Application of protein nanoparticles and composition thereof in preparation of medicines for preventing and treating tumors

Abstract

The invention discloses application of protein nanoparticles and a composition thereof in preparation of medicines for preventing and treating tumors. The amino acid sequence of the protein nanoparticle is shown as SEQ ID NO: 12, the protein nanoparticle is combined with hPDL1-NP protein nanoparticles obtained by self-assembly of fusion proteins shown as SEQ ID NO: 20 and 21 and Ad-Re-NP protein nanoparticles obtained by self-assembly of fusion proteins shown as SEQ ID NO: 22 and 25, and the vaccine for preventing and treating tumors is prepared. The method has the obvious advantages that the tumor load is obviously reduced, the T cell infiltration is increased, and the antibody titer is maintained and increased. A novel prevention and control tool is provided for CRC high-risk groups (such as familial adenomatous polyposis patients), and a new path is opened up for immune combined treatment of metabolism-related tumors such as liver cancer and pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the field of cancer treatment, and more particularly to the use of protein nanoparticles in the preparation of drugs for preventing and treating tumors. Background Art

[0002] Colorectal cancer is the third most common malignancy worldwide, with a very low five-year survival rate for patients in advanced stages. Traditional therapies (surgery, chemotherapy, and radiotherapy) are effective for early-stage patients but are less effective in advanced stages. According to molecular classifications, up to 95% of patients are classified as microsatellite stable (MSS-CRC). Immune checkpoint inhibitors (such as PD-1 / PDL1 monoclonal antibodies) are only effective in patients with microsatellite instability-high (MSI-H) chromosomal abnormalities, with response rates reaching 30% to 40%. However, the response rate for MSS-CRC patients to immune checkpoint inhibitors is less than 5%. The prognosis for MSS-CRC patients is significantly worse than that for MSI-H patients. Currently, there are no targeted therapies for MSS-CRC patients, and standard treatments have a very low response rate, with a median expected survival of only 6 to 7 months. The prognosis is extremely poor, and more effective treatment options are urgently needed.

[0003] The goal of immunotherapy is to awaken and maintain the body's natural immune response to tumors. Patients' responses to immunotherapy vary depending on the immune characteristics of their tumors. Therefore, tumors are categorized as "cold" or "hot" based on their response to immune checkpoint blockade (ICB). Cold tumors are resistant to immunotherapy due to a lack of immune cell infiltration, while hot tumors respond well to immunotherapy due to their highly active immune cells.

[0004] In recent years, the synergistic effect of metabolic disruption and immunosuppression in the tumor microenvironment has become a research focus. Studies have shown that PCSK9 leads to cholesterol accumulation by degrading LDL-R, while also weakening TCR signaling in CD8+ T cells. The PDL1 / PD-1 pathway directly induces T cell exhaustion. While single immune checkpoint blockade cannot reverse tumor microenvironment-mediated immunosuppression, inhibiting PCSK9 can restore the antigen presentation capacity of T cells and produce a synergistic effect with PDL1 blockade, transforming "cold tumors" into "hot tumors" and achieving therapeutic effects.

[0005] Mutated tumor cells express abnormal proteins not found in normal cells, known as neoantigens. Immunotherapy targeting neoantigens can overcome the body's immune tolerance, thereby generating a stronger anti-tumor immune response. Vaccines based on neoantigens are personalized immunotherapies designed to target tumor-specific mutations (neoantigens). Their core goal is to precisely target tumors by activating the patient's own T cell immune response, thereby activating the cold tumor immune microenvironment and enhancing treatment efficacy.

[0006] Helicobacter pylori Ferritin (HPF) is a nanocage structure (about 12 nm in diameter) self-assembled from 24 subunits. It has been used in COVID-19 vaccines (displaying conserved epitopes of the S protein) and anti-cancer vaccines (targeting MICA / B stress proteins). Its self-assembly properties and stability make it an ideal antigen delivery platform. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a method for preparing a drug for preventing and treating tumors by using protein nanoparticles and a composition thereof.

[0008] The first object of the present invention is to provide a fusion protein.

[0009] The second object of the present invention is to provide protein nanoparticles obtained by self-assembly of the fusion protein.

[0010] The third object of the present invention is to provide a fusion protein composition.

[0011] The fourth object of the present invention is to provide a composition of protein nanoparticles.

[0012] The fifth object of the present invention is to provide the use of the fusion protein, the protein nanoparticles, the composition or the other composition in the preparation of drugs for preventing and treating tumors.

[0013] The sixth object of the present invention is a biomaterial.

[0014] A seventh object of the present invention is to provide a composition.

[0015] The eighth object of the present invention is to provide the use of the composition in preparing drugs for preventing and treating tumors.

[0016] In order to achieve the above object, the present invention is implemented through the following technical solutions: The present invention claims protection for the following products: A fusion protein N-hPCSK9-NP, whose amino acid sequence is shown in SEQ ID NO: 12.

[0017] The protein nanoparticles are obtained by self-assembly of the fusion protein.

[0018] A fusion protein composition comprising the fusion protein N-hPCSK9-NP and one or both of the following composition 1 or composition 2: Composition 1 contains: a fusion protein SD-hPDL1 having an amino acid sequence as shown in SEQ ID NO: 20 and a fusion protein GV-HPF having an amino acid sequence as shown in SEQ ID NO: 21, which can form hPDL1-NP protein nanoparticles; Composition 2 contains: the fusion protein SD-HPF with an amino acid sequence as shown in SEQ ID NO: 22 and the fusion protein GV-Ad-Re with an amino acid sequence as shown in SEQ ID NO: 25, which can form Ad-Re-NP protein nanoparticles.

[0019] Preferably, the molar ratio of the fusion protein having the amino acid sequence shown in SEQ ID NO: 20 to the fusion protein having the amino acid sequence shown in SEQ ID NO: 21 is 1:1.8-2.2.

[0020] As a specific embodiment, the molar ratio of the fusion protein with the amino acid sequence shown in SEQ ID NO: 20 to the fusion protein with the amino acid sequence shown in SEQ ID NO: 21 is 1:2.

[0021] Preferably, the molar ratio of the fusion protein having an amino acid sequence as shown in SEQ ID NO: 22 to the fusion protein having an amino acid sequence as shown in SEQ ID NO: 25 is 1:3.6-4.4.

[0022] As a specific embodiment, the molar ratio of the fusion protein with the amino acid sequence shown in SEQ ID NO: 22 to the fusion protein with the amino acid sequence shown in SEQ ID NO: 25 is 1:4.

[0023] Preferably, the fusion protein is contained, and the following composition 1 and composition 2: Composition 1 contains: a fusion protein having an amino acid sequence as shown in SEQ ID NO: 20 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 21; Composition 2 contains: a fusion protein with an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein with an amino acid sequence as shown in SEQ ID NO: 25.

[0024] A protein nanoparticle composition comprising the protein nanoparticle and one or two of the following protein nanoparticles 1 or 2: Protein nanoparticle 1: hPDL1-NP protein nanoparticles obtained by self-assembly of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 20 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 21; Protein nanoparticle 2: The fusion protein with the amino acid sequence shown in SEQ ID NO: 22 and the fusion protein with the amino acid sequence shown in SEQ ID NO: 25 are self-assembled, namely, Ad-Re-NP protein nanoparticles.

[0025] Preferably, the protein nanoparticles are contained, and the following protein nanoparticles 1 or protein nanoparticles 2: Protein nanoparticle 1: obtained by self-assembly of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 20 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 21; Protein nanoparticle 2: obtained by self-assembly of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 25.

[0026] Preferably, the mass ratio of the protein nanoparticles, and the protein nanoparticles 1 to the protein nanoparticles 2 is 1:0.9-1.1:0.9-1.1.

[0027] As a specific embodiment, the mass ratio of the protein nanoparticles, and protein nanoparticles 1 and protein nanoparticles 2 is 1:1:1.

[0028] The present invention also claims protection for the use of the above-mentioned fusion protein, the protein nanoparticle, the composition of the fusion protein or the composition of the protein nanoparticle in the preparation of drugs for preventing and treating tumors.

[0029] The present invention also claims protection for a biological material, which is any one of the following: (1) A nucleic acid molecule encoding a fusion protein having an amino acid sequence as shown in SEQ ID NO. 12; (2) nucleic acid molecules encoding the components of the composition; (3) an expression cassette containing the nucleic acid molecule described in (1) or (2); (4) A recombinant vector containing the nucleic acid molecule described in (1) or (2) or the expression cassette described in (3); (5) A recombinant microorganism containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4); (6) A cell line containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4).

[0030] Preferably, the tumor is a solid tumor.

[0031] As a specific embodiment, the tumor is colon cancer.

[0032] Further claimed is a composition comprising the protein nanoparticles and an adjuvant.

[0033] Preferably, the adjuvant is aluminum hydroxide adjuvant.

[0034] Preferably, the mass ratio of the composition to the adjuvant is 1:0.9-1.1.

[0035] As a specific embodiment, the mass ratio of the composition to the adjuvant is 1:1.

[0036] The present invention also claims to protect the use of the above-mentioned composition in the preparation of drugs for preventing and treating tumors.

[0037] Preferably, the tumor is a solid tumor.

[0038] As a specific embodiment, the tumor is colon cancer.

[0039] Compared with the prior art, the present invention has the following beneficial effects: This study uses Helicobacter pylori ferritin (HPF) as a carrier, coupled to the human PCSK9 catalytic domain, the extracellular domain of PDL1, and a tumor-associated antigen, to form a triple-target composite vaccine, avoiding conformational conflicts. The resulting vaccine significantly reduced tumor burden and increased T cell infiltration in a gene-edited humanized mouse model.

[0040] This study proposes a novel, integrated intervention strategy combining a PCSK9 vaccine, a PDL1 vaccine, and a neoantigen vaccine for CRC, aiming to disrupt the precancerous immunosuppressive microenvironment of CRC through immune regulation. Compared to monotherapy, this approach offers significant advantages: significantly reduced tumor burden, increased T cell infiltration, and sustained increases in antibody titers. This approach not only provides a novel prevention and control tool for high-risk CRC patients (such as those with familial adenomatous polyposis), but also opens new avenues for combined immunotherapy for metabolic-related tumors such as liver and pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the construction of N-hPCSK9-NP protein nanoparticles.

[0042] Figure 2 SDS-PAGE electrophoresis analysis results of the fusion protein 3×Epitope-HPF designed with different sequences.

[0043] Figure 3 This is the structural model diagram of N-hPCSK9-NP protein nanoparticles predicted by Alphafold2.

[0044] Figure 4 This is the purification diagram of N-hPCSK9-NP protein nanoparticle chromatography.

[0045] Figure 5 The reducing SDS-PAGE electrophoresis results of N-hPCSK9-NP protein nanoparticles.

[0046] Figure 6 This is an electron micrograph of N-hPCSK9-NP protein nanoparticles.

[0047] Figure 7 Schematic diagram of the construction of hPDL1-NP protein nanoparticles.

[0048] Figure 8 This is the structural model diagram of hPDL1-NP protein nanoparticles predicted by Alphafold2.

[0049] Figure 9 This is the purification diagram of hPDL1-NP protein nanoparticle chromatography.

[0050] Figure 10 The reducing SDS-PAGE electrophoresis results of hPDL1-NP protein nanoparticles.

[0051] Figure 11 This is an electron micrograph of hPDL1-NP protein nanoparticles.

[0052] Figure 12 Schematic diagram of the construction of Ad-Re-NP protein nanoparticles.

[0053] Figure 13 This is the structural model diagram of Ad-Re-NP protein nanoparticles predicted by Alphafold2.

[0054] Figure 14 This is the purification diagram of Ad-Re-NP protein nanoparticle chromatography.

[0055] Figure 15 The results of reducing SDS-PAGE electrophoresis of Ad-Re-NP protein nanoparticles.

[0056] Figure 16 This is an electron microscope image of Ad-Re-NP protein nanoparticles.

[0057] Figure 17 For the detection of hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice; 863, 864 and 865 are hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice; P is the positive control; B6 is a wild-type C57BL / 6J mouse; N is a blank control, a control without template; the DL2000 markers are: 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, respectively.

[0058] Figure 18 Schematic diagram of the process for immunizing mice with vaccines prepared with various nanoparticles.

[0059] Figure 19 The following are the test results of IgG antibody levels in each group of mice; A is the dynamic change diagram of hPDL1-specific IgG antibody levels; B is the dynamic change diagram of hPCSK9-specific IgG antibody levels; C is the test results of hPDL1-specific IgG antibody levels two weeks after the third immunization; D is the test results of hPCSK9-specific IgG antibody levels two weeks after the third immunization.

[0060] Figure 20 Schematic diagram of the process of immunizing mice with tumors using vaccines prepared with various nanoparticles.

[0061] Figure 21 The changes in tumor volume of mice in each group.

[0062] Figure 22 Figure 3 is the tumor volume measurement of mice in each group.

[0063] Figure 23 is the weight of mice in each group.

[0064] Figure 24 Figure 3 is the immunohistochemical staining of CD3 in tumor sections of mice in each group.

[0065] Figure 25 Multiple immunofluorescence analysis of CD8, CD4 and IFN-γ in tumor sections of mice in each group.

[0066] Figure 26 The changes in body weight of mice in each group.

[0067] Figure 27 The following are the HE staining results of various organs (heart, liver, spleen, lung and kidney) of mice in each group. DETAILED DESCRIPTION The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0068] The GvTagOpti / Sdcatcher (Gv / Sd) system based on isopeptide bonds is described in Chinese patent CN113621031A. It is a combination of peptide-linked adapters that utilizes spontaneous isopeptide bonds for protein covalent self-assembly.

[0069] hPD1 / hPDL1 / hPCSK9-C57BL / 6 humanized mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0070] Example 1 Construction of PCSK9 epitope-based nanoparticles (N-hPCSK9-NP protein nanoparticles) 1. Experimental Methods 1. Construction of fusion protein In order to display the three epitopes of PCSK9 on the surface of HPF 24-mer nanoparticles, the three epitopes of PCSK9 (amino acid sequences as shown in SEQ ID NO: 1 to 3) were connected in series in different orders through a glycine-serine (GSG) linker to form a fusion protein 3×Epitope. Helicobacter pylori ferritin (HPF, amino acid sequence as shown in SEQ ID NO: 4) was linked at the C-terminus to express the fusion protein 3×Epitope-GSG-HPF. The construction diagram is shown in FIG. Figure 1 As shown, Among them, based on the different arrangement orders of PCSK9 epitopes, the obtained 3×Epitopes are as follows (from N-terminus to C-terminus): Sequence 1 (V1): Epitope 1 - GSG - Epitope 2 - GSG - Epitope 3 (SEQ ID NO: 5); Sequence 2 (V2): Epitope 2 - GSG - Epitope 3 - GSG - Epitope 1 (SEQ ID NO: 6); Sequence 3 (V3): Epitope 2 - GSG - Epitope 1 - GSG - Epitope 3 (SEQ ID NO: 7); Sequence 4 (V4): Epitope 3 - GSG - Epitope 1 - GSG - Epitope 2 (SEQ ID NO: 8); Sequence 5 (V5): Epitope 3 - GSG - Epitope 2 - GSG - Epitope 1 (SEQ ID NO: 9); The amino acid sequences of the corresponding fusion proteins 3×Epitope-GSG-HPF are shown in SEQ ID NOs: 10 to 14.

[0071] 2. Construction of recombinant vector 6×His and a translation stop codon were added to the 3' end of five different fusion proteins 3×Epitope-GSG-HPF, and then their encoding genes were cloned between the NcoI and XhoI restriction sites of the prokaryotic expression vector pET-28a to construct five different recombinant plasmids pET-28a-3×Epitope-GSG-HPF-His.

[0072] 3. Construction of recombinant strains Five different recombinant plasmids, pET-28a-3×Epitope-GSG-HPF-His, were then transformed into BL21 competent cells and cultured overnight at 37°C. Positive clones were screened and identified by PCR. The positive clones were sequenced, and the strains with correct sequencing were retained to obtain the recombinant strains.

[0073] 4. Expression of fusion protein (1) Obtaining inclusion body proteins The recombinant strains that were sequenced correctly were re-plated on LB medium. After culturing overnight, single colonies were picked from the plate and added to 2 ml of LB medium containing the corresponding antibiotics. The culture was activated at 37°C overnight.

[0074] The next day, 1 ml of activated expression bacteria was inoculated into 1 L of LB medium and cultured at 37 °C with shaking until the OD 600 =0.6. Add isopropyl β-D-thiogalactopyranoside (IPTG) to 1 mM to induce target protein expression. After 3-4 hours of induction, harvest the cells by centrifugation at 5000 rpm / min for 15 minutes. Resuspend the cells in a small volume of culture supernatant, transfer the supernatant to a pre-weighed 50 ml centrifuge tube, and pellet the cells by centrifugation. Record the wet weight of the cell pellet and freeze at -80°C until needed.

[0075] Resuspend cells in 30 ml of lysis buffer (50 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 5% glycerol, 0.1 mM DTT, 0.1 M NaCl) and disrupt with high pressure until clear. Add pure Triton X-100 to a final concentration of 1%. Solubilize and wash cell membrane proteins by pipetting or gentle sonication. Let the lysate sit on ice for 10 minutes, then centrifuge at 10,000 rpm for 15 minutes to pellet inclusion bodies, and discard the supernatant. Wash inclusion bodies by adding 30 ml of lysis buffer containing 1% Triton X-100. Pipette or gently sonicate, let it sit on ice for 10 minutes, then centrifuge at 10,000 rpm for 15 minutes to pellet inclusion bodies, and discard the supernatant. Wash inclusion bodies again by adding 30 ml of lysis buffer. Pipette or gently sonicate, let it sit on ice for 10 minutes, then centrifuge at 10,000 rpm for 15 minutes to pellet inclusion bodies, and discard the supernatant. At this point, the purity of inclusion bodies reached about 90%.

[0076] (2) Dissolution of inclusion body proteins The washed inclusion bodies were resuspended in denaturation buffer (25 mM Tris-HCl, 500 mM NaCl, 8 M urea, 1 mM DTT, pH 7.5) to denature and dissolve them; after denaturation, the cells were centrifuged at 10,000 rpm for 15 minutes to remove residual insoluble matter.

[0077] (3) Renaturation of inclusion body proteins Adjust the concentration of denatured inclusion body protein to 1 mg / ml, transfer it into a dialysis bag, and dialyze it as follows: Initial dialysis: Place the dialysis bag in a refolding buffer containing 1M urea (1M urea, 25 mM Tris-HCl, 500 mM NaCl, 0.1 mM EDTA, 1 mM L-arginine, 5% glycerol) that is 100 times the volume of the protein solution. Dialyze at 4°C for 8 hours to allow the denaturant to slowly diffuse out of the dialysis bag and the protein to begin refolding.

[0078] Dialyze again: Follow the above method to replace the refolding buffer (25 mM Tris-HCl, 500 mM NaCl, 0.1 mM EDTA, 1 mM L-arginine, 10% glycerol) and dialyze at 4°C for 16 hours. The dialysate does not contain denaturant.

[0079] Final dialysis: Follow the above method to replace the refolding buffer (25 mM Tris-HCl, 150 mM NaCl) and dialyze at 4°C for 16 hours.

[0080] 5. Protein purification Select a Superose 6 increase molecular sieve column and purify on an AKTA purifier. Buffer: 25 mM Tris-HCl, 150 mM NaCl, flow rate: 0.6 ml / min. Use an AKTA purifier for molecular sieve (gel filtration chromatography) purification.

[0081] 6. Protein identification The eluted peaks were collected, which were N-hPCSK9-NP protein nanoparticles, and analyzed by reducing SDS-PAGE electrophoresis. The morphology of the nanoparticles was observed by transmission electron microscopy.

[0082] 2. Experimental Results The SDS-PAGE electrophoresis analysis results of the fusion protein 3×Epitope-HPF (i.e., N-hPCSK9-NP protein nanoparticles) obtained by five different sequence designs are shown in Figure 2The results showed that only the fusion protein 3×Epitope-HPF based on sequence 3 (V3) (amino acid sequence as shown in SEQ ID NO: 12) obtained 3×Epitope-HPF protein with high expression and fewer protein bands, so it was selected for subsequent experiments.

[0083] Figure 3 This is the structural model diagram of N-hPCSK9-NP protein nanoparticles predicted by Alphafold2 (surface pattern diagram colored using pymol software). Figure 4 This is a purification spectrum of N-hPCSK9-NP protein nanoparticles after chromatography on a pre-packed column Siperose6Increase10 / 300 GL. The curve in the figure is the absorption peak at 280nm. The results show that the elution peak of N-hPCSK9-NP protein nanoparticles is around 9ml.

[0084] Figure 5 The results of reducing SDS-PAGE electrophoresis of the purified N-hPCSK9-NP protein nanoparticles (amino acid sequence is shown in SEQ ID NO: 12), which show that the expression purity of the N-hPCSK9-NP protein nanoparticles is high.

[0085] The results of the transmission electron microscope are shown in the figure below. Figure 6 As shown, the results showed that N-hPCSK9-NP protein nanoparticles (amino acid sequence as shown in SEQ ID NO: 12) were uniform in size and had a structurally and morphologically stable nanoparticle structure.

[0086] Example 2 Preparation of Nanoparticles Based on the Extracellular Domain of Human PDL1 (hPDL1-NP Protein Nanoparticles) 1. Experimental Methods 1. Design of fusion protein In order to utilize the GvTagOpti / Sdcatcher (Gv / Sd) system, hPDL1 (human PDL1 extracellular domain sequence aa1-aa238) was linked to the surface of HPF 24-mer nanoparticles to construct the fusion protein SD-hPDL1 and GV-HPF. The construction diagram is shown in FIG. Figure 7 shown.

[0087] The amino acid sequence of SD is shown in SEQ ID NO: 15: SGETGQSGNTTIEEDSTTHVKFSKRDINGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDSTLIVGDDPI; The amino acid sequence of GV is shown in SEQ ID NO: 16: KVGNTIVMVDKLKEVPTP.

[0088] (1) Construction of fusion protein SD-hPDL1 An SD peptide segment was linked to the N-terminus of hPDL1 (amino acid sequence as shown in SEQ ID NO: 17) via a linker (GSG) to obtain the fusion protein SD-hPDL1 (amino acid sequence as shown in SEQ ID NO: 18). In order to express the obtained fusion protein SD-hPDL1 in 293F cells, an Sp signal peptide was linked to the N-terminus of the SD peptide segment via a linker (GSG) to obtain Sp- GSG- SD-hPDL1 (amino acid sequence as shown in SEQ ID NO: 19), wherein the coding sequence of hPDL1 was codon-optimized for human hosts, and the optimized nucleotide sequence of the coding gene is shown in SEQ ID NO: 20.

[0089] (2) Construction of fusion protein GV-HPF The GV peptide segment was connected to the N-terminus of HPF via a linker (GSG) to obtain the fusion protein GV-HPF (amino acid sequence shown in SEQ ID NO: 21).

[0090] 2. Construction of recombinant expression vector The 3' end of the coding gene of Sp-GSG-SD-hPDL1 was also designed with a termination codon TAATAA. By setting XholⅠ and XbaⅠ restriction sites at the 5' and 3' ends respectively, it was ligated between the XholⅠ and XbaⅠ restriction sites of the pcDNA3.1-GFP vector to obtain the recombinant expression vector pcDNA3.1-SD-hPDL1.

[0091] The 3' end of the gene encoding the fusion protein GV-HPF was also designed with the gene encoding 6×his. Then, the NcoⅠ and XholⅠ restriction sites designed at the 5' and 3' ends were connected between the NcoⅠ and XholⅠ restriction sites of pET28a to obtain the recombinant expression vector pET28a-GV-HPF.

[0092] The recombinant expression vectors pcDNA3.1-SD-hPDL1 and pET28a-GV-HPF were transformed into DH5α and BL21 competent cells, respectively. The cells were cultured overnight at 37°C, and positive clones were screened and identified by PCR. Positive clones were sequenced, and the strains with correct sequencing results were retained. The recombinant strains DH5α-pcDNA3.1-SD-hPDL1 and BL21-pET28a-GV-HPF containing the recombinant vectors were obtained and stored.

[0093] 3. Expression and purification of fusion protein GV-HPF The recombinant strain BL21-pET28a-GV-HPF was transferred to 500 ml of LB medium containing the corresponding antibiotics and continued to be cultured until the OD was approximately 0.6. IPTG was added at a final concentration of 1 mM and protein expression was induced at 16°C and 220 rpm / min. After 18 h, the cells were collected by centrifugation and resuspended in a buffer solution of pH 7.5, 20 mM Tris-HCl, and 50 mM NaCl. The cells were dissolved and broken by high pressure, and the supernatant was collected by centrifugation to obtain a crude GV-HPF protein extract.

[0094] The crude GV-HPF protein extract was heated in a 70°C water bath for 15 minutes to denature and precipitate the heat-sensitive impurities. The precipitate was removed by centrifugation at 12,000 rpm for 10 minutes. The tiny precipitate was then filtered with a 0.45 μm filter head. The supernatant was concentrated through a 100 KD cutoff concentrator to obtain the purified fusion protein GV-HPF.

[0095] 4. Expression and purification of fusion protein SD-hPDL1 The recombinant strain DH5α-pcDNA3.1-SD-hPDL1 was transferred to 500 ml of LB medium containing the corresponding antibiotics and continued to be cultured until the OD was about 0.6. The recombinant expression vector pcDNA3.1-SD-hPDL1 was extracted and transfected into 150 ml of 293F cells (cell density was about 1 × 10 7 cells / mL), cultured at 80 rpm / min for 3 h, then supplemented with 350 mL of culture medium to 500 mL, and VPA was added at a final concentration of 3.5 mM at 110 rpm / min to induce protein expression. 25 mL of commercial PFF05 was added at 24 h and 96 h, respectively. After 120 h, the supernatant was collected by centrifugation to obtain a crude SD-hPDL1 protein extract.

[0096] The crude SD-hPDL1 protein extract was added to a nickel column and passed through the column three times. Then, 30 mM imidazole was added and passed through the column once and 50 mM imidazole twice to elute the impurities. Then, 500 mM imidazole was passed through the column and the effluent was collected. The effluent was first passed through a 100 KD cut-off concentrator to remove impurity proteins larger than 100 KD, and then concentrated using a 10 KD cut-off concentrator to obtain the purified fusion protein SD-hPDL1.

[0097] 5. Self-assembly of hPDL1-NP protein nanoparticles The fusion protein GV-HPF and the fusion protein SD-hPDL1 were incubated at a mass ratio of 1:2 in a buffer solution of pH 7.5, 20 mM Tri-HCl, and 50 mM NaCl at 16°C overnight. After incubation and binding, hPDL1 nanoparticles (hPDL1-NP) were obtained and purified by chromatography.

[0098] After molecular sieve chromatography on a Siperose6 Increase10 / 300 GL column (GE), the elution peak was collected and analyzed by reducing SDS-PAGE electrophoresis. The morphology of hPDL1-NP nanoparticles was observed by transmission electron microscopy.

[0099] 2. Experimental Results Figure 8 This is the PDL1-NP nanoparticle structure model diagram predicted by Alphafold2 (surface model diagram colored using pymol software). The fusion protein GV-HPF and the fusion protein SD-hPDL1 are covalently bound to form nanoparticles.

[0100] Figure 9 This is a purification chart of hPDL1-NP protein nanoparticles by chromatography on a pre-packed column Siperose6 Increase10 / 300 GL. The curve in the figure is the absorption peak at 280nm. The results show that the elution peak of hPDL1-NP protein nanoparticles is around 9.5ml.

[0101] Figure 10 These are the reducing SDS-PAGE electrophoresis results of the purified fusion protein GV-HPF, fusion protein SD-hPDL1 and prepared hPDL1-NP protein nanoparticles. The results show that the purified fusion protein GV-HPF and SD-hPDL1 have a good binding effect.

[0102] Figure 11 The results of hPDL1-NP protein nanoparticles taken under a transmission electron microscope show that the hPDL1-NP protein nanoparticles are uniform in size and have a structurally and morphologically stable nanoparticle structure.

[0103] Example 3 Preparation of Antigen Peptide-Based Nanoparticles (Ad-Re-NP Protein Nanoparticles) 1. Experimental Methods 1. Design of fusion protein In order to utilize the GvTagOpti / Sdcatcher (Gv / Sd) system, Adpgk (Ad) polypeptide and Reps1 (Re) polypeptide were linked to the surface of HPF 24-mer nanoparticles to construct the fusion proteins SD-HPF and GV-Ad-Re. The construction diagram is shown in FIG. Figure 12 shown.

[0104] (1) Construction of fusion protein SD-HPF The SD peptide segment was connected to the N-terminus of HPF via a linker (GSG) to obtain the fusion protein SD-HPF (amino acid sequence shown in SEQ ID NO: 22).

[0105] (2) Construction of fusion protein GV-Ad-Re The amino acid sequence of the Adpgk (Ad) polypeptide is shown in SEQ ID NO: 23: ASMTNMELM; The amino acid sequence of the Reps1 (Re) polypeptide is shown in SEQ ID NO: 24: AQLANDVVL.

[0106] From the N-terminus to the C-terminus, the GV peptide, linker (GSG), Ad polypeptide, linker (GSG) and Re polypeptide were connected in sequence to obtain the fusion protein GV-Ad-Re (amino acid sequence as shown in SEQ ID NO: 25), which was directly synthesized by a biological company.

[0107] 2. Construction of recombinant expression vector The fusion protein SD-HPF coding gene was also designed with the 6×his coding gene at the 3' end, and then connected to the NcoⅠ and XholⅠ restriction sites of pET28a by designing NcoⅠ and XholⅠ restriction sites at the 5' and 3' ends to obtain the recombinant expression vector pET28a-SD-HPF.

[0108] Transform BL21 competent cells with the recombinant expression vector pET28a-SD-HPF and culture overnight at 37°C. Positive clones were screened and identified by PCR. Positive clones were sequenced, and the strains with correct sequencing results were retained to obtain the recombinant strain BL21-pcDNA3.1-SD-HPF containing the recombinant vector and stored.

[0109] 3. Expression and purification of fusion protein SD-HPF The recombinant strain BL21-pcDNA3.1-SD-HPF was inoculated again into 5 ml of kanamycin LB liquid medium and cultured at 37°C, 220 rpm / min overnight. It was then transferred to 500 ml of culture medium and cultured until the OD was approximately 0.6. IPTG was added at a final concentration of 1 mM at 16°C, 220 rpm / min to induce protein expression. After 18 h, the cells were collected by centrifugation and resuspended in a buffer solution of pH 7.5, 20 mM Tris-HCl, and 50 mM NaCl. The cells were dissolved and broken by high-pressure dissolution, and the supernatant was collected by centrifugation to obtain a crude extract of the fusion protein SD-HPF.

[0110] The crude extract of the fusion protein SD-HPF was heated in a 70°C water bath for 15 minutes to denature and precipitate the heat-sensitive impurities. The precipitate was removed by centrifugation at 12,000 rpm for 10 minutes. The tiny precipitate was then filtered with a 0.45 μm filter head. The supernatant was concentrated through a 100 KD cutoff concentrator to obtain the purified fusion protein SD-HPF.

[0111] 4. Self-assembly of Ad-Re-NP protein nanoparticles The fusion protein GV-Ad-Re and the fusion protein SD-HPF were incubated at 16°C overnight in a buffer solution of pH 7.5, 20 mM Tri-HCl, and 50 mM NaCl, at a substance amount ratio of 4:1.

[0112] After incubation and binding, Ad-Re-NP protein nanoparticles were obtained and purified by chromatography. After molecular sieve chromatography on a Siperose 6 Increase 10 / 300 GL column (GE), the elution peak was collected and analyzed by reducing SDS-PAGE electrophoresis. The morphology of the nanoparticles was observed by transmission electron microscopy.

[0113] 2. Experimental Methods Figure 13 This is the structural model diagram of Ad-Re-NP protein nanoparticles predicted by Alphafold2 (surface model diagram colored using pymol software).

[0114] Figure 14 This is a purification spectrum of Ad-Re-NP protein nanoparticles after chromatography on a pre-packed column Siperose6 Increase10 / 300 GL. The curve in the figure is the absorption peak at 280nm. The results show that the elution peak of Ad-Re-NP protein nanoparticles is between 9 and 9.5ml.

[0115] Figure 15 These are the reducing SDS-PAGE electrophoresis results of the purified fusion protein GV-HPF, fusion protein SD-hPDL1 and prepared hPDL1-NP protein nanoparticles. The results show that the purified fusion protein GV-HPF and SD-hPDL1 can bind well.

[0116] Figure 16 The results of hPDL1-NP protein nanoparticles taken under a transmission electron microscope show that the hPDL1-NP protein nanoparticles are uniform in size and have a structurally and morphologically stable nanoparticle structure.

[0117] Comparative Example 1 Preparation of Helicobacter pylori Ferritin-Based Nanoparticles (HPF Protein Nanoparticles) 1. Experimental Methods The preparation of the fusion protein SD-HPF in Reference Example 3 was as follows: 1. Construction of fusion protein SD-HPF The SD peptide segment was connected to the N-terminus of HPF via a linker (GSG) to obtain the fusion protein SD-HPF (amino acid sequence shown in SEQ ID NO: 22).

[0118] 2. Construction of recombinant expression vector The fusion protein SD-HPF coding gene was also designed with the 6×his coding gene at the 3' end, and then connected to the NcoⅠ and XholⅠ restriction sites of pET28a by designing NcoⅠ and XholⅠ restriction sites at the 5' and 3' ends to obtain the recombinant expression vector pET28a-SD-HPF.

[0119] Transform BL21 competent cells with the recombinant expression vector pET28a-SD-HPF and culture overnight at 37°C. Positive clones were screened and identified by PCR. Positive clones were sequenced, and the strains with correct sequencing results were retained to obtain the recombinant strain BL21-pcDNA3.1-SD-HPF containing the recombinant vector and stored.

[0120] 3. Expression and purification of fusion protein SD-HPF The recombinant strain BL21-pcDNA3.1-SD-HPF was inoculated again into 5 ml of kanamycin LB liquid medium and cultured at 37°C, 220 rpm / min overnight. It was then transferred to 500 ml of culture medium and cultured until the OD was approximately 0.6. IPTG was added at a final concentration of 1 mM at 16°C, 220 rpm / min to induce protein expression. After 18 h, the cells were collected by centrifugation and resuspended in a buffer solution of pH 7.5, 20 mM Tris-HCl, and 50 mM NaCl. The cells were dissolved and broken by high-pressure dissolution, and the supernatant was collected by centrifugation to obtain a crude extract of the fusion protein SD-HPF.

[0121] The crude extract of the fusion protein SD-HPF was heated in a 70°C water bath for 15 minutes to denature and precipitate the heat-sensitive impurities. The precipitate was removed by centrifugation at 12,000 rpm for 10 minutes. The tiny precipitate was then filtered using a 0.45 μm filter head. The supernatant was concentrated through a 100 KD cutoff concentrator to obtain the purified fusion protein SD-HPF, which self-assembled to obtain HPF protein nanoparticles.

[0122] 2. Experimental Results SDS-PAGE electrophoresis analysis showed that HPF protein nanoparticles were successfully prepared.

[0123] Example 4 Construction of PD1 / PDL1 / PCSK9-trigene humanized mice Traditional mouse models are difficult to simulate the interaction between the human immune system and targets due to species differences. The homology between mouse PCSK9 and human PCSK9 is only 78%, and the affinity of the PD-1 / PDL1 pathway is two orders of magnitude lower, resulting in deviations in the prediction of vaccine antibody cross-reactivity.

[0124] Therefore, the extracellular domains of the mouse Pdcd1 (PD-1) and Cd274 (PDL1) genes were replaced with human sequences, retaining the transmembrane and intracellular segments to ensure signal transduction fidelity; a liver-specific promoter (ApoE enhancer) was used to drive human PCSK9 overexpression to construct a PD1 / PDL1 / PCSK9-three-gene humanized mouse model to simultaneously evaluate the vaccine's blocking efficacy on human immune checkpoints (PD-1 / PDL1), its regulatory effects on metabolic reprogramming (PCSK9-LDL-R axis), and its ability to reshape the tumor immune microenvironment (T cell infiltration).

[0125] 1. Experimental Methods PD1 / PDL1 / PCSK9-trigene humanized mice (hPD1 / hPDL1 / hPCSK9-C57BL / 6) were constructed by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0126] Using CRISPR / Cas9 gene editing technology, the extracellular region from exon2 to exon4 of the PDL1 gene (MGI: 1926446, NCBIGene: 60533) of C57BL / 6 mice was replaced with the corresponding human PDL1 gene fragment (NCBI Gene ID: 29126). At the same time, the intracellular portion of the mouse gene was completely retained to ensure the correct transmission of intracellular signals, thus obtaining a PDL1 humanized mouse model.

[0127] Using CRISPR / Cas9 gene editing technology, the extracellular region from exon2 to exon3 of the PD1 gene (MGI: 104879, NCBIGene: 18566) of C57BL / 6 mice was replaced with the corresponding human gene fragment (NCBI Gene ID: 5133). At the same time, the intracellular part of the mouse gene was completely retained to ensure the correct transmission of intracellular signals, thus obtaining a PD1 humanized mouse model.

[0128] Using CRISPR / Cas9 gene editing technology, the coding region of the PCSK9 gene (MGI: 2140260, NCBIGene: 100102) of C57BL / 6 mice was fully humanized. That is, the CDS of the mouse PCSK9 gene after the ATG of the exon1 promoter was replaced with the CDS of the human PCSK9 gene (NCBI Gene ID: 255738). A PCSK9 humanized mouse model was constructed. In this model, there was no significant change in LDLR levels in the liver and LDL-C levels in the blood.

[0129] Finally, the PDL1 humanized mouse model, PD1 humanized mouse model, and PCSK9 humanized mouse model were bred to obtain the PD1 / PDL1 / PCSK9-triple gene humanized mouse (hPD1 / hPDL1 / hPCSK9-C57BL / 6 mouse).

[0130] Tail samples from hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice were collected and genomic DNA was extracted. PCR amplification of this genomic DNA was then performed using the primers listed in Table 1, with genomic DNA from wild-type C57BL / 6J mice used as a control. In Table 1, KI represents the size of the amplified product from hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice; WT represents the size of the amplified product from wild-type C57BL / 6J mice.

[0131] Table 1:

[0132] 2. Experimental Results See the results Figure 17 The results showed that hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice were successfully prepared.

[0133] Example 5 Nanoparticle immunization of mice to produce specific IgG antibodies 1. Experimental Methods The N-hPCSK9-NP protein nanoparticles prepared in Example 1, the hPDL1-NP protein nanoparticles prepared in Example 2, and the HPF nanoparticles prepared in Comparative Example 1 were mixed with aluminum hydroxide adjuvant in a volume ratio of 1:1 to obtain N-hPCSK9-NP protein vaccine, hPDL1-NP protein vaccine, and HPF protein vaccine, respectively.

[0134] Female hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice aged 6 to 8 weeks were randomly divided into 6 groups, with 4 mice in each group. Each mouse was immunized by subcutaneous injection of 100 μl of the system on the back. Figure 18This is a schematic diagram of the immunization process, and the specific groups are as follows: PBS group: inoculated with PBS as control; HPF group: 20 μg of HPF protein vaccine; hPDL1-NP group: vaccinated with 20 μg of hPDL1-NP protein vaccine; N-hPCSK9-NP group: 20 μg of N-hPCSK9-NP protein vaccine; hPDL1-NP+N-hPCSK9-NP group: vaccinated with hPDL1-NP protein vaccine and N-hPCSK9-NP protein vaccine, 20 μg each.

[0135] The same dose of immunization was given again in the second and third weeks after the initial immunization. Blood was collected from the orbital venous plexus every two weeks starting from the first immunization (mice were fasted overnight before blood collection). After standing, the serum was separated by centrifugation at 3000 rpm for 15 minutes at 4°C.

[0136] Detect hPCSK9-specific IgG antibodies and hPDL1-specific IgG antibodies in serum. The specific detection method is as follows: hPCSK9 and hPDL1 (Suzhou Jinan Protein Technology Co., Ltd.) were diluted to 5 μg / ml using ELISA coating buffer (Beijing Solaibao Technology Co., Ltd.). 100 μl was plated per well of a 96-well plate and incubated overnight at 4°C. Blocking was performed with 5% skim milk at room temperature for 1 hour. Sample serum was diluted in PBS containing 5% BSA in seven 10-fold dilutions, starting at 1:30, and incubated at 37°C for 2 hours. Washes were performed three times with PBST, using 200 μl per well. HRP-conjugated goat anti-mouse secondary antibody (IgG, 1:10,000) was then added to each well, using 100 μl per well, and incubated at 37°C for 1 hour. Washes were performed seven times with PBST, using 200 μl per well. TMB substrate (100 μl / well) was added and incubated at room temperature. After substrate color development, 100 μl / well of stop solution was added to terminate the reaction. Absorbance was measured at 450 nm using a microplate reader.

[0137] 2. Experimental Methods The results are as follows Figure 19 The results showed that A was a dynamic change graph of hPDL1-specific IgG antibody levels; B was a dynamic change graph of hPCSK9-specific IgG antibody levels; C was the hPDL1-specific IgG antibody level detection result two weeks after the third immunization; D was the hPCSK9-specific IgG antibody level detection result two weeks after the third immunization.

[0138] Regarding the level of hPDL1-specific IgG antibodies, there was no significant difference between the N-hPCSK9-NP protein vaccine administered alone (N-hPCSK9-NP group) and the PBS group and the HPF group; however, after the N-hPCSK9-NP protein vaccine was administered in combination with the hPDL1-NP protein vaccine (hPDL1-NP+N-hPCSK9-NP group), the level of hPDL1-specific IgG antibodies was significantly increased compared with the hPDL1-NP protein vaccine administered alone (hPDL1-NP group).

[0139] Example 6 Effects of Nanoparticles on Tumor-Bearing Mice 1. Experimental Methods The N-hPCSK9-NP protein nanoparticles prepared in Example 1, the hPDL1-NP protein nanoparticles prepared in Example 2, the Ad-Re-NP protein nanoparticles prepared in Example 3, and the HPF nanoparticles prepared in Example 2 were mixed with aluminum hydroxide adjuvant in a 1:1 ratio to obtain N-hPCSK9-NP protein vaccine, hPDL1-NP protein vaccine, Ad-Re-NP protein vaccine, and HPF protein vaccine, respectively.

[0140] Female hPD1 / hPDL1 / hPCSK9-C57BL / 6 mice aged 6 to 8 weeks were randomly divided into 6 groups, with 4 mice in each group. Each mouse was immunized by subcutaneous injection of 100 μl of the system on the back. Figure 20 This is a schematic diagram of the immunization process, and the specific groups are as follows: PBS group: inoculated with PBS as control; HPF group: 20 μg of HPF protein vaccine; Ad-Re-NP group: vaccinated with 20 μg of Ad-Re-NP protein vaccine; hPDL1-NP group: vaccinated with 20 μg of hPDL1-NP protein vaccine; N-hPCSK9-NP group: 20 μg of N-hPCSK9-NP protein vaccine; Ad-Re-NP+hPDL1-NP+N-hPCSK9-NP group: vaccinated with Ad-Re-NP protein vaccine, hPDL1-NP protein vaccine and N-hPCSK9-NP protein vaccine, 15 μg each.

[0141] The same dose of immunization was given again in the second and third weeks after the initial immunization. In the fourth week, each animal was inoculated with 3×10 6 MC38-hPD1 / hPDL1 / hPCSK9 tumor cells (MC38 tumor cell line overexpressing human PD1, hPDL1, and hPCSK9 proteins) were used for tumor bearing.

[0142] After 7-10 days of tumor growth, the tumor volume (V = 0.5 × long diameter × short diameter) was measured. 2 The dynamic growth of tumors in each group was monitored longitudinally (tumor volume of mice was measured every 2-3 days, and the survival status of mice was observed and body weight was measured).

[0143] After about one month of tumor bearing, the mice were killed by cervical dislocation and the tumors were removed for comparative observation of tumor size and subsequent weight measurement, and the inhibition rate was calculated (inhibition rate formula: inhibition rate = 1-tumor weight of control group / tumor weight of treatment group, with the HPF group as the control group).

[0144] Some tumors were embedded in paraffin and sections were prepared for immunohistochemistry and multiplex immunofluorescence analysis.

[0145] 2. Experimental Results 1. Effects of protein vaccines prepared with nanoparticles on tumors The effect of tumor volume on each group of mice is shown in Figures 21 to 23 As shown in Table 2; the results showed that compared with the PBS group and the HPF group, each nanoparticle vaccine significantly inhibited the volume and weight of the tumor, and the combined use of the three nanoparticle vaccines (Ad-Re-NP+hPDL1-NP+N-hPCSK9-NP group) had a significantly stronger inhibitory ability on subcutaneous tumors in mice than using any of them alone.

[0146] Table 2

[0147] Using efficacy synergy analysis (MuSyC framework, β parameter), the core idea is: the MuSyC model separates efficacy (α) and potency (β) synergy, and β>0 indicates efficacy synergy (breaking the upper limit of single-drug efficacy).

[0148] The specific calculation method is: β value = inhibition rate of the combination group - best single-drug inhibition rate (N-hPCSK9-NP single-drug group), that is, β = 87.2% - 64.0% = 23.2%. If β > 0, then efficacy synergy exists. β > 0 indicates that the combination therapy has exceeded the upper limit of the efficacy of N-hPCSK9-NP single-drug through complementary mechanisms.

[0149] Immunohistochemical staining of mouse tumor CD3 in each group Figure 24 Mouse tumor sections were subjected to multiple immunofluorescence analysis to stain mouse CD8, mouse CD4, and mouse IFN-γ. Figure 25 The results showed that compared with the PBS group and the HPF group, each nanoparticle vaccine promoted T cell infiltration and effector cytokine release in the tumor, and the combined use of the three nanoparticle vaccines was significantly more effective than using any of them alone.

[0150] In summary, the combined use of three nanoparticle vaccines (Ad-Re-NP+hPDL1-NP+N-hPCSK9-NP group) can effectively inhibit tumor growth, play a role in treating tumors, and have synergistic efficacy.

[0151] 2. Safety of protein vaccines prepared with nanoparticles The body weight of mice in each group Figure 26 As shown; various organs of mice (heart, liver, spleen, lung, kidney) were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced ​​for HE staining. The results are shown in Figure 26 .

[0152] The results showed that the various groups of nanoparticle vaccines had no significant effect on the weight of mice and no significant effect on their internal organs.

Claims

1. A fusion protein, characterized in that Its amino acid sequence is shown in SEQ ID NO:

12.

2. Protein nanoparticles obtained by self-assembly of the fusion protein according to claim 1.

3. A fusion protein composition, characterized in that: Containing the fusion protein of claim 1, and one or both of the following composition 1 or composition 2: Composition 1 contains: a fusion protein having an amino acid sequence as shown in SEQ ID NO: 20 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 21; Composition 2 contains: a fusion protein having an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein having an amino acid sequence as shown in SEQ ID NO:

25.

4. The composition according to claim 3, characterized in that Containing the fusion protein of claim 1, and the following composition 1 and composition 2: Composition 1 contains: a fusion protein having an amino acid sequence as shown in SEQ ID NO: 20 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 21; Composition 2 contains: a fusion protein having an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein having an amino acid sequence as shown in SEQ ID NO:

25.

5. A composition of protein nanoparticles, characterized in that: Containing the protein nanoparticles according to claim 2, and one or two of the following protein nanoparticles 1 or protein nanoparticles 2: Protein nanoparticle 1: obtained by self-assembly of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 20 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 21; Protein nanoparticle 2: obtained by self-assembly of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein having an amino acid sequence as shown in SEQ ID NO:

25.

6. The composition according to claim 5, characterized in that Containing the protein nanoparticles according to claim 2, and the following protein nanoparticles 1 or protein nanoparticles 2: Protein nanoparticle 1: obtained by self-assembly of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 20 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 21; Protein nanoparticle 2: obtained by self-assembly of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 22 and a fusion protein having an amino acid sequence as shown in SEQ ID NO:

25.

7. Use of the fusion protein according to claim 1, the protein nanoparticles according to claim 2, the composition according to claim 3 or 4, or the composition according to claim 5 or 6 in the preparation of a drug for preventing and treating tumors.

8. A biomaterial, characterized in that It is any of the following: (1) A nucleic acid molecule encoding a fusion protein having an amino acid sequence as shown in SEQ ID NO. 12; (2) Nucleic acid molecules encoding the components of the composition of claim 3; (3) an expression cassette containing the nucleic acid molecule described in (1) or (2); (4) A recombinant vector containing the nucleic acid molecule described in (1) or (2) or the expression cassette described in (3); (5) A recombinant microorganism containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4); (6) A cell line containing the nucleic acid molecule described in (1) or (2), the expression cassette described in (3), or the recombinant vector described in (4).

9. A composition, characterized in that Contains the composition according to claim 5 and an adjuvant.

10. Use of the composition according to claim 9 in preparing drugs for preventing and treating tumors.

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