimental results are as follows Figure 2 As shown in Figure B, both MOF Vac and SU-MOF Vac induced significantly stronger fluorescence signals of the antigen (OVA-CY3) compared to Vac. Furthermore, compared to MOF Vac, SU-MOF Vac significantly increased the permeability of OVA by 2.1 times.

[0075] Assessment of antigen transcellular transport using a human gut M-cell-like model Caco-2 cells (5 × 10⁻⁶) 5 RAJI cells (2.5 × 10⁶ cells / well) were seeded in the upper membrane chamber of a Transwell and cultured for 14 days. Subsequently, RAJI cells (2.5 × 10⁶ cells / well) were seeded in the upper membrane chamber of a Transwell and cultured for 14 days. 5 The cells were seeded in the lower chamber ( / well) and incubated for 7 days. The indicated vaccine was added to the upper chamber (MOF / SU-MOF: 50 μg / mL, OVA-CY3: 100 μg / mL, CpG: 100 μg / mL). After incubation in the dark for 1 hour, the fluorescence intensity of the RAJI cells in the lower chamber was detected by flow cytometry (Beckman Cytoflex S), and the fluorescence intensity of the corresponding lower chamber supernatant was detected by a multi-functional microplate reader (PerkinElmerEnSpire).

[0076] Experimental results are as follows Figure 2 As shown in CE, compared with Vac, both MOF Vac and SU-MOF Vac significantly increased the fluorescence intensity of OVA-CY3 in lower chamber RAJI cells and their culture supernatant. Figure 2CE). Compared with MOF Vac, SU-MOF Vac further increased the fluorescence intensity of OVA-CY3 in lower chamber RAJI cells and their culture supernatant by approximately 60% ( Figure 2 (CE), suggesting that nano-spiky particles can enhance transcellular transport of antigens in M ​​cells.

[0077] The results showed that SU-MOF Vac with nanospikes can achieve stronger intestinal retention and mucosal penetration by enhancing interfacial anchoring.

[0078] Example 4: SU-MOF Vac enhances antigen endocytosis and transcytosis in M ​​cells and alleviates goblet cell-mediated immunosuppression. Constructing mouse small intestinal organoids enriched with M cells Mouse intestinal organoids were developed using MasterAim. ® Mouse intestinal organoids were cultured in complete culture medium. Intestinal tissue from C57BL / 6j mice was isolated, peripheral blood vessels and adipose tissue were removed, and the tissue was minced into 1-2 mm pieces. The pieces were then washed with antibiotic-containing DPBS (penicillin 1,500 U / mL, streptomycin 1,500 µg / mL, amphotericin B 500 µg / mL). The tissue fragments were then placed in MasterAim... ® In intestinal tissue digestion medium, incubate on ice for 30 minutes. After centrifugation, resuspend in pre-chilled DPBS and filter four times through a 70 µm pore size filter to enrich crypts. The enriched crypts are seeded into Matrigel and incubated in MasterAim. ® Mouse intestinal organoids were cultured in complete culture medium (purchased from Amin Medical). Stimulation with nuclear factor-κB receptor activator ligand (RANKL, 500 ng / mL) for 5 days was performed to obtain small intestinal organoids enriched with M cells.

[0079] Experimental results are as follows Figure 3 As shown in Figure A, glycoprotein 2 was positive (Gp2) after RANKL stimulation. + The proportion of cells (which are classic surface markers of M cells) increased significantly from 3.7% to 14.6%. Figure 3 A) indicates that mouse small intestinal organoids enriched with M cells were successfully constructed.

[0080] Single-cell RNA sequencing (scRNA-seq) analysis of small intestinal organoids enriched with M cells Small intestinal organoids enriched with M cells were treated with MOF Vac and SU-MOF Vac (MOF / SU-MOF: 50 μg / mL, FSP: 100 μg / mL, CpG: 100 μg / mL) for 24 hours, respectively. The organoids were then collected and processed using MasterAim. ®The cells were digested using ExpressEnzyme (purchased from AminMed). Single cells were resuspended for RNA-Seq library construction. The libraries were sequenced on a GeneMind SURFSeq 5,000. Sequencing data were analyzed using the Seurat R package. After quality control, the count data were normalized, and the top 2,000 hypervariable genes were selected for subsequent comprehensive gene analysis.

[0081] Experimental results are as follows Figure 3 As shown in Figure BG, after visualizing the scRNA-seq data of the MOF Vac and SU-MOF Vac groups, the total cells within the organoids were divided into 10 different subtypes, including stem cells, transitamplifying cells, enterocyte progenitors, enterocytes, paneth cells, enteroendocrine cells, M cells, goblet cells, tuft cells, and T cells. Figure 3 BC). Compared with the MOF Vac group, the proportion of goblet cells in the SU-MOF Vac group was significantly reduced by 39.5% (BC). Figure 3 DE, with no significant changes in the proportions of other cells. Reactome enrichment analysis showed that in M ​​cells, pathways associated with Rat sarcoma virus homolog guanosine triphosphatases (Rho GTPases) were significantly activated. Figure 3 F). Among all the upregulated genes associated with Rho GTPase signaling pathway activation, CDC42 was upregulated most significantly. Figure 3 G). This suggests that CDC42 may play a key role in SU-MOF Vac-mediated antigen endocytosis / transcytosis in M ​​cells.

[0082] Analysis of CDC42 activation in M ​​cells Single cells obtained from small intestinal organoid digestion enriched with M cells were stained with anti-Gp2-PE antibody to label the M cells. After incubation with the indicated vaccine (antigen: FSP) for 30 minutes, the cells were fixed and permeabilized using a Cyto-Fast™ Fix / Perm Buffer Set. Staining with anti-CDC42 antibody and FITC-labeled secondary antibody was then performed for Gp2 analysis. + CDC42 + cell.

[0083] Experimental results are as follows Figure 3 As shown in Figure H, compared with PBS, SU-MOF Vac significantly increased the activated CDC42 in M ​​cells by 57.1% ( Figure 3 H), indicating that nano-spiky thorns can activate CDC42 in M ​​cells.

[0084] Assess the endocytosis and transcellular transport of antigens loaded via M cells. To assess antigen endocytosis in M ​​cells, single cells obtained from the digestion of small intestinal organoids enriched with M cells were stained with anti-Gp2-PE antibody to label M cells and resuspended in ultra-low adsorption 96-well plates (Corning, 3474). Subsequently, they were incubated for 1 hour under light-protected conditions with different vaccines (FITC-labeled OVA) / ML141 (5 μM), and the fluorescence intensity of OVA-FITC in M ​​cells was detected by flow cytometry. To assess transcellular transport of antigens in M ​​cells, a human intestinal M cell-like model was established as described above. OVA-CY3 (100 μg / mL) was added to the upper chamber and incubated for 30 minutes. After discarding the supernatant, Caco-2 cells were incubated with the indicated material (MOF / SU-MOF: 50 μg / mL) / CDC42 inhibitor ML141 (5 μM) for 1 hour. RAJI cells and their supernatant were collected from the lower chamber for fluorescence intensity quantification.

[0085] Experimental results are as follows Figure 3 As shown in IK, ML141 treatment completely blocked SU-MOF Vac-mediated antigen endocytosis and transcellular transport in M ​​cells. Figure 3 These data indicate that SU-MOF Vac promotes antigen endocytosis and transcellular transport in M ​​cells by activating CDC42.

[0086] goblet cell assessment C57BL / 6j mice were orally administered different vaccines (0.2 mg MOF / SU-MOF, 20 μg FSP, and 20 μg CpG) every 3 days for a total of 4 times. Subsequently, the mice were orally administered castor oil (400 μL per mouse) and fasted for 12 hours. A 4 cm segment of small intestine near the cecum was harvested, and the number of goblet cells per villus was assessed by iodate-Schiff (PAS) staining. The remaining segment of small intestine near the cecum was ligated and injected with dextran-FITC (2 mg / mouse) for 30 minutes; after washing with PBS, the cells were stained with mucin 2 (Muc2-CY3) to visualize goblet cell-associated antigen channels (GAPs). After repeated oral immunization (antigen: OVA) according to the aforementioned protocol, the mice were sacrificed, and mesenteric lymph nodes (mLN) were isolated and single-cell suspensions were prepared for flow cytometry (FCM) analysis. Cells were used to detect CD103.+ DCs (CD103 antibody, CD11c antibody) and Treg cells (CD3 antibody, CD4 antibody, FoxP3 antibody).

[0087] Experimental results are as follows Figure 3 As shown in LO, compared with the PBS group, the SU-MOF Vac group reduced the number of goblet cells and GAPs by 41.4% and 52.9%, respectively. Figure 3 (LM). Furthermore, compared to the PBS group, the SU-MOF Vac group reduced CD103 in mLN. + The proportions of DCs and Treg cells decreased by 50.6% and 14.4%, respectively. Figure 3 NO). The above results indicate that SU-MOF Vac can induce a reduction in the number of goblet cells and GAPs, and further alleviate the effects of CD103. + DCs-Treg cell axis-mediated immunosuppression.

[0088] These results indicate that SU-MOF Vac with a spiky surface topology can not only promote antigen endocytosis / transcellular transport in M ​​cells by activating CDC42, but also alleviate goblet cell-CD103 syndrome. + DCs-Treg cells, an immunosuppressive axis, mediate immune tolerance, potentially activating antigen-specific CD8 cells through a dual mechanism. + T cell response.

[0089] Example 5: SU-MOF Vac induces robust intestinal mucosal immune activation in vivo. After repeated oral immunization (antigen: OVA) according to the aforementioned protocol, mice were sacrificed, and mLN and spleen were isolated and single-cell suspensions were prepared for flow cytometry (FCM) analysis. Cells were used to detect DC activation status (CD11c antibody, CD80 antibody, CD86 antibody, SIINFEKL-H2K) in mLN. b Antibodies). Subsequently, CD8 antibodies were sorted from spleen cells. + T cells (Beckman, Moflo XDP high-speed cell sorting system) were used and restimulated with SIINFEKL for 36 hours to evaluate antigen-specific antitumor CD8. + T cell response. Cells were stained with perforin and granzyme-B to analyze cytotoxic CD8. + T cells. The remaining cells were co-cultured with B16-OVA cells for 24 hours (CD8). +T cell / B16-OVA cell ratio = 20 / 1. Early apoptosis of B16-OVA cells was characterized by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylimidazolium carbocyanin (JC-1) staining, and the secretion of interferon-γ (IFN-γ) in the supernatant was detected by ELISA kit.

[0090] Experimental results are as follows Figure 4 As shown in AF, compared with PBS, both Vac and MOF Vac had no significant effect on DC activation; while SU-MOF Vac activated CD80 / 86 and SIINFEKL-H2K. b They increased by 177.8% and 255.5% respectively. Figure 4 (AB), indicating a potent DC activation effect. Furthermore, compared to PBS, MOF Vac and SU-MOF Vac, respectively, reduced CD8+ activation. + Perforin (Perf) / granzyme B (Gran-B) expression increased by 67.9% and 117% in T cells, respectively. Figure 4 C). Considering that mitochondrial membrane potential (MMP) can predict early apoptosis, JC-1 staining was further used to assess CD8+ in SU-MOF Vac treatment. + The effect of T cells on MMPs in B16-OVA cells. Results showed that, compared to the PBS group, only the SU-MOF Vac group exhibited a significantly increased J-monomer / J-aggregate ratio (1.4-fold increase). Figure 4 D). Similarly, the Cell Count Kit-8 (CCK-8) assay showed that the SU-MOFVac group had CD8... + T cells showed a 3.2-fold increase in cytotoxicity against B16-OVA cells. Figure 4 E). In addition, CD8 + The amount of IFN-γ secreted by T cells also increased by 70.4%. Figure 4 F).

[0091] Oral administration of SU-MOF Vac can effectively activate the anti-tumor CD8 in the intestinal mucosa. + The T-cell response suggests that it has potential anti-tumor preventive efficacy.

[0092] Example 6: SU-MOF Vac-induced immunoprophylaxis against Lynch syndrome-associated colorectal cancer Will Msh2 flox / flox mice and Lgr5-CreERT2 + / - Mouse hybridization to obtain Lgr5-CreERT2 + / - ; Msh2flox / flox Mice (denoted as LS-CRC mice). The genotypes of LS-CRC mice were identified by PCR, as shown in Table 1.

[0093] Table 1. PCR identification primer sequence list

[0094] Six- to eight-week-old LS-CRC mice were orally administered different vaccines (antigen: FSP) every three days for a total of six times. Five days after the last immunization, LS-CRC mice were intraperitoneally injected with tamoxifen (TMF) (2.5 mg per mouse, dissolved in corn oil) for five consecutive days. To accelerate the carcinogenesis and progression of LS-CRC, mice were given drinking water containing sodium dextran sulfate (DSS) (w / v: 1%) for seven days, followed by a 14-day supply of DSS-free water. During the six consecutive DSS treatment cycles, the occurrence and development of LS-CRC were non-invasively monitored using a small animal endoscopy system (Yuyan Instruments, YAN-E30). Mice were sacrificed at the end of the treatment, and the colon was harvested for gross observation of tumor nodules. Colon tissue was used for H&E staining analysis and Msh2 immunohistochemical analysis.

[0095] Experimental results are as follows Figure 5 As shown in the AI, to simulate the clinical scenario of LS-CRC, a conditional knockout mouse model of LS-CRC was first established, and... Msh2 flox / flox mice and Lgr5-CreERT2 + / - Mouse hybridization to obtain Lgr5-CreERT2 + / - ; Msh2 flox / flox Mice (LS-CRC mice). After 5 consecutive days of TMF induction, Msh2 in intestinal stem cells of LS-CRC mice was conditionally inactivated ( Figure 5 A); subsequently, DSS stimulation was used to promote the carcinogenesis and progression of LS-CRC. Agarose gel electrophoresis and immunohistochemical staining analysis jointly verified the successful establishment of the LS-CRC mouse model. Figure 5 BC). To evaluate the immunoprophylactic effect of SU-MOFVac on LS-CRC, LS-CRC mice were orally immunized (antigen: FSP) every 3 days for a total of 6 immunizations. On day 7 post-immunization, LS-CRC mice were subjected to continuous TMF induction and intermittent DSS treatment for 4 months. Figure 5 D). During this period, the occurrence and progression of LS-CRC were non-invasively monitored using a small animal endoscopy system. One month after DSS treatment, a certain number of polyps appeared in the colon of the PBS, Vac, and MOF Vac groups, while almost no visible polyps were observed in the SU-MOF Vac group. Figure 5 E). By 4 months of DSS treatment, numerous and relatively large tumor nodules were widely distributed in the colon of the PBS and Vac groups. Figure 5 E). In comparison, the tumor nodule volume was relatively smaller in the MOF Vac and SU-MOF Vac groups, and the tumor-suppressing effect of SU-MOF Vac was significantly enhanced compared to MOF Vac. Figure 5 E).

[0096] LS-CRC mice were subsequently sacrificed, and their colons were harvested for further analysis. Gross examination of the colons of SU-MOF Vac-immunized mice revealed a significantly fewer tumor nodules compared to other groups. Figure 5 FG). Furthermore, no large tumor nodules (2-4 mm) were observed in the SU-MOF Vac group, while larger nodules were observed in the PBS, Vac, and MOF Vac groups. Notably, four out of five mice immunized with SU-MOF Vac developed only smaller tumor nodules (<1 mm), and one mouse showed no visible tumor nodules at all. Figure 5 H). Histopathological analysis of the colon showed numerous adenocarcinoma lesions with gastrointestinal intraepithelial neoplasia in the PBS, Vac, and MOF Vac groups, while no obvious lesions were observed in the SU-MOF Vac group. Figure 5 I).

[0097] This indicates that SU-MOF Vac can serve as a potent and safe oral vaccine for the immunization of LS-CRC.

[0098] Example 7: Biosafety evaluation of SU-MOF Vac The in vivo biosafety of SU-MOF Vac was systematically evaluated through H&E staining of major organs, detection of serum biochemical indicators, and long-term weight monitoring.

[0099] Experimental results are as follows Figure 6 As shown in the AC results, oral administration of SU-MOF Vac did not cause significant side effects in mice. This indicates that SU-MOF Vac has good in vivo biocompatibility.

[0100] This application achieves immunoprophylaxis against LS-CRC by regulating microfolded cells (M cells) and goblet cells through a mechano-biological approach. Urchin-like MOFs (denoted as SU-MOFs) are synthesized by reacting zinc ions with 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid under hydrothermal conditions. Then, FSPs and a Toll-like receptor 9 agonist (CpG1826) are loaded via electrostatic interactions and / or van der Waals forces to prepare MOF Vac / SU-MOF Vac. After oral administration, SU-MOF Vac enhances intestinal retention and intestinal epithelial penetration through its surface nanospikes. Furthermore, it activates CDC42 via a mechano-biological approach, promoting antigen endocytosis / transcellular transport via M cells and improving goblet cell-CD103 expression. + The DC-Treg cell immunosuppressive axis. In addition, SU-MOF Vac can activate intestinal mucosal immunity in vivo and can prevent the occurrence and development of LS-CRC.

[0101] The terms "comprising" and "having," and any variations thereof, in the specification, claims, and accompanying drawings of this application are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such processes, methods, products, or devices. The terms "first," "second," and "third," etc., are used to distinguish different objects, etc., and do not indicate a sequence, nor do they limit "first," "second," and "third" to different types.

[0102] In the description of the embodiments of this application, terms such as "exemplary," "for example," or "for instance" are used to indicate examples, illustrations, or explanations. Any embodiment or design described as "exemplary," "for example," or "for instance" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of terms such as "exemplary," "for example," or "for instance" is intended to present the relevant concepts in a concrete manner.

[0103] In the description of the embodiments of this application, unless otherwise stated, " / " means "or". For example, A / B can mean A or B. The "and / or" in the text is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone. In addition, in the description of the embodiments of this application, "multiple" means two or more.

[0104] In some processes described in the embodiments of this application, multiple operations or steps are included in a specific order. However, it should be understood that these operations or steps may not be executed in the order they appear in the embodiments of this application, or they may be executed in parallel. The sequence number of the operation is only used to distinguish different operations, and the sequence number itself does not represent any execution order. In addition, these processes may include more or fewer operations, and these operations or steps may be executed sequentially or in parallel, and these operations or steps may be combined.

[0105] The above are merely preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. An oral frameshift peptide neoantigen vaccine, characterized in that, The oral frameshift peptide neoantigen vaccine has a delivery carrier with a sea urchin-like metal-organic framework and a sea urchin-like spine structure on its surface.

2. The oral frameshift peptide neoantigen vaccine as described in claim 1, characterized in that, It contains two frameshift peptides, one of which has the amino acid sequence shown in SEQ ID NO: 1, and the other has the amino acid sequence shown in SEQ ID NO:

2.

3. The oral frameshift peptide neoantigen vaccine as described in claim 1, characterized in that, The oral frameshift peptide neoantigen vaccine promotes the endocytosis and transcytosis of antigens via microfolded cells by activating cell division cycle protein 42.

4. The oral frameshift peptide neoantigen vaccine as described in claim 1, characterized in that, The oral frameshift peptide neoantigen vaccine can alleviate goblet cell-CD103. + Dendritic cell-regulatory T cell immunosuppressive axis-mediated immune tolerance activates antigen-specific CD8. + T cell response.

5. A method for preparing an oral frameshift peptide neoantigen vaccine as described in claim 1, characterized in that, Includes the following steps: An oral frameshift peptide neoantigen vaccine was obtained by mixing a sea urchin-shaped metal-organic framework with a frameshift peptide neoantigen in ultrapure water.

6. The method for preparing the oral frameshift peptide neoantigen vaccine as described in claim 5, characterized in that, The preparation method of sea urchin-shaped metal-organic frameworks includes the following steps: Zn(NO3)2·6H2O, 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid, trifluoroacetic acid, anhydrous ethanol and N , N - Dimethylformamide was used for ultrasonic incubation and washing to obtain a sea urchin-like metal-organic framework.

7. The method for preparing the oral frameshift peptide neoantigen vaccine as described in claim 6, characterized in that, The mass ratio of Zn(NO3)2·6H2O to 3,3''-dihydroxy-2',5'-dimethyl-[1,1':4',1''-terphenyl]-4,4''-dicarboxylic acid is 1:(0.3~0.4).

8. The method for preparing the oral frameshift peptide neoantigen vaccine as described in claim 5, characterized in that, The method of mixing sea urchin-shaped metal-organic frameworks with frameshift peptide neoantigens in ultrapure water to obtain an oral frameshift peptide neoantigen vaccine comprises: The sea urchin-like metal-organic framework was mixed with a first frameshift peptide neoantigen, a second frameshift peptide neoantigen, a Toll-like receptor-9 agonist and a solvent, and then freeze-dried to obtain an oral frameshift peptide neoantigen vaccine. The amino acid sequence of the first frameshift peptide neoantigen is shown in SEQ ID NO: 1; The amino acid sequence of the second frameshift peptide neoantigen is shown in SEQ ID NO: 2; The sequence of the Toll-like receptor-9 agonist is shown in SEQ ID NO:

3.

9. The method for preparing the oral frameshift peptide neoantigen vaccine as described in claim 8, characterized in that, The mass ratio of the sea urchin-like metal-organic framework, the first frameshift peptide neoantigen, and the second frameshift peptide neoantigen is 20:(0.9~1.1):(0.9~1.1).

10. The use of an oral frameshift peptide neoantigen vaccine as described in any one of claims 1 to 4, or an oral frameshift peptide neoantigen vaccine prepared by the method described in any one of claims 5 to 9, in the preparation of a medicament for treating Lynch syndrome-related colorectal cancer.

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