Engineered biomimetic dendritic cell vesicle-coupled antigenic peptide tumor vaccine, preparation method therefor, and use thereof

Abstract

Provided are an engineered biomimetic dendritic cell vesicle-coupled antigenic peptide tumor vaccine, a preparation method therefor, and use thereof, which belong to the technical field of biomedicine. The provided engineered biomimetic dendritic cell vesicle is internally loaded with an agonist of a stimulator of interferon genes, and an outer membrane surface of said vesicle is coupled with a melanoma antigenic peptide gp100. Also provided are a preparation method for the engineered biomimetic dendritic cell vesicle, and use of the engineered biomimetic dendritic cell vesicle or the preparation method in the preparation of a melanoma vaccine. Further provided is a melanoma vaccine, which comprises the engineered biomimetic dendritic cell vesicle.

Description

An engineered biomimetic dendritic cell vesicle coupled antigen peptide tumor vaccine, and a preparation method and application thereof

[0001] The present application claims priority to the Chinese patent application No. 2024113572905, filed on September 26, 2024, and entitled "An engineered biomimetic dendritic cell vesicle coupled antigen peptide tumor vaccine, and a preparation method and application thereof", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and in particular relates to an engineered biomimetic dendritic cell vesicle coupled antigen peptide tumor vaccine, and a preparation method and application thereof. BACKGROUND

[0003] The core goal of melanoma vaccines is to enhance immune responses to kill tumors, establish long-lasting anti-tumor immune memory, and avoid adverse reactions. However, most of the current melanoma vaccines are still in the clinical research stage, facing two major challenges: one is how to accurately select effective tumor antigens, and the other is how to overcome the highly immunosuppressive microenvironment established by tumor cells. Vaccines that simply rely on cells or their lysates have limited effects in generating immune effects, such as insufficient immunogenicity or potential tumorigenic risk. Therefore, it is particularly crucial to find effective antigen sources and innovative vaccine carriers.

[0004] Dendritic cells (DCs) are professional antigen-presenting cells that can sense various pathogens to stimulate strong and appropriate immune responses. DCs can produce all signals to activate T cells and play an extremely important role in the initiation, programming, and regulation of tumor immune responses. Nano-sized DC-derived vesicles can mimic the physiological functions of parent cells and present tumor antigens as vaccine carriers, which have excellent application prospects. However, there are still many technical difficulties in the development and application of DC vesicles, such as the need for effective separation and purification of specific types of DC vesicles from the multiple types of vesicles (such as exosomes and microvesicles) released by DCs. At the same time, DC vesicles have poor stability during storage and transportation, and are prone to degradation or loss of activity. Therefore, how to overcome the application bottlenecks of DC vesicles has become a key problem that needs to be solved urgently. SUMMARY

[0005] In view of this, one of the purposes of the present application is to provide an engineered biomimetic dendritic cell vesicle and a preparation method thereof, which can be easily recognized by antigen-presenting cells (APCs) and presented to T cells by DCs, and can stimulate the promotion of immune cells in the tumor immune microenvironment to enhance immune responses.

[0006] The second purpose of the present application is to provide an application of the engineered biomimetic dendritic cell vesicle.

[0007] The third object of the present application is to provide a melanoma vaccine.

[0008] To achieve the above-mentioned objects, the present application provides the following technical solutions.

[0009] The present application provides an engineered bionic dendritic cell vesicle, the inside of the engineered bionic dendritic cell vesicle carries an agonist of interferon gene stimulator receptor, and the outer membrane surface of the engineered bionic dendritic cell vesicle is coupled with melanoma antigen peptide gp100.

[0010] The present application also provides a preparation method of the above-mentioned engineered bionic dendritic cell vesicle, comprising the following steps: mixing dendritic cells with a buffer containing an agonist to obtain a cell resuspension; performing extrusion treatment on the cell resuspension to obtain a bionic dendritic cell vesicle; and mixing and stirring the bionic dendritic cell vesicle with a buffer containing melanoma antigen peptide gp100 to obtain an engineered bionic dendritic cell vesicle.

[0011] The present application also provides an application of the above-mentioned engineered bionic dendritic cell vesicle or the above-mentioned preparation method in the preparation of a melanoma vaccine.

[0012] The present application also provides a melanoma vaccine, which comprises the above-mentioned engineered bionic dendritic cell vesicle. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is the determination result of DCNV obtained in Comparative Example 1 in Example 7, wherein A is the particle size distribution diagram determined by DLS method, and B is the morphological diagram taken under transmission electron microscope (scale: 100 nm);

[0014] Figure 2 is the determination result of g-DCNV obtained in Comparative Example 3 in Example 7, wherein A is the particle size distribution diagram determined by DLS method, and B is the morphological diagram taken under transmission electron microscope (scale: 100 nm);

[0015] Figure 3 is the determination result of di-DCNV obtained in Comparative Example 2 in Example 7, wherein A is the particle size distribution diagram determined by DLS method, and B is the morphological diagram taken under transmission electron microscope (scale: 100 nm);

[0016] Figure 4 is the determination result of g-diDCVac prepared in Example 1, wherein A is the particle size distribution diagram determined by DLS method, and B is the morphological diagram taken under transmission electron microscope (scale: 100 nm);

[0017] Figure 5 is the detection result of Example 8, wherein A is the particle size Size result of the melanoma vaccine of the present application under the storage conditions of 4℃ and -80℃ determined by DLS method, and B is the Zeta potential change trend result of different groups (n=3, Mean±SD);

[0018] Figure 6 is the result of confocal microscope imaging of the uptake of DC2.4 to dendritic cell vesicles of different groups, the scale bar is 100 pm;

[0019] Figure 7 is the result of flow cytometry detecting the effect of PBS buffer, different concentrations of melanoma antigen peptide gp100 and positive control drug LPS on primary mouse bone marrow-derived dendritic cells (n = 3, Mean ± SD; *P < 0.05, ****P < 0.0001);

[0020] Figure 8 is the result of flow cytometry detecting the effect of PBS buffer, different groups of vaccines and positive control drug IL-2 on primary mouse spleen lymphocytes (n = 3, Mean ± SD; *P < 0.05, ***P < 0.001, ****P < 0.0001);

[0021] Figure 9 is the result of anti-tumor efficacy evaluation of each treatment group in the tumor treatment model; wherein A is the body weight change curve of tumor-bearing mice (Bodyweight-Time (d); B is the tumor volume change curve (Tumor volumn-Time (d)) (n = 5, Mean ± SD; *P < 0.05, ****P < 0.0001);

[0022] Figure 10 is the result of immunohistochemical staining of tumor tissues of mice in each group; wherein the upper row of figures is the result of Ki67 tumor proliferation detection of different groups; the lower row of figures is the result of TUNEL cell apoptosis detection of different groups (200x, scale bar is 100 pm);

[0023] Figure 11 is the result of H&E staining of heart, liver, spleen, lung and kidney tissues of mice in each group (200x, scale bar is 100 pm). DETAILED DESCRIPTION

[0024] The present application provides an engineered bionic dendritic cell vesicle, the inside of the engineered bionic dendritic cell vesicle carries an agonist of interferon gene stimulator receptor, and the outer membrane surface of the engineered bionic dendritic cell vesicle is coupled with melanoma antigen peptide gp100.

[0025] The engineered biomimetic dendritic cell vesicle provided in the application can trigger the super-activation of DC and other immune cells in the lymph node site, promote the antigen presentation process of DC, and enhance the killing ability of effector T cells. On the other hand, the agonist of the stimulator of interferon genes loaded in the vesicle can not only activate immune cells (such as DC and macrophages), but also enhance their recognition ability to pathogens and abnormal cells, and induce various immune cells to secrete type I interferons (IFN-α and IFN-β), promote tumor cell apoptosis and immune cell immune memory effect, and prevent tumor recurrence and metastasis. The engineered biomimetic dendritic cell vesicle provided in the application provides a new direction and possibility for future tumor vaccine research and application. In addition, the melanoma vaccine provided in the application can effectively inhibit tumor growth and promote tumor tissue apoptosis.

[0026] In some embodiments of the application, the amino acid sequence of the melanoma antigen peptide gp100 is IMDQVPFSV (SEQ ID NO. 1). In some embodiments of the application, the dendritic cells include DC2.4 dendritic cells. DC2.4 cells are immortalized mouse dendritic cells produced by transducing bone marrow isolates of C57BL / 6 mice with a retroviral vector expressing murine granulocyte-macrophage CSF (GM-CSF) and MYC and RAF oncogenes. DC2.4 cells exhibit the cell morphology of dendritic cells and possess the expression of specific markers, as well as the ability to phagocytose and present exogenous antigens on MHC class I and II molecules. The application uses DC2.4 as raw material to prepare engineered nanovesicles, which can produce nanoscale vesicles with uniform particle size on a large scale. In some embodiments of the application, the agonist of the stimulator of interferon genes (STING) includes diABZI, which can induce type I interferon response and inflammatory cytokines, and promote the antigen cross-presentation function of DC.

[0027] In some embodiments of the application, the particle size of the engineered biomimetic dendritic cell vesicle is 50-300 nm, and in one embodiment, the particle size of the engineered biomimetic dendritic cell vesicle is 50 nm, 80 nm, 110 nm, 140 nm, 170 nm, 200 nm, 230 nm, 260 nm, 280 nm or 300 nm. The particle size defined in the application has normal distribution characteristics, the Zeta potential is negative, and has good storage stability after structure characterization and stability test. In addition, the particle size of the engineered biomimetic dendritic cell vesicle defined in the application is more easily taken up by DC in vivo, thereby achieving the purpose of targeting the tumor-draining lymph node of the antigen peptide-coupled engineered DC vesicle tumor vaccine.

[0028] In some embodiments of the present application, the melanoma antigen peptide gp100 is gp100 modified by distearoyl phosphatidyl ethanolamine (DSPE) and PEG1000 at the C-terminus. The modification of DSPE and PEG1000 at the C-terminus of gp100 in the present application can facilitate the coupling of gp100 to the outer membrane surface of the engineered biomimetic dendritic cell vesicle and enhance its water solubility. The sequence of the modified gp100 is IMDQVPFSV-C(DSPE-PEG1000-Mal). As an antigen peptide, gp100 is connected to the outer membrane surface of the engineered biomimetic dendritic cell vesicle through a cholesterol bond, which triggers the superactivation of various immune cells such as DCs in the lymph node, promotes the antigen presentation program of DCs, and enhances the killing ability of effector T cells.

[0029] The present application also provides a preparation method of the above-mentioned engineered biomimetic dendritic cell vesicle, comprising the following steps: mixing dendritic cells with a buffer containing an agonist to obtain a cell suspension; performing extrusion treatment on the cell suspension to obtain a biomimetic dendritic cell vesicle; and mixing and stirring the biomimetic dendritic cell vesicle with a buffer containing melanoma antigen peptide gp100 to obtain an engineered biomimetic dendritic cell vesicle.

[0030] In some embodiments of the present application, the dendritic cells are DC2.4 dendritic cells, and the buffer containing an agonist is a PBS buffer. In some embodiments of the present application, the density of dendritic cells in the cell suspension is 2.0 x 10 6 to 5.0 x 10 6 per mL. In an embodiment, the density of dendritic cells in the cell suspension is 2.0 x 10 6 per mL, 2.5 x 10 6 per mL, 3.0 x 10 6 per mL, 3.5 x 10 6 per mL, 4.0 x 10 6 per mL, 4.5 x 10 6 per mL, or 5.0 x 10 6 per mL. In some embodiments of the present application, the concentration of the agonist in the cell suspension is greater than 0 and less than or equal to 2.54 μg / mL. In an embodiment, the concentration of the agonist in the cell suspension is 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL, 2.5 μg / mL, or 2.54 μg / mL.

[0031] In some embodiments of the present application, the extrusion treatment is repeatedly extruding the cell resuspension solution through polycarbonate membranes with pore sizes of 3.0 μm, 0.6 μm and 0.2 μm in sequence; the number of times of the repeated extrusion is 1, 3, 5, 7, 9 or 10. In some embodiments of the present application, the buffer containing melanoma antigen peptide gplOO is PBS buffer, and after mixing the biomimetic dendritic cell vesicle with the buffer containing melanoma antigen peptide gplOO, the concentration of the melanoma antigen peptide gplOO is greater than 0 and less than or equal to 5 μg / mL, and in an embodiment of the present application, after mixing the biomimetic dendritic cell vesicle with the buffer containing melanoma antigen peptide gplOO, the concentration of the melanoma antigen peptide gplOO is 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL, 2.0 μg / mL, 2.5 μg / mL, 3.0 μg / mL, 3.5 μg / mL or 4 μg / mL. In an embodiment of the present application, the stirring is performed by using a magnetic stirrer, the stirring speed is 500 rpm, and the stirring time is 5 h.

[0032] The present application also provides a use of the above-mentioned engineered biomimetic dendritic cell vesicle or the above-mentioned preparation method in the preparation of a melanoma vaccine.

[0033] The present application also provides a melanoma vaccine comprising the above-mentioned engineered biomimetic dendritic cell vesicle.

[0034] In some embodiments of the present application, the concentration of the engineered biomimetic dendritic cell vesicles in the melanoma vaccine is 200-400 μg / mL. In one embodiment, the concentration of the engineered biomimetic dendritic cell vesicles in the melanoma vaccine is 200 μg / mL, 240 μg / mL, 280 μg / mL, 300 μg / mL, 320 μg / mL, 360 μg / mL or 400 μg / mL. The melanoma vaccine provided by the present application containing the engineered biomimetic dendritic cell vesicles can significantly enhance the immune killing ability of lymphocytes. In the present application, the melanoma vaccine preferably further comprises trehalose. In some embodiments of the present application, the mass concentration of trehalose in the melanoma vaccine is 2%-5%. In one embodiment, the mass concentration of trehalose in the melanoma vaccine is 2%, 3%, 4% or 5%. In the present application, trehalose as a vesicle cryoprotective agent can achieve the purpose of long-term preservation of the engineered biomimetic nanodendritic cell vesicles, and thus trehalose can protect the immune effect of the melanoma vaccine of the present application, laying a solid foundation for the clinical application of DC vesicles. The melanoma vaccine of the present application can be stored for 1 month under refrigeration at 4°C and at least 1 month under freezing at -80°C. In the embodiments of the present application, the stability of the melanoma vaccine is detected, and the results show that the melanoma vaccine maintains good stability under the conditions of storage and freeze-thawing at 4°C and -80°C, proving that the particle size distribution is uniform and no large changes occur in the long range of particle size.

[0035] In some embodiments of the present application, the melanoma vaccine is used to immunize the body by subcutaneous injection. In some embodiments, the number of injections includes 5 times, and the interval time between adjacent two immunizations is 3 days. In some embodiments of the present application, the immunization dose of the melanoma vaccine is calculated according to the body weight, and the dose of the melanoma vaccine injected per g of body weight is greater than 0 and less than or equal to 0.67 μg. In one embodiment, the dose of the melanoma vaccine injected per g of body weight is 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg or 0.6 μg. After the melanoma mouse model is immunized with the melanoma vaccine of the present application, the tumor volume is significantly reduced compared with other groups, and the survival period of the mouse model is longer.

[0036] The technical solutions provided by the present application are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0037] In the following examples, all the conventional methods are used unless otherwise specified.

[0038] In the following examples, all the materials and reagents used are commercially available unless otherwise specified.

[0039] Example 1

[0040] (1) Put dendritic cells DC2.4 cells (purchased from the Chinese Academy of Sciences Cell Bank) into a culture bottle for culture, and the culture medium in the culture bottle is RPMI-1640 culture medium (add 10% fetal bovine serum and 1% penicillin-streptomycin), and the culture condition is 5% CO2, 37°C. After DC2.4 is fully grown, the culture medium is discarded, and after washing with PBS buffer, digestion is carried out to obtain a digestion product; centrifuge the digestion product, collect the precipitate, and the dispersed dendritic cells are obtained; resuspend the precipitate in PBS buffer containing STING agonist diABZI to obtain a cell suspension, and the cell density of dendritic cells DC2.4 in the cell suspension is 4.0×10 6 ~ 5.0×10 6 / mL, and the concentration of diABZI in the cell suspension is 1.27 μg / mL.

[0041] (2) The cell suspension obtained in step (1) is passed through polycarbonate membranes with pore sizes of 3.0 μm→0.6 μm→0.2 μm in sequence using a gas-tight extruder (one extrusion process), and the process is repeated 3 times to obtain biomimetic dendritic cell vesicles loaded with adjuvant diABZI.

[0042] (3) Add PBS buffer containing melanoma antigen peptide gp100 (IMDQVPFSV-C(DSPE-PEG1000-Mal)) to the biomimetic dendritic cell vesicles obtained in step (2), and adjust the concentration of gp100 to 2 μg / mL. Stir for 5 h using a magnetic stirrer at 500 rpm to obtain engineered biomimetic dendritic cell vesicles, and the particle size of the engineered biomimetic dendritic cell vesicles is 242.4 nm.

[0043] Example 2

[0044] (1) Put dendritic cells DC2.4 cells (purchased from the Chinese Academy of Sciences Cell Bank) into a culture bottle for culture, and the culture medium in the culture bottle is RPMI-1640 culture medium (add 10% fetal bovine serum and 1% penicillin-streptomycin), and the culture condition is 5% CO2, 37°C. After DC2.4 is fully grown, the culture medium is discarded, and after washing with PBS buffer, digestion is carried out to obtain a digestion product; centrifuge the digestion product, collect the precipitate, and the dispersed dendritic cells are obtained; resuspend the precipitate in PBS buffer containing STING agonist diABZI to obtain a cell suspension, and the cell density of dendritic cells DC2.4 in the cell suspension is 2.0×10 6 ~ 3.0×10 6 / mL, and the concentration of diABZI in the cell suspension is 2.54 μg / mL.

[0045] (2) The cell suspension obtained in step (1) is passed through polycarbonate membranes with pore sizes of 3.0 μm→0.6 μm→0.2 μm in sequence using a gas-tight extruder, and extruded repeatedly 5 times to obtain adjuvant-loaded biomimetic dendritic cell vesicles.

[0046] (3) PBS buffer containing melanoma antigen peptide gp100 (IMDQVPFSV) is added to the biomimetic dendritic cell vesicles obtained in step (2), and the concentration of gp100 is adjusted to 1 μg / mL. An engineered biomimetic dendritic cell vesicle with a particle size of 210 nm is obtained by stirring at 500 rpm for 5 h using a magnetic stirrer.

[0047] Example 3

[0048] (1) The dendritic cells DC2.4 cells (purchased from the Chinese Academy of Sciences Cell Bank) are cultured in a culture flask, and the culture medium in the culture flask is RPMI-1640 medium (added with 10% fetal bovine serum and 1% penicillin-streptomycin), and the culture condition is 5% CO2, 37°C. After the DC2.4 is fully grown, the culture medium is discarded, and the cells are washed with PBS buffer, then digested to obtain a digestion product; the digestion product is centrifuged, and the precipitate is collected as dispersed dendritic cells; the precipitate is resuspended in PBS buffer containing STING agonist diABZI to obtain a cell suspension, and the cell density of the dendritic cells DC2.4 in the cell suspension is 4.0×10 6 ~5.0×10 6 μg / mL, and the concentration of diABZI in the cell suspension is 2.32 μg / mL.

[0049] (2) The cell suspension obtained in step (1) is passed through polycarbonate membranes with pore sizes of 3.0 μm→0.6 μm→0.2 μm in sequence using a gas-tight extruder, and extruded repeatedly 6 times to obtain adjuvant-loaded biomimetic dendritic cell vesicles.

[0050] (3) PBS buffer containing melanoma antigen peptide gp100 (IMDQVPFSV-C(DSPE-PEG1000-Mal)) is added to the biomimetic dendritic cell vesicles obtained in step (2), and the concentration of gp100 is adjusted to 0.5 μg / mL. An engineered biomimetic dendritic cell vesicle with a particle size of 250 nm is obtained by stirring at 500 rpm for 5 h using a magnetic stirrer.

[0051] Example 4

[0052] A melanoma vaccine, the engineered biomimetic dendritic cell vesicles obtained in Example 1 are dispersed in PBS (pH 7.4) buffer, and trehalose is added to the buffer to obtain a melanoma vaccine, the concentration of the engineered biomimetic dendritic cell vesicles obtained in Example 1 in the melanoma vaccine is 200 μg / mL, and the mass concentration of trehalose is 2%.

[0053] Example 5

[0054] A melanoma vaccine, the engineered biomimetic dendritic cell vesicles obtained in Example 2 are dispersed in PBS (pH 7.4) buffer, and trehalose is added to the buffer to obtain a melanoma vaccine, the concentration of the engineered biomimetic dendritic cell vesicles obtained in Example 2 in the melanoma vaccine is 400 μg / mL, and the mass concentration of trehalose is 3%.

[0055] Example 6

[0056] A melanoma vaccine, the engineered biomimetic dendritic cell vesicles obtained in Example 1 are dispersed in PBS (pH 7.4) buffer to obtain a melanoma vaccine, the concentration of the engineered biomimetic dendritic cell vesicles obtained in Example 1 in the melanoma vaccine is 200 μg / mL.

[0057] Comparative Example 1

[0058] The difference from Example 1 is that after obtaining the dispersed dendritic cells according to step (1), step (2) is directly performed without step (3), to obtain dendritic cell vesicles without loading the adjuvant diABZI, denoted as DCNV.

[0059] Comparative Example 2

[0060] The difference from Example 1 is that only step (1) and step (2) are performed without step (3), to obtain biomimetic dendritic cell vesicles loaded with the adjuvant diABZI, denoted as di-DCNV.

[0061] Comparative Example 3

[0062] The difference from Example 1 is that after obtaining the dispersed dendritic cells according to step (1), step (2) and step (3) are directly performed, to obtain engineered biomimetic dendritic cell vesicles only coupled with the melanoma antigen peptide gp100, denoted as g-DCNV.

[0063] Example 7

[0064] The morphology of the engineered biomimetic dendritic cell vesicles prepared in Example 1 (denoted as g-diDCVac), the DCNV prepared in Comparative Example 1, the di-DCNV prepared in Comparative Example 2 and the g-DCNV prepared in Comparative Example 3 was observed under a transmission electron microscope, and the particle size of different vesicles was determined by dynamic light scattering (DLS) method. The results are shown in Figures 1-4, which show that the engineered biomimetic dendritic cell vesicles prepared in the present application have a morphology of empty vesicle membrane and a normal distribution of particle size.

[0065] Example 8

[0066] The Zeta potential of the engineered biomimetic dendritic cell vesicles prepared in Example 1 (denoted as g-diDCVac), the DCNV prepared in Comparative Example 1, the di-DCNV prepared in Comparative Example 2 and the g-DCNV prepared in Comparative Example 3 was investigated by a Zeta potential analyzer. The results are shown in Figure 5B. The melanoma vaccine prepared in Example 4 was aliquoted in 3 mL centrifuge tubes and stored in a -80°C refrigerator for 27 days, and the melanoma vaccine prepared in Example 6 was aliquoted in 3 mL centrifuge tubes and stored in a 4°C refrigerator for 27 days. The particle size of the vaccines was monitored by electric DLS method at time points of 0, 3, 6, 9, 12, 15, 21 and 27 days to investigate the stability of the vaccines under storage conditions of 4°C and -80°C, respectively. The results are shown in Figure 5A. Both Figure 5A and Figure 5B show that the melanoma vaccines and the engineered biomimetic dendritic cell vesicles prepared in the present application have good stability. The above tests were all set up in triplicate, and the results are represented by mean ± standard deviation (Mean ± SD).

[0067] Example 9

[0068] To evaluate the uptake of the engineered biomimetic dendritic cell vesicles prepared in Example 1 (denoted as g-diDCVac), the DCNV prepared in Comparative Example 1, the di-DCNV prepared in Comparative Example 2 and the g-DCNV prepared in Comparative Example 3 by dendritic cells (DCs), DC2.4 was inoculated in a confocal dish at 1 × 10 4 After the DC2.4 adhered to the wall, staining was performed (with PBS buffer as a control group).

[0069] (1) Staining of dendritic cell vesicles in different groups

[0070] 500 μL of dendritic cell vesicles in different groups were taken, 1 μL of DIO dye and staining enhancer were added, and after being fully vortexed, 500 μL of RPMI 1640 complete medium was added, and the mixture was incubated in the dark for 15 min. Subsequently, the complete medium in the confocal dish was removed, and the above-mentioned incubated solution was added to the confocal dish, and after 4 h of co-incubation, the confocal dish was washed with PBS buffer for 2 times.

[0071] (2) DC2.4 lysosome staining

[0072] Take 1 μL Lyso-Tracker Red and add to 15 mL of warm cell culture medium, mix well to obtain the working solution of Lyso-Tracker Red. Discard the liquid in the confocal dish, add the prepared and pre-incubated at 37°C Lyso-Tracker Red staining working solution, and incubate with the cells at 37°C for 40 min. Then discard the liquid in the confocal dish, wash the confocal dish twice with PBS buffer, and then add 500 μL of DAPI staining solution to cover the sample. Place at room temperature for 3 min. Remove the DAPI staining solution, and wash the confocal dish twice with PBS buffer, each for 3 min.

[0073] (3) DC2.4 nucleus staining

[0074] Remove the fixing solution, wash the confocal dish twice with PBS buffer, then add 500 μL of DAPI staining solution to cover the sample. Place at room temperature for 3 min. Remove the DAPI staining solution, and wash the confocal dish twice with PBS buffer, each for 3 min.

[0075] Then observe the uptake of DC2.4 to the vesicles of dendritic cells in different groups by confocal microscopy, and the results are shown in Figure 6. It is found that compared with the PBS group, DC2.4 can effectively uptake the vesicles of dendritic cells in each group.

[0076] Example 10

[0077] Investigation of the maturation of mouse bone marrow-derived dendritic cells by different concentrations of tumor antigen peptide gp100

[0078] In order to screen the best concentration of melanoma antigen peptide to promote the maturation of bone marrow-derived dendritic cells, flow cytometry was used to detect the effect of different concentrations of tumor antigen peptide gp100 (IMDQVPFSV-C(DSPE-PEG1000-Mal)) on mouse bone marrow-derived dendritic cells, and the detection index was the proportion of bone marrow-derived dendritic cell maturation markers CD11c, CD80, and CD86 positive cells.

[0079] Extract dendritic cells from mouse bone marrow, and the cells were adjusted to 1 × 10 6Cells were seeded per well in 6-well plates and cultured in RPMI 1640 complete medium (RPMI-1640 + 10% FBS + 1% penicillin-streptomycin + 10 ng / mL IL-4 + 20 ng / mL GM-CSF). Different concentrations of tumor antigen peptide gp100 (0 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL) and negative and positive control drugs (PBS, 1 μg / mL LPS) were added to each well, making the culture volume 2 mL per well. After co-incubation for 24 h, bone marrow-derived dendritic cells were gently scraped from the wells using a cell scraper, centrifuged at 4°C, and the supernatant was discarded. The cells were resuspended in 100 μL PBS, and 1 μL each of CD11c, CD80, and CD86 flow cytometry antibodies were added. The mixture was vortexed thoroughly and co-incubated at 4°C in the dark for 30 min. After incubation, cells were centrifuged at 4°C using a low-temperature centrifuge. The supernatant was discarded, and the cells were resuspended in pre-cooled PBS. The cell suspension was transferred to flow cytometry tubes, with each tube containing 700 μL of cell suspension. The expression of surface markers CD11c, CD80, and CD86 on bone marrow-derived dendritic cells was detected by flow cytometry (FCM) to screen for the optimal concentration of melanoma antigen peptides that promote the maturation of bone marrow-derived dendritic cells. The results are shown in Figure 7. Based on the flow cytometry results, CD11c expression in each group was statistically analyzed. + CD80 in cell population + With CD86 + The results showed that 2 μg / mL of tumor antigen peptide gp100 could activate high levels of bone marrow-derived dendritic cells and enhance their antigen-presenting capacity. (All the above experiments were conducted in triplicate, and the results are expressed as mean ± standard deviation (Mean ± SD). *P<0.05, ****P<0.0001).

[0080] Example 11

[0081] Evaluation of the differentiation effect of different groups of vaccines on mouse spleen-derived lymphocytes

[0082] To assess the effect of the vaccine on the differentiation of mouse spleen-derived lymphocytes, flow cytometry was used to detect the expression of CD3, CD4, and CD8 in lymphocytes after vaccine stimulation (the detection index was the proportion of lymphocyte differentiation markers CD3, CD4, and CD8 positive cells). Specifically:

[0083] According to the vaccine preparation method in Example 4, the engineered biomimetic dendritic cell vesicles in Example 4 were replaced with gp100, DCNV obtained in Comparative Example 1, di-DCNV obtained in Comparative Example 2, and g-DCNV obtained in Comparative Example 3, respectively, to obtain gp100 vaccine, DCNV vaccine, diDCNV vaccine, and g-DCNV vaccine.

[0084] Lymphocytes were extracted from the spleen of mice and the cells were sorted at a ratio of 1×10⁻⁶. 6 Cells were seeded per well in 6-well plates and cultured in RPMI 1640 complete medium (RPMI-1640 + 10% FBS + 1% penicillin-streptomycin). Different vaccine groups (g-diDCVac, gp100 vaccine, DCNV vaccine, diDCNV vaccine, and g-DCNV vaccine obtained in Example 4) and negative and positive control drugs (PBS, 10 ng / mL IL-2) were added to each well, making the culture volume 2 mL per well. After co-incubation for 48 h, lymphocytes were gently scraped from the wells using a pipette, centrifuged at 4°C, and the supernatant was discarded. The cells were resuspended in 100 μL PBS, and 1 μL each of CD3, CD4, and CD8 flow cytometry antibodies were added. The mixture was vortexed thoroughly and co-incubated at 4°C in the dark for 30 min. After incubation, cells were centrifuged at 4°C using a low-temperature centrifuge. The supernatant was discarded, and the cells were resuspended in pre-cooled PBS. The cell suspension was transferred to flow cytometry tubes, with each tube containing 700 μL of cell suspension. The expression of lymphocyte surface markers CD3, CD4, and CD8 was detected by flow cytometry (FCM) to assess the activation of lymphocytes by different vaccine groups. The results are shown in Figure 8. Based on the flow cytometry results, CD3 expression was statistically analyzed in each group. + CD4 in T lymphocyte population + With CD8 + The proportion of T lymphocytes showed that CD4+ cells were present in the g-diDCVac group. + / CD8 + The proportion of T lymphocytes was the lowest, indicating that CD8+ cells were present in the g-diDCVac group. + The highest proportion of T lymphocytes further indicates that the g-diDCVac group can significantly induce lymphocytes to convert to CD8. + T lymphocyte differentiation and efficient activation of lymphocyte immune killing function (all the above experiments were conducted in triplicate, and the results are expressed as mean ± standard deviation (Mean ± SD), *P<0.05, ***P<0.001, ****P<0.0001).

[0085] Example 12

[0086] Evaluation of the antitumor efficacy of different groups of vaccines in in vivo therapeutic experiments on mice with melanoma

[0087] (1) Healthy 5-week-old female C57BL / 6 mice were selected, and a mouse melanoma model was established by subcutaneous injection of B16 cells into the right back of the mice. Each mouse was injected with 1.0 × 10⁻⁶ cells. 6B16 cells, recorded as Day 0. The mice were randomly divided into groups, the groups were PBS, DCNV vaccine group, gp100 vaccine group, di-DCNV vaccine group, g-DCNV vaccine group, g-diDCVac vaccine group (obtained in Example 4), 5 mice in each group, and the corresponding group of vaccines was injected on the 3rd, 6th, 9th, 12th, and 15th days, wherein the preparation methods of the DCNV vaccine, the gp100 vaccine, the di-DCNV vaccine, and the g-DCNV vaccine were the same as in Example 11. The concentration of the gp100 antigen peptide in the vaccine containing the gp100 antigen peptide was 5 μg per mouse, the concentration of the vesicle in each group of vaccines was 200 μg / mL, the injection dose was 200 μL per mouse, the weight of each mouse was 14-19 g, and the mice were sacrificed by cervical dislocation on the 24th day.

[0088] (2) The weight of the mice was measured every 3 days, and the long diameter and short diameter of the tumor were measured, and the tumor volume was calculated. The results are shown in Figure 9, and the tumor of the g-diDCVac group of mice was significantly smaller than that of the other groups, indicating that g-diDCVac had a good anti-tumor effect on mouse melanoma (*P<0.05, ****P<0.0001).

[0089] (3) The tumor tissues of the mice in each group were taken for TUNEL and Ki67 immunohistochemical staining sections. Ki67 is a nuclear proliferation antigen of cells, and the proliferation of cells is reacted. The TUNEL method is a method for detecting cell apoptosis. The results are shown in Figure 10 (photographs taken under a microscope), and the TUNEL staining results of the tumor tissue immunohistochemistry are basically consistent with the tumor proliferation antigen Ki67 results, further indicating that the melanoma vaccine of the application can effectively inhibit tumor growth and promote tumor tissue apoptosis, and has a good anti-tumor effect on mouse melanoma.

[0090] (4) The heart, liver, spleen, lung, and kidney of the mice in each group were taken for H&E staining sections, and the results are shown in Figure 11 (photographs taken under a microscope), proving that the melanoma vaccine of the application has good safety during the treatment of melanoma mice.

[0091] The above only describes the preferred embodiments of the application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the application, and these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. An engineered, biomimetic dendritic cell vesicle, characterized in that, The inside of the engineered biomimetic dendritic cell vesicle is loaded with an agonist of stimulator of interferon genes, and the outer membrane surface of the engineered biomimetic dendritic cell vesicle is coupled with melanoma antigen peptide gp100.

2. The engineered, bionic dendritic cell vesicle of claim 1, wherein, The amino acid sequence of the melanoma antigen peptide gp100 is IMDQVPFSV.

3. The engineered, bionic dendritic cell vesicle of claim 1, wherein, The agonist of stimulator of interferon genes comprises diABZI.

4. The engineered, bionic dendritic cell vesicle of claim 1, wherein, The particle size of the engineered biomimetic dendritic cell vesicle is 50-300 nm.

5. The engineered, biologic dendritic cell-like vesicle of claim 1, wherein, The melanoma antigen peptide gp100 is gp100 modified at the C-terminal with distearoyl phosphatidyl ethanolamine and PEG1000.

6. The engineered, biologic dendritic cell-like vesicle of claim 5, wherein, The sequence of the gp100 modified at the C-terminal with distearoyl phosphatidyl ethanolamine and PEG1000 is IMDQVPFSV-C(DSPE-PEG1000-Mal).

7. The engineered, bionic dendritic cell vesicle of claim 1, wherein, The dendritic cell comprises a DC2.4 dendritic cell.

8. The method of producing the engineered, biomimetic dendritic cell vesicle of any one of claims 1-7, wherein, The method comprises the following steps: mixing a dendritic cell with a buffer containing an agonist to obtain a cell suspension; performing extrusion treatment on the cell suspension to obtain a biomimetic dendritic cell vesicle; and mixing and stirring the biomimetic dendritic cell vesicle with a buffer containing melanoma antigen peptide gp100 to obtain an engineered biomimetic dendritic cell vesicle.

9. The production method according to claim 8, characterized by, The buffer comprises a PBS buffer.

10. The method of claim 8, wherein, The density of the dendritic cells in the cell resuspension is 2.0 x 10 6 ~ 5.0 x 10 6 ~ 5.0 x 10 11. The preparation method according to claim 8, characterized in that, The concentration of the agonist in the cell suspension is greater than 0 and less than or equal to 2.54 μg / mL.

12. The method of claim 8, wherein, After the biomimetic dendritic cell vesicle is mixed with the buffer containing melanoma antigen peptide gp100, the concentration of the melanoma antigen peptide gp100 is greater than 0 and less than or equal to 5 μg / mL.

13. Use of the engineered biomimetic dendritic cell vesicle of any one of claims 1-7 or the preparation method of any one of claims 8-12 in the preparation of a melanoma vaccine.

14. A melanoma vaccine, characterized by comprising a peptide of SEQ ID NO: 1 or a salt thereof. The melanoma vaccine comprises the engineered biomimetic dendritic cell vesicle of any one of claims 1-7.

15. The melanoma vaccine of claim 14, wherein the melanoma vaccine is characterized by, The concentration of the engineered biomimetic dendritic cell vesicle in the melanoma vaccine is 200-400 μg / mL.

16. The melanoma vaccine of claim 14, wherein the melanoma vaccine is characterized by, The melanoma vaccine further comprises trehalose.

17. The melanoma vaccine of claim 16, wherein the melanoma vaccine is characterized by, The mass concentration of the trehalose in the melanoma vaccine is 2%-5%.

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