Nanoparticle vaccine based on HER2 target spot and application thereof
The HER2 epitope sequence was displayed by HBc VLPs of the core virus-like particle nanoparticle HBc VLPs, and a nanoparticle vaccine was prepared, solving the problems of weak immunogenicity and toxic side effects of the existing HER2-targeted vaccine, and achieving efficient and safe tumor treatment.
Patent Information
- Application Number
- CN202510451188.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-08-19
AI Technical Summary
Existing HER2-targeted tumor vaccines such as polypeptide vaccines are weak in immunogenicity, and monoclonal antibody treatment has problems such as short half-life, frequent administration of large doses and toxic side effects, which limits its application in cancer treatment.
The HER2 epitope sequence was accurately displayed by hepatitis B core virus-like particle nanoparticle HBc VLPs as a vector, and a nanoparticle vaccine was prepared, which used its high immunogenicity and specific identification of HER2-positive tumor cells.
It achieves an efficient and safe tumor immune response, reduces side effects, and provides a more effective HER2-positive tumor treatment plan.
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Figure CN120501848A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a HER2-targeted nanoparticle vaccine and applications thereof. Background Art
[0002] In recent years, the incidence and mortality rates of cancer patients have shown a steadily increasing trend, making it a significant and significant contributor to malignant tumors, and its incidence continues to rise. As the second leading cause of cancer-related death in women, cancer poses a particularly serious threat to women's health. Statistics show that approximately 20% to 30% of cancer patients overexpress the HER-2 gene and protein, highlighting the complexity and diversity of cancer.
[0003] Currently, surgery, radiotherapy, chemotherapy, and endocrine therapy are common treatments for cancer patients. Although advances in medical technology have significantly improved early screening, diagnosis, and treatment for cancer patients, some patients have poor tolerance to radiotherapy and chemotherapy and are unable to withstand their side effects, which often leads to treatment failure and poses a huge challenge to their recovery.
[0004] In recent years, immunotherapy (active immunity) has attracted widespread attention for its unique advantages. Research has shown that immunotherapy can achieve meaningful and lasting therapeutic effects with minimal, controllable toxicity, providing a new treatment approach for cancer patients. Tumor vaccines, as a key component of immunotherapy, aim to activate the patient's immune system to prevent and treat cancer, offering new possibilities for cancer prevention and treatment.
[0005] Human epidermal growth factor receptor-2 (HER2), a key transmembrane tyrosine kinase receptor, plays a significant role on the surface of tumor cells. This receptor not only regulates normal cell growth, but also, if aberrantly amplified or overexpressed, can trigger tumor growth and spread. Notably, HER2 is upregulated in approximately 20% to 30% of cancer patients, a characteristic often considered a warning sign of a poor prognosis.
[0006] Currently, HER2-targeted therapeutic strategies, including trastuzumab and pertuzumab, have become the mainstay of clinical cancer treatment. However, due to the relatively short half-life of monoclonal antibodies, high doses and frequent administration are typically required, which not only increases the economic burden of treatment but also makes it difficult to effectively prevent disease recurrence. Furthermore, potential side effects such as cardiotoxicity and gastrointestinal discomfort during treatment limit their long-term application.
[0007] Given this, researchers are actively pursuing strategies to develop novel anti-HER2 vaccines, hoping to provide more effective and safe treatment options. Among the numerous HER2-targeting cancer vaccine studies, HER2 peptide vaccines have garnered significant attention. However, challenges such as weak immunogenicity remain. Summary of the Invention
[0008] Virus-like particles (VLPs) offer significant advantages over monomeric protein antigens or DNA vaccines. The key to developing an effective anti-HER2 vaccine is inducing a robust and durable anti-tumor immune response and overcoming tolerance to autoantigens. Achieving this goal relies on the precise delivery of antigenic epitopes and the use of effective adjuvants.
[0009] HBc-VLPs are icosahedral particles composed of HBcAg monomers. Their assembly method presents two forms, T=3 or T=4, depending on the number of monomers involved. T represents the number of small equilateral triangles divided into one face of the icosahedron. Specifically, T=3 type particles have 90 spikes, while T=4 type has 120. The top region of these spikes, namely the immunodominant region (MIR), is a key target area for genetic manipulation and can be optimized by replacing or inserting exogenous antigen epitope peptides.
[0010] Studies have shown that antigenic proteins on the surface of various virus particles can be displayed in an orderly and repetitive manner on the spike sites on the surface of HBcAg, forming pathogen-associated molecular patterns (PAMPs). This pattern can effectively cross-link B cell receptors (BCRs) and transmit strong activation signals to B cells, thereby enhancing specific humoral immune responses. It is worth noting that the full length of the HBcAg monomer is 183 amino acids. Although only a truncated monomer containing 140-150 amino acids at the N-terminus is required to form virus-like particles, the key to its self-assembly lies in the cysteines at positions 48 and 61 that form a disulfide bond to connect four α-helices to form a central bundle.
[0011] In addition, the C-terminus of HBcAg contains approximately 40 amino acids that constitute an arginine-rich protamine-like protein domain, which has a high affinity for RNA and is therefore also known as the RNA binding region. Although removal of this region does not affect the self-assembly of HBc-VLPs, it may lead to poor folding of VLPs.
[0012] VLPs have become a hot topic in the vaccine field due to their unique immunological properties, including highly repetitive surface geometry, conformational similarity to native viruses, and ability to induce both innate and adaptive immune responses. They can generate high titers of antibodies, helping the body defend against viral infection. Furthermore, their superior immunogenicity, safety, structural stability, and homogeneity make them ideal vaccine candidates.
[0013] The HBV core virus-like particle complex can be produced at low cost and on a large scale, and has reversible assembly and disassembly characteristics. Currently, HBV core virus-like particles have been widely used in many fields such as vaccine development, nanobiotechnology, drug delivery, biomimetic synthesis, bioimaging and cell targeting. As a carrier carrying antigen epitopes, HBV core virus-like particles display antigens on the surface of their nanoparticles through self-assembly, thereby enhancing immune activity. This naturally derived nanoparticle has biocompatibility, hydrophilicity and self-assembly properties, providing an important platform for vaccine development. When vaccinated, vaccines in the form of nanoparticles can produce more effective immune effects than monomeric, soluble antigens. This is due to the affinity between the surface of the nanoparticles and the cell membrane, which enables immune cells to more effectively take up anchored antigens.
[0014] In light of this, the present invention proposes a HER2-targeted nanoparticle vaccine and its innovative application. This vaccine precisely binds the HER2 epitope sequence HER-2 (597-626aa) to the outer surface of hepatitis B virus-like particles (HBV-like particles), thereby successfully creating a cancer vaccine targeting human epidermal growth factor receptor-2 (HER2). This invention aims to provide an effective and safe treatment option for cancer patients and provide important insights and references for subsequent research.
[0015] The present invention proposes a nanoparticle vaccine based on the HER2 target, and the nanoparticle vaccine based on the HER2 target comprises:
[0016] HER-2 gene-related antigen sequence HER2 and hepatitis B core virus-like particle nanoparticles HBc VLPs;
[0017] Wherein, the amino acid sequence of the HER-2 gene-related antigen sequence HER2 is shown as SEQ ID No. 1;
[0018] The amino acid sequence of the hepatitis B core virus-like particle nanoparticle HBc VLPs is shown in SEQ ID No. 2.
[0019] Preferably, the amino acid sequence of the HER2 target-based nanoparticle vaccine is shown as SEQ ID No. 3.
[0020] Preferably, the HER2 target-based nanoparticle vaccine further comprises a linker;
[0021] The amino acid sequence of the linker is EAAAKKK.
[0022] Preferably, the amino acid sequence of the HER2 target-based nanoparticle vaccine is shown as SEQ ID No. 4.
[0023] The present invention also provides a biomaterial, which comprises the DNA molecule shown by SEQ ID No. 5 or the DNA molecule shown by SEQ ID No. 7.
[0024] The present invention also provides an application of the above-mentioned HER2-targeted nanoparticle vaccine or the above-mentioned biomaterial, wherein the application is any of the following:
[0025] A1) Use in the preparation of a medicament for treating or preventing tumors;
[0026] A2) Use in the preparation of drugs for treating or preventing diseases caused by tumors.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] This invention constructs a nanoparticle vaccine targeting HER2, using hepatitis B virus-like particles (HBc VLPs) as a carrier. The HER2 gene-associated antigen sequence is effectively displayed on the nanoparticle surface, thereby achieving specific recognition and immune response against HER2-positive tumor cells. This vaccine not only has high stability and immunogenicity, but also features a simple preparation process and low cost, promising broad application prospects.
[0029] The present invention also proposes a biomaterial containing specific DNA molecules that encode the HER2-targeting nanoparticle vaccine of the present invention, providing a convenient and flexible approach for vaccine preparation. Furthermore, the present invention proposes the use of this vaccine in the preparation of a drug for treating or preventing tumors and related diseases, offering a new option in the field of tumor immunotherapy.
[0030] The HER2-targeting nanoparticle vaccine of this invention is highly specific and targeted, precisely attacking HER2-positive tumor cells without damaging normal cells. Therefore, compared with traditional chemotherapy and radiotherapy, this vaccine has fewer side effects and is better tolerated, potentially providing a safer and more effective treatment option for cancer patients.
[0031] In summary, the present invention provides a nanoparticle vaccine based on the HER2 target, its biomaterials, and applications, which have the advantages of simple preparation, low cost, strong specificity, and high safety, and provide new ideas and directions for the development of the field of tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference symbols are used throughout the drawings to represent the same components. In the drawings:
[0033] Figure 1 The immunoblotting results of the recombinant proteins of the present invention are shown in Figure 1, where 1 is HER2-HBc VLPs ① and 2 is HER2-HBc VLPs ②.
[0034] Figure 2 The results of high-performance liquid chromatography (HPLC) of the examples of the present invention are shown, wherein 1 is HER2-HBc VLPs ①, and 2 is HER2-HBc VLPs ②;
[0035] Figure 3 The morphology of the nanoparticle vaccine obtained by transmission electron microscopy in the embodiment of the present invention is shown in Figure 1, where 1 is HER2-HBc VLPs ① and 2 is HER2-HBc VLPs ②;
[0036] Figure 4 The particle size results obtained by the laser particle size analyzer in the embodiment of the present invention are shown in FIG1 , where 1 is HER2-HBc VLPs ① and 2 is HER2-HBc VLPs ②.
[0037] Figure 5 This is the absorbance result diagram of the embodiment of the present invention;
[0038] Figure 6 The results of tumor volume changes after treatment in the embodiment of the present invention are shown. DETAILED DESCRIPTION
[0039] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, unless there is a conflict, the embodiments of the present disclosure and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.
[0040] Example 1 Preparation of recombinant nanoparticle vaccine
[0041] 1. Materials
[0042] Using genetic engineering techniques, the HER2 gene-associated antigen sequence was screened and recombinant protein was prepared using hepatitis B virus-like particles (HBc VLPs) as a carrier backbone to prepare a nanoparticle tumor vaccine. HER2 is represented by aa 597-626 of the HER2 gene, and the amino acid sequence of HER2 is shown in SEQ ID No. 1; the amino acid sequence of the HBc VLPs protein is shown in SEQ ID No. 2.
[0043] SEQ ID No. 1:
[0044] VARCPSGVKPDLSYMPIWKFPDEEGACQPL.
[0045] SEQ ID No. 2:
[0046] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLEDQASRDLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC.
[0047] The HER2-expressed protein was directly linked to the HBc VLPs protein, ultimately obtaining HER2-HBc VLPS protein sequence ①. HER2-HBc VLPS protein sequence ① is nanoparticle vaccine ①, and the amino acid sequence of nanoparticle vaccine ① is shown in SEQ ID No. 3.
[0048] The HER2-expressed protein and the HBc VLPs protein were linked via a rigid linker (EAAAKKK) to ultimately generate HER2-HBc VLPs protein sequence ②. HER2-HBc VLPs protein sequence ② is nanoparticle vaccine ②, and the amino acid sequence of nanoparticle vaccine ② is shown in SEQ ID No. 4.
[0049] SEQ ID No.3:
[0050] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLED VARCPSGVKPDLSYMPIWKFPDEEGACQPL QASRDLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC.
[0051] SEQ ID No.4:
[0052] MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALFRDALESPEHCSPHHTALRQATLCWGELMXLATWVGANLED EAAAKKKVARCPSGVKPDLSYMPIWKFPDEEGACQPLEAAAKKK QASRDLVVSYVNTYMGLKFRQLLWFHISCLTFGRETVIEYLVSFGVWIRTPPAYRPTNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRTQSRESQC.
[0053] 2. Preparation of recombinant nanoparticle vaccines
[0054] The spliced fragments, SEQ ID No. 3 and SEQ ID No. 4, were ligated into the NdeI / xhoI region downstream of the T7 promoter in the pET 28a plasmid and transfected into BL21(DE3) E. coli to create an E. coli expression system containing the target genes. Following prokaryotic expression, the proteins were purified by ammonium sulfate precipitation, followed by Sepharose S1000 purification, DEAE ion exchange chromatography, protein dialysis, and concentration to obtain nanoparticle vaccines ① and ②.
[0055] The specific process includes:
[0056] (1) Escherichia coli BL21 (DE3) was cultured in LB liquid medium at 37°C for 8-10 h;
[0057] (2) Add 0.2 mol / L IPTG and induce expression at 26°C for 16 h;
[0058] (3) Collecting bacteria: centrifuge the expressing bacteria at 4000 rpm / min for 10 min, discard the supernatant, and resuspend the bacterial pellet in 20 ml of 10 mM Tris-HCl;
[0059] (4) Ultrasonic disruption: Place the resuspended bacterial solution in an ice water bath, set the power to 250W, turn on the ultrasound for 3 seconds, turn off the ultrasound for 3 seconds, and repeat 20 cycles;
[0060] (5) Ammonium sulfate precipitation: Centrifuge at 10,000 rpm / min for 10 min. Transfer the supernatant to a centrifuge cup. Depending on the volume of the supernatant obtained, slowly add 20% saturated ammonium sulfate under magnetic stirring. React at 4°C for 5-10 min. Centrifuge at 9,000 rpm / min for 10 min. Discard the supernatant and resuspend the precipitate in 10 mM Tris-HCl, pH = 8.0.
[0061] (6) Anion exchange chromatography purification using a DEAE resin column with a mobile phase of 10 mM Tris-HCl (pH 8.0) buffer and elution with a sodium chloride gradient (100, 200, 500 mM sodium chloride);
[0062] (7) Dialysis: Based on the results of protein electrophoresis, the proteins were combined and transferred into a dialysis bag (3.5 kDa). The dialysis bag was tied tightly and placed into a clean tray after 10 h of dialysis. PEG 20000 was sprinkled on the bottom and surface until the volume was concentrated to about half. The purified nanoparticle vaccine was obtained.
[0063] Example 2
[0064] The nanoparticle vaccines ① and ② prepared in Example 1 were tested.
[0065] (1) The nanoparticle vaccines ① and ② prepared by the present invention were analyzed by immunoblotting for recombinant protein, as shown in FIG. Figure 1 As shown, HER2-HBc VLPs nanoparticles can specifically bind to HER2 antibodies and develop color, with clear specific target bands appearing at 24KDa and 26KDa, respectively, which are consistent with the expected protein molecular weight.
[0066] (2) The purity of HBc VLPs was tested by high performance liquid chromatography. Figure 2 As shown, both nanoparticle vaccines ① and ② showed a single chromatographic peak, indicating that they were of high purity and met the quality standards.
[0067] (3) To further characterize the morphology and particle size of the nanoparticle vaccine, we used transmission electron microscopy and laser particle size analyzer for measurement. Figure 3 As shown in Figure 2, the nanoparticle vaccine presents a spherical structure; the results of the laser particle size analyzer are shown in Figure 2. Figure 4 As shown, the particle size is relatively uniform, with an average particle size of about 30 nm, confirming the uniformity and stability of the nanoparticle vaccine.
[0068] Example 3
[0069] The effects of the nanoparticle vaccines ① and ② prepared in Example 1 were evaluated.
[0070] Female Balb / c mice aged 6-8 weeks (5 mice per group) were used as experimental subjects and TUBO breast cancer cells were injected subcutaneously on the right side of the back (5×10 5 cells), and a unilateral tumor model was successfully established.
[0071] To further investigate the impact of different treatment options on tumor growth, unilateral tumor models in each group were treated with HER2-HBc VLPs①, HER2-HBc VLPs②, and PBS. The specific treatment strategy involved subcutaneous immunization of mice every 7 days with a single treatment dose of 50 μg for 3 consecutive weeks. To ensure the accuracy and reliability of the experimental data, tail vein blood samples were collected from mice on days 0 and 28 before treatment and analyzed using an indirect ELISA.
[0072] After coating with HER2-Fc antigen (100 ng / well), serum from the third treatment was diluted 1:200 as the primary antibody. Serum from mice on day 0 before treatment was used as a negative control. During sample loading, 100 μL of sample was added to each well, followed by incubation at 37°C for 0.5 h and five washes with PBST. Next, an enzyme-linked goat anti-mouse IgG-HRP conjugate diluted to an appropriate concentration was added as the secondary antibody (1:2000) and incubated in a 37°C oven for 0.5 h. Following incubation, the sample and conjugate were discarded, and the plate was washed again five times with PBST. Subsequently, 50 μL of chromogen A and chromogen B were added to each well, followed by sealing film and incubation in a 37°C oven for 20 min. Finally, 50 μL of stop solution was added to each well, and the absorbance at 450 nm was immediately measured using a microplate reader. The experimental results showed that the recombinant protein HER2-HBc VLPs② significantly induced anti-HER2 IgG response, with the endpoint titer reaching 31,000. Figure 5 .
[0073] On the 14th day after TUBO cell inoculation, when the tumor was palpable, the treatment was initiated. Figure 6. The observation results showed that the tumor growth rate in the PBS group was significant, and its volume had reached the preset endpoint standard of 1000 cubic millimeters on the 28th day after the tumor was stimulated. Compared with the PBS group, the growth rate of mice treated with HER2-HBc VLPs① group and HER2-HBc VLPs② group showed a significant delay trend. Moreover, compared with the treatment with HER2-HBc VLPs① group, the HER2-HBc VLPs② group showed higher superiority in tumor volume control. Specifically, the tumor volume of the HER2-HBc VLPs② group was relatively small, and it also showed a more obvious delay trend in the growth rate of the tumor. It was not until the 40th day after the tumor was stimulated that its volume reached the endpoint standard.
[0074] In summary, both HER2-HBc VLPs① and HER2-HBc VLPs② nanoparticle vaccines demonstrated promising tumor suppression effects, with HER2-HBc VLPs② demonstrating superior efficacy in inducing anti-HER2 IgG responses and controlling tumor volume. This discovery provides a novel, more effective therapeutic strategy for HER2-positive breast cancer and holds broad application prospects.
[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered by the scope of protection of the claims of the present invention.
Claims
1. A nanoparticle vaccine based on HER2 target, characterized in that: The nanoparticle vaccine based on the HER2 target includes: HER-2 gene-related antigen sequence HER2 and hepatitis B core virus-like particle nanoparticles HBc VLPs; Wherein, the amino acid sequence of the HER-2 gene-related antigen sequence HER2 is shown as SEQ ID No. 1; The amino acid sequence of the hepatitis B core virus-like particle nanoparticle HBc VLPs is shown in SEQ ID No.
2.
2. The HER2-targeted nanoparticle vaccine according to claim 1, characterized in that: The HER2 target-based nanoparticle vaccine is spherical and has a particle size of 30 nm.
3. The HER2-targeted nanoparticle vaccine according to claim 1, characterized in that: The amino acid sequence of the HER2 target-based nanoparticle vaccine is shown in SEQ ID No.
3.
4. The HER2-targeted nanoparticle vaccine according to claim 1, characterized in that: The HER2 target-based nanoparticle vaccine further includes a linker; The amino acid sequence of the linker is EAAAKKK.
5. The HER2-targeted nanoparticle vaccine according to claim 6, characterized in that: The amino acid sequence of the HER2 target-based nanoparticle vaccine is shown in SEQ ID No.
4.
6. A use of the HER2-targeted nanoparticle vaccine according to any one of claims 1 to 5 or the biomaterial according to claim 6, characterized in that: The application is any of the following: A1) Use in the preparation of a medicament for treating or preventing tumors; A2) Use in the preparation of drugs for treating or preventing diseases caused by tumors.