A chimeric hepatitis b virus-like particle vaccine expressing melanoma antigens

Abstract

The present application relates to a kind of hepatitis B virus-like particles (HBc VLPs) targeted to M2 type macrophages and specifically express melanoma antigen MAGEA10, the recombinant hepatitis B virus-like particles include HBcAg shell structure, surface specifically expresses the specific peptide segment capable of targeting M2 type macrophages, and carries MAGEA10 tumor antigen polypeptide.The present application also relates to the application of the hepatitis B virus-like particles in treating melanoma.

Description

[0001] Involves sequence lists

[0002] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Technical Field

[0003] This invention relates to a hepatitis B virus-like particle that can be used for tumor immunotherapy and drug delivery. Background Technology

[0004] Melanoma antigen A10 (MAGEA10) is a tumor antigen homologous to both humans and mice and specifically expressed in various melanoma cell types. M2 macrophages play a role in promoting tumor growth and immunosuppression within the tumor microenvironment, making them an important target in cancer therapy. Hepatitis B virus-like particles (HBV VLPs) are nanoparticles self-assembled from the hepatitis B virus core protein (HBcAg) and are widely used in vaccine development and drug delivery systems. Combining MAGEA10 and HBcVLPs, and utilizing specifically expressed peptides targeting M2 macrophages, can achieve a novel immunotherapy approach that modulates the tumor immune microenvironment. Summary of the Invention

[0005] The present invention provides a hepatitis B virus-like particle (HBc VLP) targeting M2 macrophages, comprising nanoparticles self-assembled from hepatitis B virus core protein (HBcAg), wherein the nanoparticles express specific peptides targeting M2 macrophages on their surface.

[0006] The present invention provides hepatitis B virus-like particles (HBc VLPs) that target M2 macrophages and specifically express melanoma antigens, such as melanoma antigen MAGEA10. The HBc VLPs comprise nanoparticles self-assembled from hepatitis B virus core protein (HBcAg). The nanoparticles express specific peptides targeting M2 macrophages on their surface and carry and express melanoma antigens, such as MAGEA10 tumor antigen.

[0007] Preferably, for the hepatitis B virus-like particles, the specific peptide is bound to the hepatitis B virus core protein (HBcAg), and preferably, the specific peptide is linked to the amino terminus of the hepatitis B virus core protein.

[0008] Preferably, for the hepatitis B virus-like particles, the nanoparticles express melanoma antigens.

[0009] Preferably, for the hepatitis B virus-like particles, the amino acid sequence of the specific targeting peptide is shown in SEQ ID NO:1.

[0010] Preferably, for the hepatitis B virus-like particles, the melanoma antigen is melanoma antigen MAGEA10, and preferably, its amino acid sequence is shown in SEQ ID NO:2.

[0011] Preferably, the hepatitis B virus-like particles comprise or are composed of a fusion protein, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:3.

[0012] This invention also provides a method for preparing hepatitis B virus-like particles that target M2 macrophages and specifically express MAGEA10, comprising the following steps:

[0013] a) Construct a recombinant plasmid containing the M2 targeting peptide gene, the MAGEA10 gene, and the HBcAg gene;

[0014] b) Express the recombinant plasmid in a suitable host cell to produce hepatitis B virus-like particles;

[0015] c) The hepatitis B virus-like particles were obtained by separation and purification.

[0016] The present invention also provides a pharmaceutical composition comprising the hepatitis B virus-like particles described herein, and a pharmaceutically acceptable carrier.

[0017] Preferably, the present invention can be a vaccine, such as a therapeutic vaccine, such as an anti-tumor vaccine.

[0018] The present invention also provides the application of the hepatitis B virus-like particles described herein in the preparation of drugs for treating melanoma. Attached Figure Description

[0019] Figure 1 The results are from protein electrophoresis of chimeric hepatitis B virus-like particles.

[0020] Figure 2 It refers to the changes in the purity and yield of chimeric hepatitis B virus-like particles.

[0021] Figure 3 This is an ion exchange chromatography purification spectrum of chimeric hepatitis B virus-like particles.

[0022] Figure 4 These are gel electrophoresis images of the chimeric hepatitis B virus-like particles before and after purification by ion exchange chromatography.

[0023] Figure 5 The results are from transmission electron microscopy of chimeric hepatitis B virus-like particles.

[0024] Figure 6 The results are fluorescence results of macrophage-targeted cells that contain chimeric hepatitis B virus-like particles.

[0025] Figure 7 This is the result of tumor suppression in different control groups of chimeric hepatitis B virus-like particles. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the following detailed description is provided in conjunction with specific embodiments.

[0027] Malignant tumors, commonly known as cancer, are diseases caused by the accumulation of mutations in somatic cells to achieve immortality, invasive metastasis, and immune evasion. With the increasing aging population and unhealthy diets and lifestyles, malignant tumors have become one of the most serious threats to human health and survival worldwide, severely impacting the quality of life and life expectancy of people globally. Surgery, chemotherapy, and radiotherapy remain the mainstream clinical treatments for malignant tumors, but their application is greatly limited by side effects such as systemic toxicity, dose dependence, and secondary trauma. With the development of molecular biology and tumor immunology, tumor immunotherapy has received increasing attention and research due to its highly targeted and tumor-specific killing properties. Tumor vaccines typically involve selected exogenous tumor antigens and adjuvants that activate dendritic cells (DCs), with the main purpose of inducing tumor regression and eliminating residual tumor cells by stimulating the patient's adaptive immune system.

[0028] Ideally, the immune system, with the help of drugs and other means, can perform immune surveillance and clearance of tumors through the aforementioned pathways. However, this role is extremely limited in cancer patients, as cancer cells achieve metastatic growth through immune evasion mechanisms. In fact, the tumor microenvironment (TME) plays a crucial role in promoting or inhibiting tumor growth and progression. It contains multiple cell types and cytokines, and the components interact and coordinate their respective functions, thereby affecting multiple aspects such as tumor growth, angiogenesis, immune evasion, drug resistance, and lesion metastasis.

[0029] Given the numerous challenges in vaccine administration, developing a safer and more efficient new strategy for delivering cancer immunotherapy can not only minimize the immunotoxicity of immunotherapeutic drugs but also offer better broad-spectrum efficacy, expanding immunotherapy to a wider range of patients. Virus-like particles (VLPs) are highly homogenized, self-assembled viral proteins that are morphologically and structurally similar to natural viral particles, but with their genetic material removed, exhibiting excellent safety. Composed of hundreds to thousands of protein molecules, VLPs display major immunogenic regions (MIRs) in a highly repetitive and ordered manner, promising to induce strong cellular and humoral immune responses. Spanning from 10 nm to 1 μm in size, most VLPs have icosahedral or helical structures, making them readily uptaken by APCs and presented to T lymphocytes.

[0030] Since the 1960s, vapor-level proteinaceous (VLPs) have played a significant role in biomedical engineering as a novel biotool. VLPs can be classified into two types: encapsulated and capsid VLPs. Unlike non-encapsulated VLPs, which are composed of capsid proteins, the former consists of matrix proteins and glycoproteins contained within a lipid layer. The surface of VLPs can be genetically and chemically modified to present different epitopes, enabling functions such as long-term in vivo circulation, induction of humoral and cellular immunity; the interior can be loaded with proteins, nucleic acids, or compounds to achieve specific types of intracellular delivery. Compared with other nanoparticles, the uniform small particle size, highly repetitive epitope display, and modifiability of VLPs make them a promising and diverse strategy for developing cancer therapeutic vaccines. Utilizing VLPs to specifically deliver drugs to the tumor microenvironment (TAM) and modulate phenotypic reversal is a feasible approach for cancer vaccines. To regulate dendritic cell (DC)-specific activation and maturation, VLPs carrying antigens and conjugated with specific ligands are typically employed.

[0031] This invention uses malignant melanoma as a tumor model and hepatitis B virus core protein HBc-VLP as a vector to conduct in vitro cellular-level validation, including investigation of cytotoxicity, phagocytosis rate, targeting, and phenotypic reversal efficiency. Finally, it was successfully validated by constructing a tumor-bearing mouse model.

[0032] Based on the above scheme, the hepatitis B virus-like particles that specifically express the melanoma antigen MAGEA10 on M2 macrophages provided by the present invention have at least one of the following beneficial effects:

[0033] 1. In some embodiments of the present invention, the recombinant HBc VLPs particles have obvious specific targeting. M2 macrophages play a key role in the tumor microenvironment, promoting tumor growth and immune escape. By targeting M2 macrophages, VLPs can more effectively locate and influence the tumor microenvironment, thereby improving the specificity and effectiveness of treatment.

[0034] 2. In some embodiments of the present invention, the recombinant HBc VLPs particles targeting M2 macrophages can reduce the impact on normal tissues and cells, thereby reducing side effects during treatment and improving the patient's quality of life.

[0035] 3. In some embodiments of the present invention, recombinant HBc VLPs particles significantly enhance the anti-melanoma immune response. MAGEA10 is an antigen highly expressed in tumors such as melanoma. By specifically expressing MAGEA10 through VLPs, a strong immune response against tumor cells can be stimulated, and a specific T cell response can be induced, thereby improving the immunotherapy effect. At the same time, VLPs can effectively deliver MAGEA10 to M2 macrophages, improve antigen presentation efficiency, activate more immune cells, and enhance the overall immune response.

[0036] 4. In some embodiments of the present invention, recombinant HBc VLPs particles improve treatment efficiency. Hepatitis B virus-like particles (HBc VLPs), as multifunctional carriers, have high self-assembly ability and structural stability, can carry and protect antigens, and can be surface modified to achieve multiple functions. Due to the stability and persistence of VLPs, they can remain in vivo for a long time, continuously provide antigen stimulation, and improve the persistence of immune response and treatment effect.

[0037] 5. In some embodiments of the present invention, the prepared recombinant HBc VLPs particles are constructed by constructing recombinant plasmids through genetic engineering methods, and VLPs are expressed in Escherichia coli, yeast or mammalian cells. The production process is mature and highly scalable, suitable for large-scale production. The combination of genetic engineering technology and bioreactors makes the production cost of VLPs relatively low, which can meet clinical needs.

[0038] 6. In some embodiments of the present invention, the prepared recombinant HBc VLPs particles can also be extended to other cancers expressing MAGEA10 or similar tumor antigens, such as lung cancer and liver cancer, improving the breadth and flexibility of treatment. This technology can be used in combination with other treatment methods, such as chemotherapy, radiotherapy and other immunotherapies, to produce a synergistic effect and further improve the treatment effect.

[0039] 7. In some embodiments of the present invention, the prepared recombinant HBc VLPs particles are customized according to the patient's tumor characteristics and immune status to achieve individualized treatment and improve treatment efficacy. As non-pathogenic nanoparticles, VLPs have been verified to be safe in various vaccine and drug delivery systems and have broad prospects for clinical application.

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be further described below with reference to specific embodiments and accompanying drawings. It should be noted that the specific embodiments listed below are merely illustrative examples, and the scope of protection of this invention is not limited thereto.

[0041] Example 1

[0042] Constructing recombinant plasmids:

[0043] A recombinant plasmid containing a fusion protein (SEQ ID NO:3) targeting a specific peptide (SEQ ID NO:1) targeting M2 macrophages and the melanoma antigen MAGEA10 sequence (SEQ ID NO:2) was constructed using genetic engineering methods. The plasmid can be expressed in *E. coli*.

[0044] Synthesize specific peptides targeting M2 macrophages, the MAGEA10 gene, and the HBcAg gene, or amplify them by PCR from template DNA. Design primers containing restriction enzyme sites (EcoRI and XhoI) to ensure that the corresponding restriction enzyme sites are introduced at both ends of the PCR product. Detect the PCR products using electrophoresis.

[0045] Reaction system (50 μL):

[0046] Template DNA: 1 μL

[0047] - Forward primer (10 μM): 1 μL

[0048] - Reverse primer (10 μM): 1 μL

[0049] -dNTP mixture (10mM): 1μL

[0050] -10X PCR buffer: 5 μL

[0051] -Taq DNA polymerase: 0.25μL

[0052] -ddH2O: 40.75μL

[0053] PCR procedure: Initial denaturation: 94℃, 5 minutes

[0054] - Transformation: 94℃, 30 seconds

[0055] Annealing: Primer Tm temperature, 30 seconds

[0056] - Extension: 72℃, adjust time according to product length (typically 1kb / min)

[0057] Repeat steps 2-4 for 35 cycles.

[0058] - Final extension: 72℃, 10 minutes

[0059] Enzyme digestion:

[0060] The PCR products and vector plasmids were digested with restriction endonucleases EcoRI and XhoI, respectively. The digestion was carried out at 37°C for 2–4 hours. After digestion, the digested products were separated by agarose gel electrophoresis, and the target band was excised and purified using a gel extraction kit.

[0061] Reaction system:

[0062] - DNA (PCR product or plasmid): 1-2 μg - 10X digestion buffer: 5 μL - EcoRI enzyme: 1 μL - XhoI enzyme: 1 μL - ddH2O: Add to 50 μL

[0063] Connection reaction:

[0064] The digested M2 targeting peptide gene, MAGEA10 gene, and HBcAg gene fragments were ligated into the vector plasmid using T4 DNA ligase. The mixture was incubated overnight at 16°C.

[0065] Reaction system:

[0066] - Vector DNA (after enzyme digestion): 50-100 ng

[0067] - Inserted DNA fragment (after enzyme digestion): 3 molar amounts of vector

[0068] -10X ligation buffer: 1 μL

[0069] -T4 DNA ligase: 0.5 μL

[0070] -ddH2O: Add to 10μL

[0071] Transformation:

[0072] The ligation reaction product was transformed into competent Escherichia coli (DH5α).

[0073] Operating steps:

[0074] Take 50 μL of competent cells and add 5 μL of ligation product.

[0075] Incubate on ice for 30 minutes.

[0076] Heat shock at 42℃ for 45 seconds.

[0077] Cool immediately on ice for 2 minutes.

[0078] Add 450 μL of SOC medium and incubate at 37°C with shaking for 1 hour.

[0079] Spread onto LB agar plates containing antibiotics (such as ampicillin) and incubate overnight at 37°C.

[0080] Screening and identification: The following day, single colonies were picked and inoculated into LB liquid medium containing antibiotics, and cultured overnight at 37°C on a shaker. Plasmid DNA was extracted (using a Solarbio YZ-D6950 plasmid extraction kit).

[0081] Example 2

[0082] activation:

[0083] The recombinant plasmid was transformed into host cells BL21(DE3) Escherichia coli to induce the expression of recombinant HBc VLPs. The VLPs were purified by centrifugation, chromatography, and other methods.

[0084] Prepare 50 mL of LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride, dissolved in deionized water), place it in a 200 mL Erlenmeyer flask, seal it with a microporous filter membrane and kraft paper, and autoclave it at 120 °C for 30 min. After it returns to room temperature, add 50 μL of kanamycin stock solution (100 mg / mL) in a clean bench and shake well. Inoculate with 0.4% (200 μL) of glycerol bacteria (BL21) and incubate overnight at 37 °C and 200 rpm on a shaker.

[0085] Expansion cultivation:

[0086] Prepare 500 mL of LB medium, place it in a 2 L Erlenmeyer flask, and seal it with a microporous membrane and kraft paper. Sterilize in a benchtop rapid steam sterilizer for 30 min. Add 500 μL of kanamycin stock solution (100 mg / mL) and shake well. Add 5 mL of the above activated bacterial solution and incubate at 37 °C and 200 rpm in a constant temperature shaker for about 3 h until the OD600 is about 0.8.

[0087] Inducement:

[0088] The recombinant engineered bacteria were induced with IPTG at final concentrations of 0.1 mM, 0.5 mM, and 1.0 mM, respectively, and the bacterial strains and protein yields were recorded. The bacterial cell yields under the three conditions were 5.35 g / L, 5.04 g / L, and 5.42 g / L, respectively, and the protein yields were 5.919 mg / mL, 5.637 mg / mL, and 5.366 mg / mL, respectively. The 0.5 mM IPTG concentration, which induced the highest protein yield per bacterial strain, was selected as the induction condition. The bacteria were then cultured at 37°C and 200 rpm for approximately 4 hours to obtain BL21 *E. coli* strains that ultimately expressed the fusion protein in large quantities. The amino acid sequence of the fusion protein is shown in SEQ ID NO:3.

[0089] The bacterial strains before and after induction were analyzed by reducing SDS-PAGE protein electrophoresis. After induction, compared with the whole bacteria before induction, more target protein was expressed at about 23 kDa, which was basically consistent with the molecular weight predicted by VLP monomer.

[0090] collect:

[0091] The fermentation broth obtained from the above process was centrifuged at 4000 rpm for 30 min at 4℃. The bacterial cells were collected and weighed. 1 g of bacterial cells was used for subsequent operations, and the remaining bacterial cells were placed in a sealed bag and stored at -20℃ for later use. A lysis buffer (20 mM Tris-HCl, 3 mM EDTA, 0.1% Triton X-100, 1 mM PMSF, pH 8.0) was prepared and the bacterial cells were resuspended at a ratio of 1:20 (g / mL). The buffer was then sonicated on ice. The ultrasonic cell disruptor was set with the following parameters: ultrasonic power 300 W, 5 s operation followed by a 5 s pause, 30 min sonication, and an alarm temperature of 27℃. After the lysis buffer was collected, the lysis buffer was centrifuged at 10000 rpm for 30 min at 4℃. The supernatant was collected and the precipitate was weighed. The protein concentration in the supernatant was determined using the BCA method, and the buffer was refrigerated at 4℃. Figure 1 This is the protein electrophoresis result of the chimeric hepatitis B virus-like particles in this embodiment. It can be clearly seen that the two lanes on the left of the figure have obvious protein bands in the 22-31 kDa range compared to the control group on the far right, which indicates the successful expression of recombinant hepatitis B virus-like particles.

[0092] Example 3

[0093] Ammonium sulfate precipitates VLP proteins:

[0094] The preparation of the ammonium sulfate stock solution (3M) was carried out as follows: 198.21g of ammonium sulfate was weighed and dissolved in approximately 400mL of deionized water. 10mL of Tris stock solution (1M) was added, and the mixture was stirred until homogeneous. The pH was adjusted to approximately 7.0 with HCl, and the volume was brought to 500mL. 20mL of the supernatant from centrifugation was taken and slowly added to the ammonium sulfate stock solution while stirring until the final concentrations were 0.1M, 0.4M, 0.7M, and 1.0M, respectively. The mixture was rotated for 5 minutes to allow for complete reaction, and then allowed to stand at 4℃ for 30 minutes. The mixture was then centrifuged at 8000rpm for 5 minutes to obtain the precipitate, which was weighed. At a 0.4M ammonium sulfate concentration, the number of protein monomers and dimers in the solution further decreased. With increasing ammonium sulfate concentration, the target protein was almost completely precipitated at 0.7M, and no monomers or dimers remained in the solution at 1.0M. The final purity of the target protein gradually increased from 51.32% to approximately 90.25%.

[0095] Disassemble and redissolve:

[0096] The preparation of urea stock solution (4M) is as follows: Weigh 240.24g of urea, add approximately 800mL of deionized water and stir thoroughly to dissolve. Add 10mL of Tris stock solution (1M). Once the temperature returns to room temperature, adjust the pH to 8.0 with HCl, and finally bring the volume to 1L. Depolymerize and resuspend the (NH4)2SO4 precipitated protein in 4M urea at a 1:1 ratio (urea to lysate volume ratio). Mix thoroughly with a pipette, seal with a sealing film, and incubate overnight at room temperature on a vertical vortex mixer to obtain depolymerized VLP monomeric protein. Centrifuge at 10,000rpm for 5min and measure the precipitate mass, supernatant volume, and protein concentration. Urea can disrupt hydrogen bonds and hydrophobic bonds in protein polymers, thereby altering the protein's tertiary structure, causing protein denaturation, and resulting in the unfolding or depolymerization of protein peptide bonds. Using 4M urea, an 80% protein yield is achieved. Figure 2 The changes in purity and yield of the chimeric hepatitis B virus-like particles in this embodiment show that, after the ammonium sulfate purification step, the purity and yield of the target novel recombinant hepatitis B virus-like protein particles can both reach over 80%.

[0097] Example 4

[0098] Impurity removal:

[0099] Preparation of the buffer: Weigh 4 mol Urea (240.24 g), add approximately 800 mL of deionized water and stir thoroughly to dissolve. Add 10 mL of Tris stock solution (1 M) to a final concentration of 20 mM. After the temperature returns to room temperature, adjust the pH to 8.5 with HCl and bring the volume to 1 L. Finally, filter twice under vacuum through a 0.1 μm filter membrane. Preparation of the elution buffer: Weigh 4 mol Urea (240.24 g) and 2 mol NaCl (116.89 g), add approximately 800 mL of deionized water and stir thoroughly to dissolve. Add 10 mL of Tris stock solution (1 M) to a final concentration of 20 mM. After the temperature returns to room temperature, adjust the pH to 8.5 with HCl and bring the volume to 1 L. Finally, the system was vacuum filtered twice using a 0.1 μm filter membrane. The AKTA system was then rinsed with ultrapure water at a flow rate of 1 mL / min using a 50% B pump, with HiTrap Q XL connected to the system. The system was thoroughly rinsed to remove 20% ethanol from the system and column. The system was paused, and elution buffer was added to both pump A and pump B. The column pressure was set to 0.3 MPa, and the system was rinsed with 100% B pump until the conductivity reached equilibrium. Rinsing continued for 5-10 column volumes to ensure all impurities in the system and column were removed. Subsequently, the system was rinsed with 0% B pump until the conductivity reached equilibrium, and then rinsed for another 5-10 column volumes to remove all salts. The loading method was determined based on the actual sample volume: for volumes less than 5 mL, a loading loop of the corresponding volume was used; for volumes greater than 5 mL, pump A was used directly. When the sample UV280 began to rise to 1000, the flow-through sample was collected; when it fell back to 1000, collection was stopped. After sample loading, the elution buffer was aspirated using a 100% B pump to step-elute the protein attached to the column, and the liquid from each peak was collected. The sample volume and protein concentration at each stage were recorded. Finally, the sample was washed with ultrapure water and 20% ethanol to terminate the operation. The final purified target protein yield was 66.7%. Figure 3 This is the ion exchange chromatography purification spectrum of the chimeric hepatitis B virus-like particles in this embodiment. It clearly shows that the protein sample exhibits two peaks during the protein chromatography purification process, with the second peak being the elution peak of the novel recombinant hepatitis B virus-like protein particles, proving that the target protein can be successfully obtained through ion exchange chromatography purification. Figure 4 This is a protein gel electrophoresis image of the chimeric hepatitis B virus-like particles purified by ion exchange chromatography in this embodiment. Protein gel electrophoresis of the novel recombinant hepatitis B virus-like protein particles purified by ion exchange chromatography clearly shows bands of the target protein in the 22-31 kDa range, indicating that the target protein can be successfully obtained by ion exchange chromatography purification.

[0100] Example 5

[0101] Reassembly:

[0102] Prepare the dialysis buffer: 20 mM Tris, 0.1% Tween-80, 0.5 M NaCl, pH 7.4. Place the purified sample into a dialysis bag, add the dialysis buffer, and dialyze for 24 hours, changing the dialysis buffer every 4 hours. The VLP reassembly principle is to remove urea from the buffer system using methods such as dialysis, allowing the VLP monomers to reassemble into complete VLP particles. The assembled VLP particles have a relatively uniform size distribution, with a PDI of around 0.3. The average value of three measurements is 34.35 nm. The overall particle size is negatively charged, approximately -11.4 mV, and the electrophoretic purity of the protein is greater than 90%. Figure 5 This is the transmission electron microscopy (TEM) result of the chimeric hepatitis B virus-like particles in this embodiment. The TEM results show that the hepatitis B virus-like protein particles have a complete spherical structure and an intact self-assembled structure, indicating the successful acquisition and self-assembly of the target protein.

[0103] Example 6

[0104] M2 macrophage targeting with chimeric hepatitis B virus-like particles:

[0105] ① Female C57BL / 6 mice aged 4-6 weeks were euthanized by cervical dislocation and soaked in 75% alcohol for 5-10 minutes;

[0106] ② Using sterile surgical scissors and forceps, remove the femur and tibia of the mouse hind leg in a sterile laminar flow hood. Carefully remove excess muscle attached to the bone (be careful not to cut out the bone marrow to prevent contamination). Immerse the treated femur and tibia in RPMI-1640 medium or PBS and place them on ice to keep the bones in good condition.

[0107] ③ Use a 1mL sterile syringe to draw RPMI-1640 basic culture medium, and carefully insert the needle into the medullary cavity from one end of the bone shaft by rotating it. Rinse the bone marrow into a 15mL sterile centrifuge tube, rinsing each end twice until the bone turns white. Collect the culture medium, centrifuge at 1500rpm for 5min, and discard the supernatant.

[0108] ④ Add 3 mL of red blood cell lysis buffer to the above precipitate, gently pipette to mix evenly, suspend the precipitate cells, let stand at room temperature for 3 min to lyse the red blood cells, centrifuge again at 1500 rpm for 5 min, and discard the supernatant.

[0109] ⑤ Resuspend the cells in 3 mL of RPMI-1640 complete culture medium, pass through a cell sieve, wash the sieve with 1 mL, wash the culture dish with 1 mL, centrifuge at 1500 rpm for 5 min, discard the supernatant, and resuspend the cells in complete culture medium containing macrophage colony-stimulating factor (M-CSF, ThermoFisher, 20 ng / mL). Count the cells using a counter at a ratio of 1 × 10⁻⁶. 6Cells / wells were evenly distributed into 24-well culture plates (0.5 mL per well), and then 0.5 mL of the above-mentioned complete culture medium was added. After microscopic observation, the cells were cultured in a cell culture incubator (37℃, 5% CO2) for 48 h.

[0110] ⑥ Observe the cell state under a microscope. Gently shake the cells and aspirate the suspended cells along with the culture medium (slowly aspirate from top to bottom, retaining only adherent cells and removing granulocytes and lymphocytes). Add fresh complete culture medium and continue culturing until day 5 to obtain immature BMDCs (M0). On day 5, add culture medium containing cytokines IFN-γ (Thermo Fisher, 20 ng / mL) and LPS (100 ng / mL) to the immature macrophages to induce macrophage polarization towards M1; add culture medium containing cytokines IL-4 (Thermo Fisher, 20 ng / mL) to the immature macrophages to induce macrophage polarization towards M2. Collect M1 and M2 macrophages separately for subsequent in vitro cell function experiments.

[0111] ⑦ Digest differentiated macrophages with trypsin, count them, and then use 5 × 10⁻⁶ cells per cell. 5 Cells / wells were seeded into 24-well plates and cultured in complete culture medium for 24 hours. Macrophages were then co-incubated with Rhodamine B-labeled VLPs. After incubation, M2 macrophages were stained with F4 / 80 monoclonal antibody, and the phagocytic effect of VLPs on M2 macrophages was examined using confocal fluorescence microscopy. Compared to the non-targeted control group, the phagocytic efficiency of M2 macrophages for antigen-chimeric hepatitis B virus-like particles reached 92% when co-incubated with targeted VLP nanoparticles. Figure 6 This is the fluorescence result of macrophage targeting cells with chimeric hepatitis B virus-like particles in this embodiment. It can be seen that the novel hepatitis B virus-like particles, labeled with red fluorescence, specifically aggregate in M2 macrophages labeled with green fluorescence, indicating that the novel hepatitis B virus-like particles can successfully target M2 macrophages.

[0112] Example 7

[0113] In vivo antitumor therapy with chimeric hepatitis B virus-like particles:

[0114] Cryopreserved melanoma B16F10 cells were retrieved and subjected to routine thawing, i.e., rapid thawing in a 37°C water bath. The thawed cell suspension was transferred to a sterile centrifuge tube using a pipette, 3 mL of 1640 complete medium was added, and the cells were mixed and centrifuged at 1500 rpm for 5 min. The cell pellet was transferred to a T25 culture flask containing 6 mL of 1640 complete medium and cultured in a CO2 cell culture incubator (37°C, 5% CO2). When cell adhesion was greater than 80% (approximately 2 days), the cells were digested with 0.25% DEAE-trypsin for 1 min, observed, and collected. Cells were passaged at a 1:4 ratio or collected, and adherent cells in the logarithmic growth phase were selected for subsequent experiments. The collected B16F10 cells were washed three times with PBS, and finally resuspended in PBS and the cell concentration was adjusted to prepare a 5×10⁶ cell suspension. 6 A single-cell suspension of 5 × 10⁶ cells / mL was prepared and stored on ice for later use. Female C57BL / 6 mice aged 4–6 weeks were selected and housed in the animal facility for one week to acclimatize. After measuring their weight, 100 μL of the above-mentioned melanoma suspension (5 × 10⁶ cells / mL) was subcutaneously injected into the right axilla. 5 Mice were randomly divided into 6 groups of 6 mice each, with each group having 6 mice per group. Mouse weight and tumor volume were measured every two days. When the tumor volume reached 20-30 mm², the tumor was counted. 3 Immunotherapy was administered using chimeric hepatitis B virus-like particle (HBV-like particle) vaccine. Ultimately, compared to the saline group, the HBV-like particle vaccine achieved a 70% tumor inhibition rate. Figure 7 This embodiment presents the tumor suppression results of different control groups incorporating chimeric hepatitis B virus-like particles. It is clear that the tumor volume in mice significantly decreased after immunotherapy with the hepatitis B virus-like particle vaccine.

[0115] In summary, this invention constructs an expression vector containing MAGEA10 antigen, M2 targeting peptide, and hepatitis B virus core protein. This vector can be expressed in suitable host cells and is easily purified to form a chimeric VLP. This VLP specifically targets M2 macrophages in the tumor microenvironment, reducing the impact on normal tissues. By delivering the MAGEA10 antigen via the VLP, it stimulates a strong anti-tumor immune response, improving the efficacy of anti-tumor immunotherapy and potentially reducing tumor recurrence rates.

Claims

1. A hepatitis B virus-like particle (HBc VLP) targeting M2 macrophages, comprising nanoparticles self-assembled from hepatitis B virus core protein (HBcAg), wherein the nanoparticles express specific peptides targeting M2 macrophages on their surface.

2. A hepatitis B virus-like particle (HBc VLP) that targets M2 macrophages and specifically expresses a melanoma antigen, such as melanoma antigen MAGEA10, comprising nanoparticles self-assembled from hepatitis B virus core protein (HBcAg), the nanoparticles expressing a specific peptide targeting M2 macrophages on their surface, and the nanoparticles carrying and expressing the melanoma antigen.

3. The hepatitis B virus-like particle according to claim 1 or 2, wherein the specific peptide binds to the hepatitis B virus core protein (HBcAg), preferably, the specific peptide is linked to the amino terminus of the hepatitis B virus core protein.

4. The hepatitis B virus-like particles according to claim 1 or 2, characterized in that, The nanoparticles express melanoma antigens.

5. The hepatitis B virus-like particle according to claim 1 or 2, wherein the amino acid sequence of the specific targeting peptide is shown in SEQ ID NO:

1.

6. The hepatitis B virus-like particle according to claim 3, wherein the melanoma antigen is melanoma antigen MAGEA10, preferably, the amino acid sequence of which is shown in SEQ ID NO:

2.

7. The hepatitis B virus-like particle according to claim 1 or 2, wherein the hepatitis B virus-like particle comprises or is composed of a fusion protein, the amino acid sequence of which is shown in SEQ ID NO:

3.

8. A method for preparing hepatitis B virus-like particles that target M2 macrophages and specifically express MAGEA10, comprising the following steps: a) Construct a recombinant plasmid containing the M2 targeting peptide gene, the MAGEA10 gene, and the HBcAg gene; b) Express the recombinant plasmid in host cells to produce hepatitis B virus-like particles; c) Purify and separate the hepatitis B virus-like particles.

9. A pharmaceutical composition comprising hepatitis B virus-like particles as described in any one of claims 1-8, and a pharmaceutically acceptable carrier, the pharmaceutical composition being a vaccine, such as a therapeutic vaccine.

10. The use of hepatitis B virus-like particles according to any one of claims 1-8 in the preparation of a drug for treating melanoma.

Images