Bionic personalized in-situ tumor vaccine as well as preparation and use methods thereof

Abstract

The invention discloses a bionic personalized in-situ tumor vaccine and a preparation and use method thereof, and belongs to the technical field of material science. Through synergistic design, HBc VLPs is taken as a carrier, fusion expression of melittin, phagocytic peptide Tuftsin, tumor cell targeting peptide RGD and M2 macrophage targeting peptide M2pep is realized, and an MMP-2 restriction enzyme cutting site is designed in an MIR region, so that enzyme response cleavage is realized in a tumor microenvironment, and the tumor vaccine is formed. The invention discloses an oncolytic virus biomimetic preparation method, which fully utilizes the effect of inducing tumor cell necrosis or apoptosis of melittin and the property of promoting macrophages to secrete proinflammatory cytokines by virus-like particles so as to repolarize M2 type macrophages into M1 type macrophages, simulates the immune mechanism of oncolytic viruses, and aims to solve the problems of few tumor-associated antigens, low toxicity and the like. The immunologic adjuvant efficiency and the antigen lymph node enrichment degree are low.

Description

Technical Field

[0001] The present invention belongs to the field of material science and technology, and also relates to the field of medical technology; specifically, it relates to the development of a new type of in situ tumor vaccine of bionic oncolytic virus, which is used to improve the tumor microenvironment, induce tumor cell necrosis or apoptosis, and improve the effect of tumor immunotherapy. Background Art

[0002] Virus-like particles (VLPs) refer to spherical or tubular protein nanostructures formed by the self-assembly of viral capsid proteins, which can be prepared in a variety of biological systems, including mammals, plants, insects, and bacteria. Hepatitis B core (HBc) protein VLPs have highly appropriate immunogenicity at the levels of B cells, T cells, and cytotoxic T lymphocytes, and its monomers (HBV core proteins) can be easily obtained and purified in Escherichia coli expression systems. Insertion of foreign proteins on the major immune domains of HBc VLPs can be easily achieved through genetic engineering. In addition, HBc VLPs can be dissociated and reassembled by destroying the disulfide bonds between VLPs monomers by relying on denaturant stock solutions such as urea and guanidine. Based on the assembly and disassembly characteristics of HBc VLPs, different functionalized HBc VLP monomers can be reassembled into multifunctional hybrid HBc VLPs. Therefore, HBc VLPs are promising candidate vaccine antigens and carriers for the delivery of genes, drugs, or other therapeutic agents with good biocompatibility and non-infectiousness. (ACS Appl Mater Interfaces. 2020, 12(48): 53682-53690.) However, there are relatively few studies on VLP-based tumor vaccines, especially the lack of in-depth research on the role of the immunogenicity of VLP itself in activating tumor antigen-specific immunity.

[0003] Host defense peptides (HDPs), originally called antimicrobial peptides, are produced by all organisms as an evolutionarily conserved component of the innate immune system. These peptides often exhibit direct cytotoxic activity against bacteria, fungi, protozoa, enveloped viruses, and even cancer cells. As a natural HDP, bee venom peptide, which consists of 26 amino acids, has an excellent inhibitory effect on tumors such as gastric cancer, liver cancer, and leukemia. It can also indirectly eliminate pathogens through complex regulation of innate and adaptive immune responses, including chemical attraction of immune cells, regulation of cytokine production, induction of phagocyte differentiation, and activation of T cell responses (Peptides. 2021, 146: 170644.). However, bee venom peptide has a strong hemolytic reaction and cytotoxicity to normal cells, which makes it impossible to administer intravenously and interact with immune cells for a long time, severely limiting its clinical application.

[0004] Oncolytic virus therapy is a common in situ tumor vaccine program. Oncolytic viruses can directly infect and dissolve tumor cells in situ, use antigens released by necrotic tumor cells, and release cytokines (interleukin-12, TNF-α, etc.) to promote the maturation and antigen presentation of antigen-presenting cells to achieve anti-tumor effects. However, the normal cell toxicity caused by weak targeting and the antibody neutralization caused by the activation of the body's antiviral immunity have hindered the clinical application of oncolytic virus therapy. Based on gene fusion expression technology, HBc VLP monomers functionalized with bee venom peptide were used to construct "oncolytic" virus-like particles with the functions of "tumor cell membrane targeting and enzyme response release", overcoming the shortcomings of bee venom peptide such as low bioavailability, short biological half-life, and poor targeting, making it a new type of in situ tumor vaccine of biomimetic oncolytic virus, which plays an effective role in tumor immunotherapy. Summary of the invention

[0005] The present invention aims to solve the problems of few tumor-associated antigens, low efficiency of immune adjuvants and low enrichment of antigen lymph nodes, and provide a biomimetic personalized in situ tumor vaccine that is easy to obtain through bacterial culture, has high expression yield, and is convenient for industrial production. In the process of killing tumors, the shortcomings of bee venom peptide such as low bioavailability, short biological half-life, and poor targeting are overcome; and the immunogenicity of VLPs themselves and the danger signals and cytokines released after tumor cell necrosis are used to reverse the immunosuppressive tumor microenvironment.

[0006] The present invention provides a bionic personalized in situ tumor vaccine, which serves as an effective component of the vaccine. An oncolytic hepatitis B virus core protein virus-like particle designed by the present invention is Tuftsin-HBc-Mel VLPs; the virus-like particle can both efficiently kill tumor cells and activate immune response; the Tuftsin-HBc-Mel VLPs recombinant protein is prepared by an Escherichia coli heterologous expression system; its structure is as follows: using HBc VLPs as a basic skeleton, constructing a macrophage targeting peptide element M2pep-Tuftsin at the C-terminus of its protein monomer, constructing a tumor cell membrane targeting peptide element and an enzyme response element at its major immune region (MIR), and constructing a tumor killing element Melittin at the N-terminus of its protein monomer; the amino acid sequence of the Tuftsin-HBc-Mel VLPs is SEQ ID NO.1.

[0007] The present invention provides a recombinant plasmid, which is obtained by cloning a Tuftsin-HBc-Mel VLPs gene fragment as shown in SEQ ID NO.2 in the sequence table into the NdeI site and XhoI site of a prokaryotic expression vector pET43.1a by homologous recombination to obtain the recombinant plasmid Tuftsin-HBc-Mel-pET43.1a.

[0008] The invention provides a recombinant Escherichia coli Tuftsin-HBc-Mel-pET43.1a-BL21 containing the recombinant plasmid.

[0009] The present invention also provides a method for preparing Tuftsin-HBc-Mel VLPs expressed by recombinant Escherichia coli, comprising the following steps:

[0010] 1) The Tuftsin-HBc-Mel VLPs gene fragment as shown in SEQ ID NO.2 in the sequence listing was cloned into the pET43.1a prokaryotic expression vector to obtain the positive recombinant plasmid Tuftsin-HBc-Mel-pET43.1a;

[0011] 2) The recombinant plasmid with the correct sequence in step 1) was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant expression strain Tuftsin-HBc-Mel-pET43.1a-BL21;

[0012] 3) Cultivate the recombinant expression strain Tuftsin-HBc-Mel-pET43.1a-BL21 from step 2), add IPTG to induce expression, and obtain Tuftsin-HBc-Mel VLPs recombinant protein.

[0013] As a preferred embodiment of the method for preparing Tuftsin-HBc-Mel VLPs virus-like particles expressed by recombinant Escherichia coli, when the recombinant expression strain Tuftsin-HBc-Mel-pET43.1a-BL21 is cultured to an OD600 of 0.6 to 0.8, IPTG with a final concentration of 1 mM is added to induce expression.

[0014] Application of biomimetic personalized in situ tumor vaccine on M2 macrophage repolarization.

[0015] Application of bionic personalized in situ tumor vaccine to kill B16-OVA tumor cells.

[0016] Compared with the prior art, the present invention has the following outstanding technical effects and advantages:

[0017] 1. Constructing tumor cell membrane targeting peptide elements in the major immune region (MIR) of the HBc VLPs protein monomer enables the vaccine to specifically identify tumor cells. Compared with traditional treatment methods, it improves the targeting of tumor tissues and reduces damage to normal tissues.

[0018] 2. Constructing the tumor-killing element Melittin at the N-terminus to overcome the shortcomings of melittin such as low bioavailability, short half-life, and poor targeting. Melittin can act more effectively on tumor cells and improve the efficiency of killing tumor cells. Experiments have shown that the concentration of modified Melittin in tumor cells is significantly higher than that of unmodified melittin, and the inhibition rate of tumor cells is significantly improved.

[0019] 3. Construct a macrophage targeting peptide element M2pep-Tuftsin at the C-terminus of the protein monomer to enhance the targeting of macrophages. After macrophages take up the vaccine, they can be effectively activated and promote their immune function. Experiments show that the phagocytic activity of macrophages treated with the vaccine is significantly enhanced compared with the untreated group, and can better eliminate tumor cells.

[0020] 4. By utilizing the immunogenicity of VLPs themselves and the danger signals and cytokines released after tumor cell necrosis, the immunosuppressive tumor microenvironment can be reversed. Experiments have shown that Tuftsin-HBc-Mel VLPs can induce the repolarization of M2 macrophages to M1 macrophages, promoting anti-tumor immune response, which is difficult to achieve with existing technologies. M1 macrophages have stronger anti-tumor activity and can effectively enhance the body's immune surveillance and clearance of tumors.

[0021] 5. The vaccine is obtained by culturing recombinant E. coli, with high expression yield. It has low cost and high yield, and is easy for large-scale industrial production, making it possible for widespread clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the recombinant plasmid Tuftsin-HBc-Mel-pET43.1a provided in the examples of the present invention.

[0023] Figure 2 Schematic diagram of SDS-PAGE running of Tuftsin-HBc-Mel VLPs recombinant protein provided in the examples of the present invention.

[0024] Figure 3 The expression levels of M1 macrophage markers and M2 macrophage markers on the cell surface were detected after Tuftsin-HBc-Mel VLPs induced M2 macrophage repolarization in the embodiment of the present invention. Flow cytometry was used to study the ability of Tuftsin-HBc-Mel VLPs to inhibit the polarization of myeloid macrophages to M2. Wherein, a is CD80, a specific marker on the surface of M1 macrophages; b is CD206, a specific marker on the surface of M2 macrophages.

[0025] Figure 4The expression level changes of M1-related genes and M2-related genes after Tuftsin-HBc-Mel VLPs induced M2 macrophage repolarization in the embodiment of the present invention. Real-time quantitative PCR technology was used to analyze the effect of Tuftsin-HBc-Mel VLPs on the expression level of inflammatory cytokines expressed by myeloid macrophages. Among them, a is the M1-related gene TNF-α; b is the M1-related gene IL-6; c is the M1-related gene iNOS; d is the M1-related gene IL-1β; e is the M2-related gene Arg1; f is the M2-related gene Mrc1; g is the M2-related gene Mgl1.

[0026] Figure 5 The results of the immunotherapy of mice by Tuftsin-HBc-Mel VLPs in the embodiment of the present invention. Among them, a is a schematic diagram of the immunotherapy scheme (subcutaneous injection of tumor cells on the outer thigh of mice, and seven days later, intratumoral injection of drugs was started. The drugs were given every two days for a total of three times, and the mouse status and tumor volume changes were observed until the 30th day.); b is the monitoring data of tumor volume changes in mice in the PBS group, n=6; c is the monitoring data of tumor volume changes in mice in the Melittin group, n=6; d is the monitoring data of tumor volume changes in mice in the Tuftsin-HBc-Mel VLPs group, n=6; e is the integrated data of tumor volume changes in the three groups of mice; f is the monitoring data of weight changes in the three groups of mice; g is the statistics of survival rates of the three groups of mice; h is the actual photos of tumors in the three groups of mice after the observation period.

[0027] Figure 6 The safety evaluation results of mice after immunotherapy with Tuftsin-HBc-Mel VLPs in the examples of the present invention. H&E sections were taken and photographed for the main organs of the mice after treatment. The order of the groups from left to right is: PBS group, Melittin group, Tuftsin-HBc-Mel VLPs group. The order of organs from top to bottom is: lung, liver, spleen, kidney, heart. DETAILED DESCRIPTION

[0028] The present invention is described in detail below in conjunction with specific embodiments.

[0029] The present invention provides a Tuftsin-HBc-Mel VLPs protein expressed by recombinant Escherichia coli, which uses HBc VLPs as a basic framework, constructs a macrophage targeting peptide element M2pep-Tuftsin at the C-terminus of the protein monomer, constructs a tumor cell membrane targeting peptide element and an enzyme response element at the major immune region (MIR), and constructs a tumor killing element Melittin at the N-terminus of the protein monomer. The amino acid sequence of the Tuftsin-HBc-Mel VLPs is SEQ ID NO.1.

[0030] SEQ ID NO.1:

[0031] MYEQDPWGVKWWYGSSGSGGSGSTKPRGSGSGSSGMDIDHYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDGTSGSGSGGSGSGGGGPAGLV GGSGGGRGDGGSGSGGSSGSTGKSRELVVGYVNVNMGLKIRQILWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPGSGSGGSGSGGIGAVLKVLTTGLPALISWIKRKRQQGSGSGGHHHHHH

[0032] The invention provides a recombinant plasmid, which is obtained by cloning SEQ ID NO.2 into the NdeI site and XhoI site of a prokaryotic expression vector pET43.1a by homologous recombination to obtain the recombinant plasmid Tuftsin-HBc-Mel-pET43.1a.

[0033] SEQ ID NO.2:

[0034] atgtatgaacaagatccctggggagtaaaatggtggtacggtagcagcggctcaggtggatctggttcgaccaaaccgcgtggttctggctccggtagcagcggtatggacatcgaccattataaagagttcggcgcgagcgtggaattgctgtcgttcctgccgagcgactttttcccgagcattcgtgatctgctggataccgcttctgcactctaccgcgaggccttggagagcccggaacattgcagcccgcatcacaccgcactgcgtcaggcgattctgtgttggggtgaattaatgaacctcgctacctgggtgggttccaacttggaggacggcacttcaggcagcggttccggtggttccggctcgggcggcggtggaccggcgggtctggtgggtggctccggtggtggccgtggagatggtggcagcggcagcggtggtagcagcggcagcaccggtaaaagccgtgaacttgtagttggttatgttaatgttaacatgggtctgaagatccgccaaatcttatggtttcatatcagctgcctgacgtttggccgtgagacagttttggaatacctggtctccttcggggtgtggattcgtaccccgccggcgtatcgccctccgaatgccccaattctgtccaccctgccgggttcaggttcgggtggctctggtagcggcggtatcggtgcggttctgaaggtgctgacgaccggtttgccggcgctgattagctggatcaagcgcaaaagacaacagggtagcggctctggcggccatcaccaccaccaccactaa

[0035] Example 1 Preparation of Tuftsin-HBc-Mel VLPs Recombinant Protein

[0036] See Figure 1 and Figure 2 to provide a method for preparing Tuftsin-HBc-Mel VLPs recombinant protein, comprising the following steps:

[0037] (1) Construction of recombinant plasmid: SEQ ID NO.2 was directly synthesized by GenScript Biotech Co., Ltd. into the NdeI and XhoI restriction sites of pET43.1a vector to obtain the recombinant plasmid Tuftsin-HBc-Mel-pET43.1a; the synthesized recombinant plasmid was sequenced to ensure that the inserted sequence was accurate.

[0038] (2) Construction of recombinant expression strain: The recombinant plasmid is transformed into Escherichia coli BL21 (DE3) by chemical transformation or electroporation to obtain the recombinant expression strain Tuftsin-HBc-Mel-pET43.1a-BL21; the transformed strain is screened and identified, such as by using colony PCR to confirm that the recombinant plasmid has been successfully transferred into the host bacteria.

[0039] (3) Bacterial culture and recombinant protein purification: Cultivate the Tuftsin-HBc-Mel-pET43.1a-BL21 recombinant expression strain and induce expression with IPTG to obtain the Tuftsin-HBc-Mel VLPs recombinant protein.

[0040] Specifically, the steps of expressing and purifying the Tuftsin-HBc-Mel VLPs recombinant protein are as follows:

[0041] (3.1) Expression of recombinant protein in Tuftsin-HBc-Mel VLPs

[0042] Transform the BL21 (DE3) expression strain, shake the bacteria and concentrate by centrifugation, spread on ampicillin resistance plates, culture at 37 ° C overnight, pick colonies, inoculate into 10ml LB medium containing ampicillin, and shake the bacteria at 37 ° C 220r until the OD value of the bacterial solution is about 0.8. Transfer it to 750mL LB medium containing ampicillin, and shake the bacteria at 37 ° C 220r until the OD value of the bacterial solution is about 0.8. Add IPTG inducer with a final concentration of 0.1mM and express overnight at 30 ° C. On the third day, centrifuge at 3600rpm for 15min to collect the bacteria. Ultrasonic disruption of bacteria for 20min, 3s on, 4s off, power 125w, ultrasonic process in an ice bath, ensure that the temperature is around 4 ° C. After the end of ultrasound, 12000rpm, 15min, 4°C to collect the supernatant.

[0043] (3.2) Purification of Tuftsin-HBc-Mel VLPs recombinant protein

[0044] The supernatant was centrifuged and purified using a size exclusion chromatography column. 45 µL of each collected solution was taken and added with 15 µL of 4× protein electrophoresis loading buffer and heated at 100°C for 7 min. SDS-PAGE electrophoresis analysis was then performed. The collected solution where the target band was located was stained and observed, and the effluent that matched the molecular weight of the target protein was collected. The collected solution was concentrated using PEG20000 and then purified using an anion affinity chromatography column. Load the sample, elute with 2 column volumes at 20 mM, collect the effluent, and perform SDS-PAGE electrophoresis analysis. The results showed that a clear protein band appeared at the expected molecular weight position, and pure Tuftsin-HBc-MelVLPs were obtained. Figure 2 After concentration with PEG20000, the solution was dialyzed to replace the solvent with PBS.

[0045] Example 2 Studying the effects of different drugs on the repolarization of M2 macrophages

[0046] Primary myeloid cells were extracted from healthy mice and inoculated into differentiation medium to induce differentiation into M2 macrophages.

[0047] Set up 5 groups, each with 3 parallel groups, and each parallel group with 4×10 5 Cells, the culture system is 2mL culture medium. Among them:

[0048] Control group: divided into two parts, one part was treated with M2 macrophages only with PBS as a blank control; the other part was treated with free Tuftsin peptide to observe the effect of Tuftsin peptide alone.

[0049] PBS group: 50 μL of 1× PBS solution was added to the cell culture system in each parallel group.

[0050] Tuftsin group: 50 μL of 1 mg / mL Tuftsin solution was added to the cell culture system in each parallel group.

[0051] Experimental groups: including M2T-Tuftsin-HBc 78 group, mmp&RGD HBc VLPs group and Tuftsin-HBc-MelVLPs group;

[0052] M2T-Tuftsin-HBc 78 group: 50 μL of 1 mg / mL M2T-Tuftsin-HBc78 solution was added to the cell culture system in each parallel group.

[0053] mmp&RGD HBc VLPs group: 50 μL of 1 mg / mL mmp&RGD HBc VLPs solution was added to the cell culture system in each parallel group.

[0054] Tuftsin-HBc-Mel VLPs group: 50 μL of 1 mg / mL Tuftsin-HBc-Mel VLPs solution was added to the cell culture system in each parallel group.

[0055] The above five groups of drugs were co-cultured with differentiated M2 macrophages for 24 hours. During the co-culture process, the consistency of culture conditions was ensured, including the temperature being maintained at 37°C, the CO2 concentration of the incubator being stabilized at 5%, and contamination was avoided.

[0056] After the culture was completed, the cells were collected and flow cytometry was used to detect the expression changes and differences of M1-specific surface marker CD80 and M2-type macrophage surface marker CD206. The results are as follows Figure 3 As shown. Compared with the control group, under the induction of Tuftsin-HBc-Mel VLPs, the fluorescence intensity of the cell surface marker CD206 was significantly reduced, while the fluorescence intensity of CD80 was significantly enhanced. This shows that under the action of this drug, the expression of M2 macrophage surface markers decreased, and the expression of M1 macrophage surface markers increased, suggesting that the cell phenotype changed.

[0057] The supernatant of 24-hour culture was collected, and the expression levels of M1-related genes: TNFα, IL-6, iNOS and IL-1β, and M2-related genes: Arg-1, Mrc-1, Mgl-1 were detected by ELISA. The results of ELISA were as follows: Figure 4 As shown. Figure 4 It can be seen that in the Tuftsin-HBc-Mel VLPs-treated group, the expression levels of M1-related genes (TNF α, IL-6, iNOS and IL-1β) showed an upward trend, while the expression levels of M2-related genes (Arg-1, Mrc-1, Mgl-1) decreased; experimental data showed that under the action of Tuftsin-HBc-Mel VLPs, the gene expression profile of M2 macrophages changed significantly, which was consistent with the changes in cell surface markers, further proving that Tuftsin-HBc-Mel VLPs can successfully induce the repolarization of M2 macrophages to M1 macrophages and promote anti-tumor immune response.

[0058] Example 3 Tuftsin-HBc-Mel VLPs tumor vaccine in vivo immunization method

[0059] 1. Establishment of B16-OVA tumor-bearing mouse model: Select healthy mice of similar age and weight (6-8 weeks old, weighing about 20-25g), the mouse strain is C57BL / 6 mice. Use trypsin-EDTA solution to digest B16-OVA cells from the culture flask, incubate for 1-2 minutes, and add serum-containing culture medium to terminate digestion after the cells fall off. Collect the cells by centrifugation, resuspend them in PBS, and adjust the cell concentration to 1×10 6 / 50 μL. The above cell suspension was injected subcutaneously into the right back of the mouse, with an injection volume of 50 μL per mouse to establish a B16-OVA tumor-bearing mouse model.

[0060] 2. The mice with established B16-OVA tumor-bearing mouse model were randomly divided into three groups, with n=6 in each group. The groups were as follows:

[0061] Control group 1: PBS group, injected with an equal volume of PBS as a negative control to exclude the influence of injection operation and solvent on the experimental results.

[0062] Control group 2: Melittin group, treated with Melittin, the dose of Melittin was 100 μg / mouse.

[0063] Experimental group: Tuftsin-HBc-Mel VLPs group, treated with Tuftsin-HBc-Mel VLPs at a dose of 100 μg / mouse.

[0064] Treatment process Figure 5 As shown in a, the date of tumor cell injection was defined as day 0. On day 7, the formation of subcutaneous tumors in mice was determined by visual observation and palpation. When the tumor volume reached a measurable and obvious size, direct intratumoral administration began. In the PBS group, 100 μL of 1×PBS solution was injected intratumorally in each mouse; in the Melittin group, 100 μL of 1 mg / mL Melittin solution was injected intratumorally in each mouse; in the Tuftsin-HBc-Mel VLPs group, 100 μL of 1 mg / mL Tuftsin-HBc-Mel VLPs solution was injected intratumorally in each mouse.

[0065] 3. Measurement of tumor volume and mouse weight:

[0066] After the start of intratumoral administration, the long diameter (L) and short diameter (W) of the tumor were measured every three days, and the tumor volume was calculated according to the formula V=(L×W²) / 2. During the measurement, the mouse was gently fixed to avoid squeezing or damaging the tumor to ensure the accuracy of the measurement data. The tumor volume of each measurement was recorded and the tumor growth curve was drawn ( Figure 5be in the figure), and observe the changing trend of tumor volume in different treatment groups over time. As can be seen from the figure, as time goes by, the tumor volume of the PBS group and the Melittin group shows a continuous growth trend, while the growth of the tumor volume in the Tuftsin-HBc-Mel VLPs group is significantly inhibited. On the 16th day, the tumors of all mice were completely relieved, and the tumor volume approached zero.

[0067] At the same time, the weight of the mice was measured at the same time point, accurate to 0.1g. The change in the weight of the mice can reflect the effect of the drug on the overall health of the mice and is one of the important indicators for evaluating the safety of the drug. The weight data of each measurement was recorded and the weight change curve of the mice was drawn ( Figure 5 f in ). From Figure 5 As can be seen from figure f, the body weights of mice in the PBS and Melittin groups fluctuated during the experiment, possibly due to factors such as tumor growth and drug side effects, while the body weights of mice in the Tuftsin-HBc-Mel VLPs group were relatively stable with less fluctuation, indicating that the vaccine had little effect on the overall health of mice.

[0068] 4. Statistics of mouse survival rate:

[0069] The survival status of mice was observed, the survival rate of mice in different treatment groups was calculated, and the statistical results were presented in the form of survival curves. Figure 5 As can be seen from the graph, the survival rates of mice in the PBS group and the Melittin group gradually decreased over time. All mice in the PBS group died due to excessive tumor burden on day 21, and all mice in the Melittin group died on day 28, with a survival rate of 0% on day 30. In the Tuftsin-HBc-Mel VLPs group, all 6 mice survived, demonstrating that the vaccine has an excellent anti-tumor effect and significantly improves the survival rate of mice ( Figure 5 g in the figure).

[0070] 5. Vaccine safety assessment in vivo

[0071] One mouse was randomly selected from each group and killed. The heart, liver, spleen, lung, and kidney were dissected and sliced ​​with H&E to take photos. Figure 6 It can be seen that there is no obvious lesion in the organs of the Tuftsin-HBc-Mel VLPs group and the control group, indicating that the vaccine designed by the present invention has good safety and can be used for in vivo treatment.

[0072] The present invention discloses a method for preparing an in situ tumor vaccine based on hepatitis B virus core protein virus-like particles (HBc VLPs), and applies the vaccine to tumor immunotherapy. Hepatitis B virus core antigen (HBc Ag) is the capsid core protein of hepatitis B virus, which can self-assemble to form hepatitis B core virus-like particles (HBc VLPs). Virus-like particles are virus-derived nanoparticles without viral genomes, which can load genes and small molecule drugs with good biocompatibility and non-infectiousness into the internal cavity, or hybridize and reorganize monomers with different functions, and are a promising vaccine platform and drug carrier. Melittin is a host defense polypeptide composed of 26 amino acids, which can induce cell necrosis or apoptosis, promote the recruitment of immune cells, regulate adaptive immune responses, and have excellent inhibitory effects on tumors such as gastric cancer, liver cancer, and leukemia. The present invention uses HBc VLPs as a carrier to express melittin, tuftsin, RGD, and M2 macrophage targeting peptides through synergistic design, and designs MMP-2 cleavage sites in the MIR region to achieve enzyme response cleavage in the tumor microenvironment to form a tumor vaccine. The effect of melittin inducing tumor cell necrosis or apoptosis, and the properties of virus-like particles that promote macrophages to secrete proinflammatory cytokines and repolarize M2 macrophages to M1 macrophages are fully utilized to mimic the immunotherapy mechanism of oncolytic viruses, aiming to solve the problems of few tumor-associated antigens, low efficiency of immune adjuvants, and low antigen lymph node enrichment.

[0073] The above embodiments are only preferred embodiments of the present invention, which are used to illustrate the technical solutions of the present invention, but not to limit them. Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solutions described in the above embodiments can still be modified, or some of the technical features can be replaced by equivalents, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomimetic personalized in situ tumor vaccine, characterized in that It comprises Tuftsin-HBc-Mel VLPs protein expressed by recombinant Escherichia coli, and the amino acid sequence of the Tuftsin-HBc-Mel VLPs protein is shown in SEQ ID NO.

1.

2. A Tuftsin-HBc-Mel VLPs protein expressed by recombinant Escherichia coli, characterized in that Taking HBc VLPs as the basic framework, a macrophage targeting peptide element M2T-Tuftsin is constructed at the C-terminus of the protein monomer, a tumor cell membrane targeting peptide element and an enzyme response element are constructed at the main immune region, and a tumor killing element Melttin is constructed at the N-terminus of the protein monomer; the above gene sequences are cloned into a prokaryotic expression vector to obtain a recombinant expression vector, the recombinant expression vector is transfected into Escherichia coli BL21 (DE3), and Tuftsin-HBc-Mel VLPs protein is obtained by expression in transfected Escherichia coli BL21 (DE3).

3. A recombinant plasmid Tuftsin-HBc-Mel-pET43.1a, characterized in that: The recombinant plasmid is obtained by cloning the nucleotides shown in SEQ ID NO.2 into the NdeI site of the prokaryotic expression vector pET43.1a through a homologous recombination method.

4. A recombinant Escherichia coli Tuftsin-HBc-Mel-pET43.1a-BL21, characterized in that: Contains the recombinant plasmid according to claim 3.

5. A method for preparing Tuftsin-HBc-Mel VLPs expressed by recombinant Escherichia coli, characterized in that: The steps include: (1) The Tuftsin-HBc-Mel VLPs gene fragment shown in SEQ ID NO.2 in the sequence listing was cloned into the pET43.1a prokaryotic expression vector to obtain the positive recombinant plasmid Tuftsin-HBc-Mel-pET43.1a; (2) The recombinant plasmid obtained in step (1) and confirmed by sequencing was transformed into Escherichia coli BL21 (DE3) to obtain the recombinant expression strain Tuftsin-HBc-Mel-pET43.1a-BL21; (3) Cultivate the recombinant expression strain Tuftsin-HBc-Mel-pET43.1a-BL21 of step (2), add IPTG to induce expression, and obtain Tuftsin-HBc-Mel VLPs recombinant protein.

6. A method for preparing Tuftsin-HBc-Mel VLPs expressed by recombinant Escherichia coli as claimed in claim 5, characterized in that: In step (1), the prokaryotic expression vector is pET43.1a.

7. A method for preparing Tuftsin-HBc-Mel VLPs expressed by recombinant Escherichia coli as claimed in claim 5, characterized in that: In step (3), the recombinant expression strain Tuftsin-HBc-Mel-pET43.1a-BL21 is cultured until OD600 reaches 0.6-0.8, and IPTG with a final concentration of 1 mM is added to induce expression.

8. Application of the bionic personalized in situ tumor vaccine according to claim 1 on the repolarization of M2 macrophages.

9. Use of the bionic personalized in situ tumor vaccine according to claim 1 to kill B16-OVA tumor cells.

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