Nanoparticles targeting her2 breast cancer and uses thereof

Abstract

The application discloses a nanoparticle for targeting HER2 breast cancer and application thereof, and is characterized in that Helicobacter pylori ferritin nanoparticles (HPF) are used as a nanovaccine core, a WH peptide for targeting dendritic cells, an extracellular domain of a HER2 positive breast cancer target antigen HER2 and a T cell epitope immunogen AE37 peptide are covalently connected to the ferritin nanoparticles through a molecular glue system, and the nanoparticle for targeting HER2 breast cancer is obtained. The nanoparticle can be self-assembled and can enhance an immune response, can simultaneously induce strong humoral immunity and T cell immunity, and can further enhance an anti-tumor effect, and can be applied to the preparation of a HER2 positive breast cancer vaccine.

Description

Technical Field

[0001] This invention belongs to the field of breast cancer targeted vaccines, specifically relating to a nanoparticle that targets HER2 breast cancer and its application. Background Technology

[0002] Breast cancer is the most common malignant tumor among women, seriously threatening their health. HER2-positive breast cancer accounts for approximately 15% of all breast cancer diagnoses and is characterized by its high invasiveness and poor prognosis. Furthermore, HER2-positive breast cancer is prone to lung, bone, liver, and brain metastases, severely impacting the accessibility of treatment drugs. Although HER2-targeting monoclonal antibodies (such as trastuzumab) and antibody-drug conjugates have significantly improved patient prognosis, tumor resistance and postoperative tumor recurrence remain major clinical challenges. In recent years, immunotherapy has established a new paradigm for cancer treatment. These immunotherapies include immune checkpoint inhibitors, CAR-T cell therapy, tumor vaccines, and oncolytic virus therapy. Among them, tumor vaccines are biological agents that prevent and treat cancer by stimulating or enhancing the body's anti-tumor immune response. They typically contain tumor cell fragments and fragments containing tumor-specific antigens (TSA) or tumor-associated antigens (TAA). After entering the human body, tumor vaccines activate the host's immune system, inducing a specific immune response, overcoming immunosuppression, and enhancing resistance and attack against specific tumors. Current HER2 breast cancer vaccines primarily employ strategies such as peptide vaccines (e.g., GP2 and AE37), dendritic cell vaccines, or nucleic acid vaccines. However, these methods generally suffer from low antigen presentation efficiency, weak immunogenicity, difficulty in inducing durable immune memory, and inability to resist the tumor immunosuppressive microenvironment. Therefore, there is an urgent need to develop novel vaccines that can efficiently present immunogens, enhance the immunogenicity of tumor vaccines, and simultaneously induce potent and durable humoral and T-cell immunity for the prevention and treatment of HER2-positive breast cancer.

[0003] Nanoparticle delivery systems, as a next-generation drug and vaccine delivery platform, have demonstrated multiple advantages in the biomedical field. Previous studies have found that ACNVax vaccines based on iron nanocores enhance the uptake and presentation of HER2 epitope antigens by dendritic cells (DCs) or macrophages, and promote the activity of B cells and CD4+. +Interactive activation of T cells. Although the ACNVax nanovaccine has shown great potential in HER2 tumor vaccine applications, the potential safety of its iron nanoparticles remains to be considered in clinical application. For tumor vaccines that activate B cell immunity, tumor progression may lead to antigen loss. Simultaneously, the tumor microenvironment exhibits significant immunosuppressive properties, characterized by high PD-L1 expression, regulatory T cell (Treg) infiltration, and myeloid-derived suppressor cell (MDSC) aggregation, severely limiting the anti-tumor efficacy of B cell vaccines.

[0004] Therefore, there is an urgent need to develop HER2-targeting nanoparticle vaccines and their combinations to effectively prevent and treat HER2-induced breast cancer by enhancing the immunogenicity of tumor vaccines, overcoming the tumor immunosuppressive microenvironment, and synergistically enhancing T / B cell immunity. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the shortcomings of existing HER2 vaccines in terms of protective and therapeutic efficacy. The primary objective is to provide a nanoparticle targeting HER2 in breast cancer. This invention uses a bio-autogenously assembled Helicobacter pylori ferritin (HPF) 24-tetrameric nanovaccine core. A molecular glue system covalently links a dendritic cell-targeting WH peptide, the HER2 extracellular domain, and the T-cell epitope immunogen AE37 to the ferritin nanoparticle. Furthermore, fullerol nanoparticles (FNPs) are used as an adjuvant to enhance the vaccine's immune response. During vaccine immunization, the synergistic delivery of PD-1 and CD47 monoclonal antibodies inhibits the formation of a tumor immunosuppressive microenvironment.

[0006] Another objective of this invention is to provide a HER2 nanoparticle tumor vaccine based on self-assembled Helicobacter pylori ferritin, and to develop a "nanovice / anti-PD-1 / anti-CD47" triple immunotherapy combination strategy for HER2 breast cancer by combining it with immune checkpoint blockade therapy.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A nanoparticle targeting HER2 breast cancer uses bio-autogenously assembled Helicobacter pylori ferritin nanoparticles (HPF) as the core of a nanovaccine. Through a molecular glue system, WH peptide targeting dendritic cells, the extracellular domain of HER2-positive breast cancer target antigen, and AE37 peptide, an immunogen of T cell epitope, are covalently linked to the ferritin nanoparticles to obtain nanoparticles targeting HER2 breast cancer.

[0009] Preferably, the amino acid sequence of the ferritin nanoparticles is shown in SEQ ID NO:1, the amino acid sequence of the HER2 extracellular domain is shown in SEQ ID NO:2, the amino acid sequence of the AE37 peptide is shown in SEQ ID NO:3, and the amino acid sequence of the WH peptide is shown in SEQ ID NO:4.

[0010] Preferably, the SpyTag003 / SpyCatcher003 molecular gel covalent coupling system is used to link the extracellular domain of HER2 to the outer surface of ferritin nanoparticles. The SpyTag003 / SpyCatcher003 molecular gel system, currently the most efficient coupling system, is employed, which can complete the covalent assembly of the target immunogen within 5 minutes at room temperature.

[0011] Preferably, the nanoparticles are prepared by the following method:

[0012] (1) WH peptide and SpyTag003 peptide (ST003) were placed at the N-terminus and C-terminus of the extracellular domain of HER2, respectively, and fused with the extracellular domain of HER2 to obtain an immunogen containing ST003; SpyCatcher003 peptide (SC003) and AE37 peptide were placed at the N-terminus of ferritin nanoparticles and fused with ferritin nanoparticles to obtain a ferritin core containing SC003.

[0013] (2) After co-incubating the immunogen containing ST003 with the ferritin core containing SC003, the two will form intermolecular heteropeptide bonds through ST003 and SC003, so that the immunogen is covalently modified on the outer surface of the ferritin nanoparticles.

[0014] Preferably, the amino acid sequence of the SpyTag003 peptide is shown in SEQ ID NO:5; the amino acid sequence of the SpyCatcher003 peptide is shown in SEQ ID NO:6.

[0015] Preferably, the amino acid sequence of the immunogen containing ST003 in step (2) is shown in SEQ ID NO:7, and the amino acid sequence of the ferritin core containing SC003 is shown in SEQ ID NO:8.

[0016] Preferably, the molar ratio of the immunogen containing ST003 to the ferritin core containing SC003 is 1:1.

[0017] Application of the nanoparticles targeting HER2 breast cancer in the preparation of HER2-positive breast cancer vaccines.

[0018] Preferably, during vaccine immunization, CpG ODN 1826 is added as an adjuvant, and fullerol nanoparticles (FNPs) are used as a co-adjuvant. CpG ODN 1826 adjuvant is an in vitro synthesized oligonucleotide with immunomodulatory functions. It induces cellular and humoral immunity by specifically stimulating Toll-like receptor 9 (TLR-9), thereby enhancing the body's immune response. Fullerol helps enhance calreticulin exposure, improving the phagocytic activity of macrophages against cancer cells and vaccine immunogens. Furthermore, its combination with anti-CD47 monoclonal antibodies can enhance the anti-tumor efficacy of anti-CD47 monoclonal antibodies.

[0019] Preferably, during vaccine immunization, anti-PD-1 and anti-CD47 monoclonal antibody treatments are also administered. The anti-PD-1 monoclonal antibody binds to PD-1 on the surface of T cells, relieving T cell suppression, inhibiting the production of exhausted T cells, restoring their ability to kill tumor cells, and enabling the immune system to re-recognize and eliminate tumor cells. The anti-CD47 monoclonal antibody binds to CD47 on the tumor surface, blocking CD47-SIRPα signaling, relieving tumor immunosuppression, and promoting macrophage-mediated phagocytosis.

[0020] This invention first selects Helicobacter pylori ferritin 24-tetrameric as the core of the nanoparticle vaccine (SEQ ID NO:1). It can form a stable 24-tetrameric structure even after immunogenicity binding, allowing direct targeting of lymphoid organs via 12-15 nm nanoparticles, thus exhibiting extremely high stability and lymphoid organ targeting. Helicobacter pylori ferritin can serve as a carrier, effectively delivering antigen proteins to antigen-presenting cells. To enhance the antigen-presenting cell targeting of the nanoparticle vaccine, this invention covalently couples the dendritic cell (DC) targeting peptide WH peptide (SEQ ID NO:4) to the surface of the nanoparticle vaccine. The presence of WH peptide allows the nanoparticle vaccine to be specifically phagocytosed and presented by dendritic cells. To enhance the immunogenicity of the extracellular HER2 antigen, this invention links the AE37 peptide and WH peptide to the N-terminus of the HER2 extracellular domain of ferritin and HER2, respectively. This invention uses the extracellular domain of HER2 (SEQ ID NO:2), a target antigen of HER2-positive breast cancer, and the T-cell epitope peptide AE37 (SEQ ID NO:3) as immunogens. The extracellular domain of HER2 can stimulate strong anti-tumor humoral immunity, while the AE37 peptide can stimulate strong HER2-targeting T-cell immunity.

[0021] The SpyTag / SpyCatcher system plays a crucial role in vaccine research due to its ease of simple and efficient covalent coupling of proteins. Therefore, SpyCatcher003 is linked to the N-terminus of AE37-HPF, and the extracellular domain of SpyTag003 is fused to the C-terminus of WH-HER2. Furthermore, the secretion signal peptide (SP) is fused to the N-terminus of the HER2 gene to facilitate extracellular secretion. Adding a 6His tag to the C-terminus of both AE37-HPF and WH-HER2 facilitates protein purification using Ni-NTA affinity chromatography.

[0022] Therefore, this invention utilizes the SpyCatcher / SpyTag system to covalently couple the tumor antigen HER2 and the T-cell epitope peptide AE37 to the surface of AE37-HPF nanoparticles, achieving high-density, targeted display of the antigen on the nanocarrier surface. Furthermore, the addition of WH peptide and fullerol further enhances the antigen-presenting cell targeting and delivery of the HER2 antigen. This vaccine simultaneously synergizes with PD-1 inhibitors and CD47 antagonists during immunization. This synergistic approach of multi-epitope vaccine and dual immune checkpoint blockade provides a novel strategy for overcoming the tumor immunosuppressive microenvironment and enhancing tumor immune efficacy.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] (1) This invention provides a nanoparticle vaccine targeting HER2 breast cancer based on Helicobacter pylori ferritin 24-tetrameric, which can efficiently induce anti-tumor humoral immunity and T-cell immunity by directly targeting lymph nodes, and improve the immunogenicity of tumor antigens and CD8. + The level of T cell response ultimately achieves a highly effective anti-tumor effect.

[0025] (2) This invention utilizes the SpyCatcher003 and SpyTag003 molecular gel systems to efficiently couple the target antigen to the surface of nanoparticles, thereby achieving high-density display of the tumor immunogen HER2 on ferritin nanoparticles.

[0026] (3) In this invention, the HER2 breast cancer-specific T cell epitope peptide AE37 is covalently presented onto ferritin nanoparticle vaccines, thereby enhancing CD8. + The formation of T cell responses and anti-tumor T cell memory.

[0027] (4) This invention combines a WH-targeting peptide that enhances immunogen phagocytosis and presentation with a novel adjuvant, Fullerol nanoparticle (FNP), which greatly enhances the phagocytosis and presentation of HER2 immunogen by dendritic cells and macrophages.

[0028] (5) This invention combines a PD-1 monoclonal antibody that combats T cell depletion with a CD47 monoclonal antibody that enhances macrophage recognition of tumor cells, thereby reshaping the tumor microenvironment, restoring T cell killing activity, and preventing tumor immune escape.

[0029] (6) The present invention provides a nanoparticle that can self-assemble and enhance the immune response to target HER2 breast cancer, which can simultaneously induce strong humoral immunity and T cell immunity, thereby enhancing the anti-tumor effect. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the plasmid construction strategy for AE37-HPF-HER2-WH nanoparticles.

[0031] Figure 2 These are purification and staining images of AE37-HPF, WH-HER2, and AE37-HPF-HER2-WH proteins.

[0032] Figure 3 This is a molecular sieve diagram of the purification of the AE37-HPF-HER2-WH nanoparticle vaccine.

[0033] Figure 4 The images show the antibody titers detected after immunization with the AE37-HPF-HER2-WH nanoparticle vaccine in combination with different amounts of 0, 40, and 60 μg of FNP adjuvant.

[0034] Figure 5 CD4 count was detected after immunization with the AE37-HPF-HER2-WH nanoparticle vaccine. + T cells TNF-α + CD4 + T cells IFNγ + CD4 + T cells Granzyme B + Cytokines and CD8 + T cells IFNγ + CD8 + T cells TNF-α + CD8 + T cells Granzyme B + Cytokine secretion levels.

[0035] Figure 6 This is a schematic diagram of the immunization strategy for the AE37-HPF-HER2-WH nanoparticle vaccine.

[0036] Figure 7 It is the anti-HER2 specific IgG response in the serum of mice after vaccination.

[0037] Figure 8This is the average tumor growth curve of mice in each group after vaccination.

[0038] Figure 9 It is the average tumor weight of mice in each group after vaccination. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. All raw materials involved in the present invention can be purchased directly from the market. For process parameters not specifically specified, conventional techniques can be referred to.

[0040] Fullerol nanoparticles (FNPs) were purchased from Guangzhou Ming'an Biotechnology Co., Ltd. CpG ODN 1826 (HY-146245) was purchased from MedChemExpress. Anti-PD-1 (RMP1-14, BE0146) and anti-CD47 (MIAP301, BE0270) antibodies were purchased from BioXcell. E. coli competent cells were purchased from Shenzhen Kangti Life Science Co., Ltd. Ni Sepharose Excel packing material was purchased from Cytiva. HEK293F cells were obtained from Beijing Sinocare Biotech Co., Ltd.

[0041] pET28a plasmid (69864-3CN) was purchased from Merck. pcDNA3.1 plasmid (V79020) was purchased from Thermo Fisher Scientific. The pcDNA3.1-Intron-IRES-GFP-WPRE plasmid was obtained by inserting the IRES-GFP-WPRE sequence into the pcDNA3.1 multiple cloning site. This plasmid was reported in the literature Xiancai Ma, Fan Zou, Fei Yu, Rong Li, YaochangYuan, Yiwen Zhang, Xiantao Zhang, Jieyi Deng, Tao Chen, Zheng Song, YidanQiao, Yikang Zhan, Jun Liu, Junsong Zhang, Xu Zhang, Zhilin Peng, YuzhuangLi, Yingtong Lin, Liting Liang, Guanwen Wang, Yingshi Chen, Qier Chen, TingPan, Xin He, Hui Zhang, Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses, Immunity, Volume 53, Issue 6, 2020, Pages 1315-1330.

[0042] Example 1

[0043] Preparation method of AE37-HPF-HER2-WH nanoparticles

[0044] 1. The nucleotide sequence of Helicobacter pylori ferritin (HPF) (SEQ ID NO: 9) and the nucleotide sequence of the HER2 extracellular domain (SEQ ID NO: 10) were obtained from the NCBI database. SC003 (SEQ ID NO: 11) and AE37 (SEQ ID NO: 12) were added to the N-terminus of the HPF sequence (SEQ ID NO: 9), and a 6His tag was fused to the C-terminus for affinity chromatography purification. Subsequently, the sequence was cloned into the pET28a vector using restriction endonuclease to obtain the pET28a-SC003-AE37-HPF-6His plasmid. Similarly, the nucleotide sequences of secretory signal peptide (SP) (SEQ ID NO:13), WH (SEQ ID NO:14), ST003 (SEQ ID NO:15), and 6His (SEQ ID NO:16) were added to the N-terminus and C-terminus of the extracellular nucleotide sequence encoding HER2 (SEQ ID NO:10), respectively. These sequences were then cloned into another plasmid (pcDNA3.1-Intron-IRES-GFP-WPRE) to obtain the recombinant plasmid pcDNA3.1-Intron-SP-WH-HER2-ST003-6His-IRES-GFP-WPRE. After sequencing verification, the recombinant plasmid was used for the expression of the nanonucleus and antigen proteins.

[0045] 2. The recombinant plasmid pcDNA3.1-Intron-SP-WH-HER2-ST003-6His-IRES-GFP-WPRE was transfected into HEK293F cells. Seven days after transfection, the cell supernatant was harvested by centrifugation and then purified using Ni Sepharose Excel packing material. Impurities were removed by washing with low-concentration imidazole buffer (20 mM Tris-HCl, 50 mM NaCl, 50 mM Imidazole, pH 7.4). Subsequently, the target protein was eluted with high-imidazole buffer (20 mM Tris-HCl, 50 mM NaCl, 500 mM Imidazole, pH 7.4). The protein was then concentrated by ultrafiltration to remove imidazole, yielding the WH-HER2 antigen protein.

[0046] 3. The constructed pET28a-SC003-AE37-HPF-6His plasmid was transformed into competent *E. coli* cells (BL21, DE3), inoculated into LB medium, and cultured at 37°C with shaking at 220 rpm until the OD600 reached 0.4. 1 mM IPTG was added to the culture, and protein expression was induced at 16°C for 16 hours. Bacteria were collected by centrifugation and lysed by sonication. The supernatant was incubated with a Ni-NTA column, followed by washing with low-concentration imidazole buffer (20 mM Tris-HCl, 50 mM NaCl, 50 mM Imidazole, pH 7.4) to remove contaminating proteins, and then eluted with high-concentration imidazole buffer (20 mM Tris-HCl, 50 mM NaCl, 500 mM Imidazole, pH 7.4). The protein was concentrated by ultrafiltration to remove imidazole, yielding the AE37-HPF core protein.

[0047] 4. The two purified proteins, AE37-HPF core and WH-HER2 antigen protein, were incubated in an equimolar ratio in an enzyme-free buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.4) for 24 hours. SC003 and ST003 formed intermolecular heteropeptide bonds, and the WH-HER2 antigen was coupled to the AE37-HPF surface. The purity of the three proteins, AE37-HPF core, WH-HER2 antigen protein, and the corresponding AE37-HPF-HER2-WH ferritin nanoparticles, was verified by Coomassie blue staining (e.g., ...). Figure 2 (As shown). Furthermore, AE37-HPF-HER2-WH was subjected to molecular sieve chromatography using a Superose 6 Increase 10 / 300 GL molecular sieve column, and the elution peak of the AE37-HPF-HER2-WH teicomeric protein was collected to obtain high-purity protein (e.g., as shown). Figure 3 (As shown). After concentration, the target protein is aliquoted and stored at -80 °C.

[0048] Example 2

[0049] Fuller-pure nanoparticles (FNP) as adjuvant immunoassay

[0050] This embodiment uses AE37-HPF-HER2-WH nanoparticles as a vaccine and FNP as an adjuvant to analyze the humoral and cellular immune enhancement effects of FNP in female BALB / c mice. Female BALB / c mice were divided into three groups of four mice each. The three groups were subcutaneously immunized with 10 μg AE37-HPF-HER2-WH, 10 μg AE37-HPF-HER2-WH + 40 μg FNP, and 10 μg AE37-HPF-HER2-WH + 60 μg FNP, respectively, every 7 days for a total of three immunizations. Seven days after the last immunization, blood was collected from the mice. After being incubated at room temperature for 1 hour, the blood was centrifuged at 2800 rpm for 10 minutes at 4 °C. Serum was collected for ELISA detection of antigen-specific antibodies. After three immunizations, the nanoparticle vaccine induced an effective immune response in the mice, with a significant increase in specific antibody levels. Furthermore, the IgG titer in the 60 μg group was significantly higher than that in the group immunized with the nanovaccine alone. The combination of 60 μg FNP and the AE37-HPF-HER2-WH nanovaccine induced a strong humoral immune response, with a significant increase in specific antibody levels, such as... Figure 4 As shown. Simultaneously, mouse spleens were removed, aseptically and gently ground, and filtered through a 70 μm cell sieve to prepare a single-cell suspension. Red blood cell lysis buffer was added and incubated at room temperature for 5 min to remove red blood cells. After washing twice with PBS, the cell concentration was adjusted to 2 × 10⁶ cells / mL using RPMI-1640 medium containing 10% FBS. 6 Cells / mL. 100 μL of cell suspension was seeded into 24-well plates, and CD28 and HER2 peptide libraries were added at final concentrations as specific stimulants. After incubation for 1 h, 1 μL of LBrefeldin A (1 μg / mL) was added to block the extracellular secretion of cytokines. After another 6 h of incubation, cells were collected and surface stained for 30 min with FITC anti-mouse CD3, AF700 anti-mouse CD4, and PE-Cy7 anti-mouse CD8α flow cytometry antibodies. After cell fixation and permeabilization, cells were stained with BV421 anti-mouse IFNγ, APC anti-mouse TNF-α, and PE-conjugated anti-mouse Granzyme B antibodies. Flow cytometry analysis of stained cells was used to evaluate cellular immune levels, such as... Figure 5As shown, mouse spleen cells immunized with 60 μg FNP combined with the AE37-HPF-HER2-WH nanovaccine showed significantly increased levels of IFNγ, TNF-α, and Granzyme B cytokines secreted by CD4 and CD8-positive T cells. FNP can significantly enhance the immunogenicity of the vaccine, and the combination with the vaccine induces stronger humoral and cellular immune responses.

[0051] Example 3

[0052] BALB / c mouse immunization experiment

[0053] This invention evaluated the therapeutic effect of the AE37-HPF-HER2-WH tumor vaccine using a HER2-overexpressing 4T1 breast tumor model. Female BALB / c mice were divided into three groups of four each. Each group was subcutaneously injected with 1×102 HER2-overexpressing 4T1 breast tumor vaccine into the back of each mouse. 5 There are 1 tumor cells, which is day 0. Immunization began on day 1 after tumor inoculation. The six groups received the following treatments: 60 μg fullerol adjuvant + 30 μg CpG ODN 1826 adjuvant; antibody group (100 μg anti-CD47 and 100 μg anti-PD-1); 1 nM WH-HER2 immunogen + 60 μg fullerol adjuvant + 30 μg CpG ODN 1826 adjuvant; 1 nM AE37-HPF + 60 μg fullerol adjuvant + 30 μg CpG ODN 1826 adjuvant; 1 nM AE37-HPF-HER2-WH nanovaccine + 60 μg fullerol adjuvant + 30 μg CpG ODN 1826 adjuvant; 1 nM AE37-HPF-HER2-WH nanovaccine + 60 μg fullerol adjuvant + 30 μg CpG ODN 1826 adjuvant + 100 μg anti-CD47 and 100 μg anti-PD-1; and 100 μg anti-CD47 and 100 μg anti-PD-1. Administered 100 μg of anti-PD-1 every 7 days for a total of 3 immunizations. Tumor size was measured from the time the tumor became visible, every day until day 20. In the group receiving the nanovaccine combined with monoclonal antibody, 100 μg of anti-CD47 and 100 μg of anti-PD-1 monoclonal antibody were injected intraperitoneally three times. After euthanasia, tumor tissue was removed and tumor weight was recorded. The results indicate that the combination of AE37-HPF-HER2-WH nanoparticle vaccine with anti-PD-1 and anti-CD47 therapy effectively inhibited tumor growth, demonstrating a significant therapeutic effect on tumors.

[0054] Evaluation of treatment efficacy for HER2-positive tumors: Tumor volume = 0.5 × long diameter of tumor × short diameter of tumor × short diameter of tumor. Figure 8The average tumor growth curves of each group of mice are shown. The results of the tumor growth curves show that the tumor size of mice in the adjuvant group increased exponentially, while the AE37-HPF-HER2-WH group showed a more significant tumor inhibition effect compared with the adjuvant group and the WH-HER2 monomer group. Figure 9 The average tumor weight data for each group of mice are presented. The results show that the tumors in the AE37-HPF-HER2-WH nanoparticle vaccine group grew the slowest, and the AE37-HPF-HER2-WH nanoparticle vaccine combined with anti-PD-1 and anti-CD47 immunotherapy can significantly inhibit tumor growth.

[0055] Example 4

[0056] Antigen-specific antibody assay

[0057] Antigen-specific antibodies are an important indicator of vaccine efficacy. Therefore, it is necessary to collect mouse serum samples and measure antibody levels. Using HER2 protein as the coating antigen, the titer of anti-HER2 specific IgG antibodies in serum was determined by ELISA. 3 μg / mL HER2 protein was placed in a 96-well pre-coated plate and blocked with PBS containing 5% skim milk at 37°C for 1 hour. Immunized animal serum was serially diluted 10-fold and added to each well, then incubated at 37°C for 1 hour. After washing with PBS / T, horseradish peroxidase-conjugated goat anti-mouse IgG (1:10000) was added and incubated at 37°C for 1 hour. After color development with TMB solution for 15 minutes, color development was terminated by adding 1 M H2SO4 solution. The absorbance value was measured at 450 nm. The titer of bound antibodies in each serum sample was calculated based on the absorbance value and dilution gradient. Compared with the adjuvant group, the antibody levels in the WH-HER2 and AE37-HPF-HER2-WH vaccine groups were higher, and the IgG antibody levels in the AE37-HPF-HER2-WH group were significantly higher than those in the WH-HER2 group (e.g., Figure 7 (As shown). This indicates that AE37-HPF-HER2-WH exhibits a strong ability to induce the production of antigen-specific IgG antibodies, enhances the immunogenicity of WH-HER2, and triggers a strong humoral immune response in vivo.

[0058] In summary, this invention successfully developed a HER2 breast cancer nanovaccine based on ferritin, which efficiently binds the antigen to the surface of nanoparticles. This vaccine effectively targets immune tissues and lymphoid organs, promoting dendritic cell maturation and effectively activating humoral and cellular immune responses. Furthermore, its combined use with immune checkpoint blockade (anti-PD-1) therapy and anti-CD47 therapy enhances its anti-tumor effect and effectively inhibits tumor growth.

[0059] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0060] SEQ ID NO:1 HPF amino acid sequence

[0061] DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0062] SEQ ID NO:2 HER2 extracellular domain amino acid sequence

[0063] GTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPANASLSFLQDIQEVQGYMLIAHNRVKHVPLQRLRIVRGTQLFEDKYALAVLDNRDPLDNVTTAAPGRTPEGLRELQLRSLTEILKGGVLIRGNPQLCYQDMVLWKDVLRKNNQLAPVDMDTNRSRACPPCAPTCKDNHCWGESPEDCQILTGTICTSGCARCKGRLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALITYNTDTFESMLNPEGRYTFGASCVTTCPYNYLSTEVGSCTLVCPPNNQEVTAEDGTQRCEKCSKPCAGVCYGLGMEHLRGARAITSDNIQEFAGCKKIFGSLAFLPESFDGNPSSGVAPLKPEHLQVFETLEEITGYLYISAWPESFQDLSVFQNLRVIRGRILHDGAYSLTLQGLGIHSLGLRSLRELGSGLALIHRNTHLCFVNTVPWDQLFRNPHQALLHSGNRPEEACGLEGLVCNSLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVWKGLPREYVRGKHCLPCHPECQPQNSSETCYGSEADQCEACAHYKDSSSCVARCPSGVKPDLSYMPIWKYPDEEGICQPCPINCTHSCVDLDERGCPAEQR

[0064] SEQ ID NO:3 Amino acid sequence of AE37

[0065] LRMKGVGSPY VSRLLGICL

[0066] SEQ ID NO:4 Amino acid sequence of WH peptide

[0067] WPRFHSSVFHTH

[0068] SEQ ID NO:5 Amino acid sequence of SpyTag003 peptide

[0069] RGVPHIVMVDAYKRYK

[0070] SEQ ID NO:6 Amino acid sequence of SpyCatcher003 peptide

[0071] VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT

[0072] SEQ ID NO:7 Amino acid sequence of WH-HER2-ECD-ST003-6His

[0073] WPRFHSSVFHTHGGGGSGGGGSGGGGSGTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPANASLSFLQDIQEVQGYMLIAHNRVKHVPLQRLRIVRGTQLFEDKYALAVLDNRDPLDNVTTAAPGRTPEGLRELQLRSLTEILKGGVLIRGNPQLCYQDMVLWKDVLRKNNQLAPVDMDTNRSRACPPCAPTCKDNHCWGESPEDCQILTGTICTSGCARCKGRLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALITYNTDTFESMLNPEGRYTFGASCVTTCPYNYLSTEVGSCTLVCPPNNQEVTAEDGTQRCEKCSKPCAGVCYGLGMEHLRGARAITSDNIQEFAGCKKIFGSLAFLPESFDGNPSSGVAPLKPEHLQVFETLEEITGYLYISAWPESFQDLSVFQNLRVIRGRILHDGAYSLTLQGLGIHSLGLRSLRELGSGLALIHRNTHLCFVNTVPWDQLFRNPHQALLHSGNRPEEACGLEGLVCNSLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVWKGLPREYVRGKHCLPCHPECQPQNSSETCYGSEADQCEACAHYKDSSSCVARCPSGVKPDLSYMPIWKYPDEEGICQPCPINCTHSCVDLDERGCPAEQRGSGRGVPHIVMVDAYKRYKHHHHHH

[0074] SEQ ID NO:8 Amino acid sequence of SC003-AE37-HPF-6His

[0075] MGVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGSGLRMKGVGSPYVSRLLGICLGSGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSLEHHHHHH

[0076] SEQ ID NO:9 HPF nucleotide sequence

[0077] GACATCATTAAGTTGCTAAACGAACAAGTGAATAAGGAAATGCAATCTTCCAACTTGTATATGAGCATGAGTTCATGGTGCTATACCCATAGCTTAGATGGCGCGGGGCTTTTCTTGTTTGACCATGCGGCTGAAGAATACGAGCATGCTAAAAAGCTTATTATCTTCTTGAATGAAAACAATGTGCCTGTGCAATTGACCAGCATCAGCGCGCCTGAGCATAAGTTTGAAGGTTTGACTCAAATTTTCCAAAAAGCCTATGAACATGAGCAACACATCAGCGAGTCTATTAACAATATCGTAGATCACGCCATAAAAAGCAAAGATCATGCGACTTTCAATTTCTTGCAATGGTATGTGGCTGAACAGCATGAAGAAGAAGTGCTTTTCAAGGATATTTTGGATAAAATTGAGTTGATTGGTAATGAAAATCATGGCTTGTATTTAGCCGATCAGTATGTCAAAGGGATCGCTAAAAGCAGGAAATCT

[0078] SEQ ID NO:10 HER2 extracellular domain nucleotide sequence

[0079]

[0080] SEQ ID NO:11 SpyCatcher003 peptide nucleotide sequence

[0081] GTAACCACCTTATCAGGTTTATCAGGTGAACAAGGTCCGTCCGGTGATATGACAACTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCCGTGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACAT GGATTTCAGATGGACATGTGAAGGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACCAGACGGTTATGAGGTAGCAACTCCAATTGAATTTACAGTTAATGAGGACGGTCAGGTTACTGTAGATGGTGAAGCAACTGAAGGTGACGCTCATACT

[0082] SEQ ID NO:12 AE37 nucleotide sequence

[0083] CTAAGGATGAAAGGAGTAGGGTCACCCTATGTGAGCCGTTTGCTGGGTATCTGCCTG

[0084] SEQ ID NO:13 SP nucleotide sequence

[0085] ATGGGCATTCTCCCTAGCCCTGGAATGCCAGCCCTGCTCAGCCTGGTGTCCCTCCTGTCCGTGCTGCTGATGGGCTGCGTGGCC

[0086] SEQ ID NO:14 WH peptide nucleotide sequence

[0087] TGGCCTAGATTCCACAGCAGCGTGTTCCACACCCAC

[0088] SEQ ID NO:15 ST003 peptide nucleotide sequence

[0089] AGAGGAGTGCCACACATCGTGATGGTGGACGCCTACAAGCGGTACAAG

[0090] SEQ ID NO:16 6His nucleotide sequence

[0091] CACCACCACCACCACCAC

Claims

1. A nanoparticle targeting HER2 in breast cancer, characterized in that, Using bio-autogenously assembled Helicobacter pylori ferritin nanoparticles as the core of a nanovaccine, the targeting dendritic cell WH peptide, the extracellular domain of HER2-positive breast cancer target antigen, and the T cell epitope immunogen AE37 peptide are covalently linked to the ferritin nanoparticles through a molecular glue system, thus obtaining nanoparticles targeting HER2 breast cancer. The nanoparticles were prepared by the following method: (1) WH peptide and SpyTag003 peptide were placed at the N-terminus and C-terminus of the extracellular domain of HER2, respectively, and fused with the extracellular domain of HER2 to obtain an immunogen containing ST003; SpyCatcher003 peptide and AE37 peptide were placed at the N-terminus of ferritin nanoparticles and fused with ferritin nanoparticles to obtain a ferritin core containing SC003. (2) After co-incubating the immunogen containing ST003 with the ferritin core containing SC003, the two will form intermolecular heteropeptide bonds through ST003 and SC003, so that the immunogen is covalently modified on the outer surface of the ferritin nanoparticles. Step (2) The molar ratio of the immunogen containing ST003 to the ferritin core containing SC003 is 1:1; the amino acid sequence of the immunogen containing ST003 is shown in SEQ ID NO:7, and the amino acid sequence of the ferritin core containing SC003 is shown in SEQ ID NO:

8.

2. The use of the HER2-targeting breast cancer nanoparticles as described in claim 1 in the preparation of HER2-positive breast cancer vaccines.

3. The application according to claim 2, characterized in that, during vaccine immunization, CpG ODN 1826 is added as an adjuvant, and fullerol nanoparticles are used as an auxiliary adjuvant.

4. The application according to claim 2 or 3, characterized in that, during the vaccine immunization process, anti-PD-1 and anti-CD47 monoclonal antibody treatment is also used in combination.

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