Engineering bionic dendritic cell vesicle coupled antigen peptide tumor vaccine as well as preparation method and application thereof

Abstract

The invention provides an engineered bionic dendritic cell vesicle coupled antigen peptide tumor vaccine as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The engineered bionic dendritic cell vesicle provided by the invention is internally loaded with an agonist of an interferon gene stimulation receptor, and the surface of an outer membrane is coupled with a melanoma antigen peptide gp100. In the engineering bionic dendritic cell vesicle provided by the invention, the antigen peptide gp100 can trigger the hyperactivity of various immune cells such as DC at a lymph node part, the antigen presentation procedure of the DC is promoted, and the killing ability of effector T cells is enhanced; the interferon gene loaded in the vesicles stimulates agonists of receptors, so that immune cells can be promoted to be activated, the ability of the immune cells to recognize pathogens and abnormal cells is enhanced, meanwhile, various immune cells are induced to secrete I-type interferon, apoptosis of tumor cells is promoted, immune memory effects of the immune cells are promoted, and recurrence and metastasis of tumors are prevented.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an engineered bionic dendritic cell vesicle-coupled antigen peptide tumor vaccine and a preparation method and application thereof. Background Art

[0002] The core goal of melanoma vaccines is to enhance immune responses to kill tumors, establish lasting anti-tumor immune memory, and avoid adverse reactions. However, most melanoma vaccines are still in the clinical research stage and face 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 rely solely on cells or their lysates have limited effects in producing immune effects, such as insufficient immunogenicity or potential tumorigenic risks. Therefore, it is particularly important to find effective antigen sources and innovative vaccine carriers.

[0003] Dendritic cells (DC) are professional antigen-presenting cells that can sense a variety of pathogens to stimulate a strong and appropriate immune response. DC is able to produce all the signals that activate T cells and plays an extremely important role in the initiation, programming and regulation of tumor immune responses. Nanoscale cell vesicles derived from DC can mimic the physiological functions of parent cells and present tumor antigens as vaccine carriers, with excellent application prospects. However, it still faces many technical difficulties in the process of development and application. For example, DC can release multiple types of vesicles (such as exosomes and microvesicles), and it is particularly important to effectively separate and purify specific types of DC vesicles. 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 bottleneck of the above-mentioned DC vesicles has become a key issue that needs to be solved urgently. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an engineered bionic dendritic cell vesicle, which is easily recognized by antigen presenting cells (APC) and presented to T cells by DC, and can stimulate promoting immune cells in the tumor immune microenvironment to enhance the immune response.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an engineered bionic dendritic cell vesicle, wherein an agonist of an interferon gene stimulating receptor is loaded inside the engineered bionic dendritic cell vesicle, and a melanoma antigen peptide gp100 is coupled to the outer membrane surface of the engineered bionic dendritic cell vesicle, wherein the amino acid sequence of the melanoma antigen peptide gp100 is IMDQVPFSV (SEQ ID NO.1).

[0007] Preferably, the agonist of the interferon gene-stimulated receptor comprises diABZI.

[0008] Preferably, the particle size of the engineered bionic dendritic cell vesicles is 50 to 300 nm.

[0009] Preferably, the melanoma antigen peptide gp100 is gp100 modified at the C-terminus with distearoylphosphatidylethanolamine and PEG1000.

[0010] The present invention also provides a method for preparing the above-mentioned engineered bionic dendritic cell vesicles, comprising the following steps: mixing dendritic cells with a buffer solution containing an agonist to obtain a cell resuspension; extruding the cell resuspension to obtain bionic dendritic cell vesicles; mixing and stirring the bionic dendritic cell vesicles with a buffer solution containing a melanoma antigen peptide gp100 to obtain the engineered bionic dendritic cell vesicles.

[0011] Preferably, the density of dendritic cells in the cell resuspension is 2.0×10 6 ~5.0×10 6 / mL, and the concentration of the agonist in the cell resuspension is greater than 0 and below 2.54 μg / mL.

[0012] Preferably, after the bionic dendritic cell vesicles are mixed with the buffer solution containing the melanoma antigen peptide gp100, the concentration of the melanoma antigen peptide gp100 is greater than 0 and less than 5 μg / mL.

[0013] The present invention also provides the use of the engineered bionic dendritic cell vesicle or the preparation method in preparing a melanoma vaccine.

[0014] The present invention also provides a melanoma vaccine, which comprises the above-mentioned engineered bionic dendritic cell vesicles, and the concentration of the engineered bionic dendritic cell vesicles in the melanoma vaccine is 200-400 μg / mL.

[0015] Preferably, the melanoma vaccine further comprises trehalose, and the mass concentration of trehalose in the melanoma vaccine is 2% to 5%.

[0016] Beneficial effects of the present invention:

[0017] The engineered bionic dendritic cell vesicles provided by the present invention, on the one hand, the antigen peptide gp100 triggers the hyperactivation of various immune cells such as DC in the lymph node, promotes the antigen presentation program of DC, and enhances the killing ability of effector T cells; on the other hand, the agonist of the interferon gene stimulating receptor loaded in the vesicle can not only promote the activation of immune cells (such as DC and macrophages), but also enhance their ability to recognize pathogens and abnormal cells, and induce various immune cells to secrete type I interferon (IFN-α and IFN-β), promote tumor cell apoptosis and immune cell immune memory effect, and prevent tumor recurrence and metastasis. The engineered bionic dendritic cell vesicles provided by the present invention provide new directions and possibilities for the research and application of future tumor vaccines. In addition, the melanoma vaccine provided by the present invention can effectively inhibit tumor growth and promote apoptosis of tumor tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of the DCNV determination in Comparative Example 1 in Example 7 are shown in Figure 1, where A is the particle size distribution diagram determined by the DLS method, and B is the morphology diagram taken under a transmission electron microscope (scale: 100 nm);

[0019] Figure 2 The results of g-DCNV measurement obtained in Comparative Example 3 in Example 7, wherein A is the particle size distribution diagram measured by DLS method, and B is the morphology diagram taken under transmission electron microscope (scale: 100 nm);

[0020] Figure 3 The results of the di-DCNV determination in Comparative Example 2 in Example 7 are shown in Figure 1, where A is the particle size distribution diagram determined by the DLS method, and B is the morphology diagram taken under a transmission electron microscope (scale: 100 nm);

[0021] Figure 4 The results of g-diDCVac prepared in Example 1 are shown in Figure 1, where A is the particle size distribution diagram measured by DLS method, and B is the morphology diagram taken under transmission electron microscope (scale: 100 nm);

[0022] Figure 5 The test results of Example 8, wherein A is the particle size of the melanoma vaccine of the present invention measured by DLS method under 4°C and -80°C storage conditions, and B is the Zeta potential change trend results of different groups, (n=3, Mean±SD);

[0023] Figure 6 The results of DC2.4 uptake of vesicles from dendritic cells of different groups were taken under confocal microscopy, the scale bar is 100 μm;

[0024] Figure 7Flow cytometry was used to detect the effects 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);

[0025] Figure 8 Flow cytometry was used to detect the effects 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);

[0026] Fig. 9 The anti-tumor efficacy evaluation results of each treatment group in the tumor treatment model; A is the body weight change curve of tumor-bearing mice (Bodyweight-time (Time(d)); B is the tumor volume (Tumor volume)-time (Time(d)) change curve (n=5, Mean±SD; *P<0.05, ****P<0.0001);

[0027] Fig.10 The immunohistochemical staining results of tumor tissues of mice in each group; the upper row of pictures shows the Ki67 tumor proliferation detection results of different groups; the lower row of pictures shows the TUNEL cell apoptosis detection results of different groups (200×, scale bar is 100μm);

[0028] Fig.11 The results of H&E staining of heart, liver, spleen, lung and kidney tissues of mice in each group (200×, scale bar is 100 μm). DETAILED DESCRIPTION

[0029] The invention provides an engineered bionic dendritic cell vesicle, wherein an agonist of an interferon gene stimulating receptor is loaded inside the engineered bionic dendritic cell vesicle, and a melanoma antigen peptide gp100 is coupled to the outer membrane surface of the engineered bionic dendritic cell vesicle, wherein the amino acid sequence of the melanoma antigen peptide gp100 is IMDQVPFSV (SEQ ID NO.1).

[0030] In the present invention, the dendritic cells preferably include DC2.4 dendritic cells, which are immortalized mouse dendritic cells produced by transducing bone marrow isolates of C57BL / 6 mice with a retroviral vector expressing mouse granulocyte-macrophage CSF (GM-CSF) and MYC and RAF oncogenes, and DC2.4 cells exhibit the cell morphology of dendritic cells and have the expression of specific markers, as well as the ability to phagocytose and present exogenous antigens on MHC class I and class II molecules. The present invention uses DC2.4 as a raw material to prepare engineered nanovesicles, which can mass-produce nanoscale vesicles with uniform particle size. In the present invention, the agonist of the interferon gene stimulating receptor (STING) preferably includes diABZI, which can induce type I interferon response and inflammatory cytokines, and promote the antigen cross-presentation function of DC.

[0031] In the present invention, the particle size of the engineered bionic dendritic cell vesicles is preferably 50-300 nm, more preferably 100-280 nm, and further preferably 200-260 nm. The particle size defined in the present invention, after structural characterization and stability testing, the particle size of the nanovesicles shows a normal distribution characteristic, the Zeta potential is negative, and has good storage stability. In addition, the particle size of the engineered bionic dendritic cell vesicles defined in the present invention is more easily taken up by DC in the body, thereby achieving the purpose of targeting tumor-draining lymph nodes with the antigen peptide-coupled engineered DC vesicle-type tumor vaccine.

[0032] In the present invention, the melanoma antigen peptide gp100 is preferably a gp100 modified with distearoylphosphatidylethanolamine (DSPE) and PEG1000 at the C-terminus. The present invention performs DSPE and PEG1000 modification at the C-terminus of gp100 to promote the coupling of gp100 to the outer membrane surface of the engineered bionic 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 bionic dendritic cell vesicle through a cholesterol bond. The antigen peptide gp100 triggers the hyperactivation of various immune cells such as DC in the lymph node, promotes the antigen presentation program of DC, and enhances the killing ability of effector T cells.

[0033] The present invention also provides a method for preparing the above-mentioned engineered bionic dendritic cell vesicles, comprising the following steps: mixing dendritic cells with a buffer solution containing an agonist to obtain a cell resuspension; extruding the cell resuspension to obtain bionic dendritic cell vesicles; mixing and stirring the bionic dendritic cell vesicles with a buffer solution containing a melanoma antigen peptide gp100 to obtain the engineered bionic dendritic cell vesicles.

[0034] In the present invention, the dendritic cells are preferably DC2.4 dendritic cells, and the buffer containing the agonist is preferably PBS buffer. The density of the dendritic cells in the cell resuspension is preferably 2.0×10 6 / mL~5.0×10 6 / mL, more preferably 4×10 6 / mL~5×10 6 The concentration of the agonist in the cell resuspension is preferably greater than 0 and below 2.54 μg / mL, and more preferably 1.20-1.27 μg / mL.

[0035] In one embodiment of the present invention, the extrusion treatment is to repeatedly extrude the cell resuspension through polycarbonate membranes with pore sizes of 3.0 μm, 0.6 μm and 0.2 μm in sequence; the number of repeated extrusions is preferably 1 to 10 times. In the present invention, the buffer containing the melanoma antigen peptide gp100 is preferably a PBS buffer, and after the bionic dendritic cell vesicles are mixed with the buffer containing the melanoma antigen peptide gp100, the concentration of the melanoma antigen peptide gp100 is preferably greater than 0 and less than 5 μg / mL, more preferably 0.5 to 4 μg / mL, and further preferably 1 to 2 μg / mL. In one embodiment of the present invention, the stirring is performed using a magnetic stirrer, the stirring speed is 500 rpm, and the stirring time is 5 hours.

[0036] The present invention also provides the use of the engineered bionic dendritic cell vesicle or the preparation method in preparing a melanoma vaccine.

[0037] The present invention also provides a melanoma vaccine, the melanoma vaccine includes the above-mentioned engineered bionic dendritic cell vesicles, and the concentration of the engineered bionic dendritic cell vesicles in the melanoma vaccine is 200-400 μg / mL. In the melanoma vaccine of the present invention, the concentration of the engineered bionic dendritic cell vesicles is preferably 200-300 μg / mL. The melanoma vaccine containing engineered bionic dendritic cell vesicles provided by the present invention can significantly enhance the immune killing ability of lymphocytes. In the present invention, the melanoma vaccine preferably also includes trehalose, and the mass concentration of trehalose in the melanoma vaccine is preferably 2%-5%, more preferably 3%-4%. In the present invention, trehalose is used as a vesicle cryoprotectant to achieve the purpose of long-term preservation of engineered bionic nano dendritic cell vesicles, and trehalose can protect the immune effect of the melanoma vaccine of the present invention, laying a solid foundation for the clinical application of DC vesicles. The melanoma vaccine of the present invention can be stored for 1 month under 4°C refrigeration conditions and can be stored for at least 1 month under -80°C freezing conditions. In the embodiment of the present invention, the melanoma vaccine was tested for stability, and the results showed that the melanoma vaccine maintained good stability under 4°C and -80°C storage and freeze-thaw conditions, proving that the particle size distribution is uniform and there is no significant change in the particle size over a long range.

[0038] In the present invention, the melanoma vaccine is preferably administered subcutaneously to immunize the body. The number of injections is preferably 5, and the interval between two adjacent immunizations is preferably 3 days. The immunization dose of the melanoma vaccine is based on body weight, and preferably the dose of the melanoma vaccine injected per gram of body weight is greater than 0 and less than 0.67 μg. After immunizing the melanoma mouse model with the melanoma vaccine of the present invention, the tumor volume was significantly reduced compared with other groups, and the mouse model had a longer survival period.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Example 1

[0043] (1) Dendritic cells DC2.4 cells (purchased from the cell bank of the Chinese Academy of Sciences) were cultured in a culture flask, the culture medium in the culture flask was RPMI-1640 culture medium (added with 10% fetal bovine serum and 1% penicillin-streptomycin), and the culture conditions were 5% CO2, 37°C. After DC2.4 cells were fully grown, the culture medium was discarded, and after washing with PBS buffer, digestion was performed to obtain a digestion product; the digestion product was centrifuged, and the precipitate was collected to obtain dispersed dendritic cells; the precipitate was resuspended in PBS buffer containing the STING agonist diABZI to obtain a cell resuspension, and the cell density of the dendritic cells DC2.4 in the cell resuspension was 4.0×10 6 ~5.0×10 6 / mL, so that the concentration of diABZI in the cell resuspension is 1.27μg / mL.

[0044] (2) The cell resuspension obtained in step (1) was passed through a polycarbonate membrane with a pore size of 3.0 μm → 0.6 μm → 0.2 μm in sequence (a single extrusion process) using an airtight extruder, and the extrusion was repeated three times to obtain biomimetic dendritic cell vesicles loaded with the adjuvant diABZI.

[0045] (3) Add PBS buffer containing melanoma antigen peptide gp100 (IMDQVPFSV-C (DSPE-PEG1000-Mal)) to the bionic dendritic cell vesicles obtained in step (2), and adjust the concentration of gp100 to 2 μg / mL. Use a magnetic stirrer to stir at 500 rpm for 5 hours to obtain engineered bionic dendritic cell vesicles, wherein the particle size of the engineered bionic dendritic cell vesicles is 242.4 nm.

[0046] Example 2

[0047] (1) Dendritic cells DC2.4 cells (purchased from the cell bank of the Chinese Academy of Sciences) were cultured in a culture flask, the culture medium in the culture flask was RPMI-1640 culture medium (added with 10% fetal bovine serum and 1% penicillin-streptomycin), and the culture conditions were 5% CO2, 37°C. After DC2.4 cells were fully grown, the culture medium was discarded, and after washing with PBS buffer, digestion was performed to obtain a digestion product; the digestion product was centrifuged, and the precipitate was collected to obtain dispersed dendritic cells; the precipitate was resuspended in PBS buffer containing the STING agonist diABZI to obtain a cell resuspension, and the cell density of the dendritic cells DC2.4 in the cell resuspension was 2.0×10 6 ~3.0×10 6 / mL, so that the concentration of diABZI in the cell resuspension is 2.54μg / mL.

[0048] (2) The cell resuspension obtained in step (1) was passed through a polycarbonate membrane with a pore size of 3.0 μm → 0.6 μm → 0.2 μm in sequence using an airtight extruder, and extruded repeatedly for 5 times to obtain bionic dendritic cell vesicles loaded with adjuvant.

[0049] (3) Add PBS buffer containing melanoma antigen peptide gp100 (IMDQVPFSV) to the bionic dendritic cell vesicles obtained in step (2), and adjust the concentration of gp100 to 1 μg / mL. Stir with a magnetic stirrer at 500 rpm for 5 hours to obtain engineered bionic dendritic cell vesicles, wherein the particle size of the engineered bionic dendritic cell vesicles is 210 nm.

[0050] Example 3

[0051] (1) Dendritic cells DC2.4 cells (purchased from the cell bank of the Chinese Academy of Sciences) were cultured in a culture flask, the culture medium in the culture flask was RPMI-1640 culture medium (added with 10% fetal bovine serum and 1% penicillin-streptomycin), and the culture conditions were 5% CO2, 37°C. After DC2.4 cells were fully grown, the culture medium was discarded, and after washing with PBS buffer, digestion was performed to obtain a digestion product; the digestion product was centrifuged, and the precipitate was collected to obtain dispersed dendritic cells; the precipitate was resuspended in PBS buffer containing the STING agonist diABZI to obtain a cell resuspension, and the cell density of the dendritic cells DC2.4 in the cell resuspension was 4.0×10 6 ~5.0×10 6 / mL, so that the concentration of diABZI in the cell resuspension is 2.32μg / mL.

[0052] (2) The cell resuspension obtained in step (1) was passed through a polycarbonate membrane with a pore size of 3.0 μm → 0.6 μm → 0.2 μm in sequence using an airtight extruder, and extruded repeatedly for 6 times to obtain bionic dendritic cell vesicles loaded with adjuvant.

[0053] (3) Add PBS buffer containing melanoma antigen peptide gp100 (IMDQVPFSV-C (DSPE-PEG1000-Mal)) to the bionic dendritic cell vesicles obtained in step (2), and adjust the concentration of gp100 to 0.5 μg / mL. Use a magnetic stirrer to stir at 500 rpm for 5 hours to obtain engineered bionic dendritic cell vesicles, wherein the particle size of the engineered bionic dendritic cell vesicles is 250 nm.

[0054] Example 4

[0055] A melanoma vaccine is disclosed. The engineered bionic dendritic cell vesicles prepared in Example 1 are dispersed in a PBS (pH 7.4) buffer solution, and trehalose is added to the buffer solution to obtain a melanoma vaccine. The concentration of the engineered bionic dendritic cell vesicles obtained in Example 1 in the melanoma vaccine is 200 μg / mL, and the mass concentration of trehalose is 2%.

[0056] Example 5

[0057] A melanoma vaccine is disclosed. The engineered bionic dendritic cell vesicles prepared in Example 2 are dispersed in a PBS (pH 7.4) buffer solution, and trehalose is added to the buffer solution to obtain a melanoma vaccine. The concentration of the engineered bionic dendritic cell vesicles obtained in Example 2 in the melanoma vaccine is 400 μg / mL, and the mass concentration of trehalose is 3%.

[0058] Example 6

[0059] A melanoma vaccine is provided. The engineered bionic dendritic cell vesicles prepared in Example 1 are dispersed in a PBS (pH 7.4) buffer to obtain a melanoma vaccine. The concentration of the engineered bionic dendritic cell vesicles obtained in Example 1 in the melanoma vaccine is 200 μg / mL.

[0060] Comparative Example 1

[0061] The difference from Example 1 is that after obtaining dispersed dendritic cells according to step (1), step (2) is directly performed without step (3), and dendritic cell vesicles without adjuvant diABZI are obtained, which are recorded as DCNV.

[0062] Comparative Example 2

[0063] The difference from Example 1 is that only steps (1) and (2) are performed, and step (3) is not performed, to obtain biomimetic dendritic cell vesicles loaded with adjuvant diABZI, which are denoted as di-DCNV.

[0064] Comparative Example 3

[0065] The difference from Example 1 is that after obtaining dispersed dendritic cells according to step (1), steps (2) and (3) are directly performed to obtain engineered biomimetic dendritic cell vesicles coupled only to the melanoma antigen peptide gp100, which are denoted as g-DCNV.

[0066] Example 7

[0067] The morphologies of the engineered biomimetic dendritic cell vesicles (denoted as g-diDCVac) prepared in Example 1, the DCNV obtained in Comparative Example 1, the di-DCNV obtained in Comparative Example 2, and the g-DCNV obtained in Comparative Example 3 were observed under a transmission electron microscope, and the particle sizes of the different vesicles were measured by dynamic light scattering (DLS). The results are as follows: Figure 1-Figure 4 As shown, it is shown that the engineered bionic dendritic cell vesicles prepared by the present invention have a vacuolar membrane morphology and a normal distribution of particle sizes.

[0068] Example 8

[0069] The Zeta potential of the engineered biomimetic dendritic cell vesicles (denoted as g-diDCVac) prepared in Example 1, the DCNV obtained in Comparative Example 1, the di-DCNV obtained in Comparative Example 2, and the g-DCNV obtained in Comparative Example 3 was investigated by a Zeta potential analyzer. Figure 5 The melanoma vaccine obtained in Example 4 was divided into 3 mL centrifuge tubes and stored in a -80°C refrigerator for 27 days. The melanoma vaccine obtained in Example 6 was divided into 3 mL centrifuge tubes and stored in a 4°C refrigerator for 27 days. The particle size of the vaccine was monitored by the DLS method at 0, 3, 6, 9, 12, 15, 21, and 27 days to investigate its stability under 4°C and -80°C storage conditions. The results are shown in FIG. Figure 5 As shown in A. Figure 5 A and Figure 5 B in the figure all indicate that the melanoma vaccine and engineered bionic dendritic cell vesicles prepared by the present invention have good stability. The above experiments were all conducted in triplicate, and the results were expressed as mean±standard deviation (Mean±SD).

[0070] Example 9

[0071] In order to evaluate the uptake of the engineered biomimetic dendritic cell vesicles (g-diDCVac) obtained in Example 1, DCNV obtained in Comparative Example 1, di-DCNV obtained in Comparative Example 2, and g-DCNV obtained in Comparative Example 3 by dendritic cells (DC), DC2.4 was cultured at 1×10 4 Inoculate each cell in a confocal dish and add 1 mL of RPMI 1640 complete medium. After DC2.4 adhere to the wall, staining is performed (PBS buffer is used as the control group).

[0072] (1) Staining of dendritic cell vesicles in different groups

[0073] Take 500 μL of dendritic cell vesicles from different groups, add 1 μL of DIO dye and staining enhancer, vortex thoroughly, then add 500 μL of RPMI 1640 complete medium, and incubate in the dark for 15 minutes. Then remove the complete medium in the confocal dish, add the above incubation solution to the confocal dish, incubate for 4 hours, and wash the confocal dish twice with PBS buffer.

[0074] (2) DC2.4 lysosome staining

[0075] Take 1 μL of Lyso-Tracker Red and add it to 15 mL of warm cell culture medium. Mix well to obtain Lyso-Tracker Red working solution. Discard the liquid in the confocal dish, add the prepared Lyso-Tracker Red staining working solution pre-incubated at 37°C, and incubate with the cells at 37°C for 40 minutes. Then discard the liquid in the confocal dish, wash the confocal dish twice with PBS buffer, and add paraformaldehyde to fix for 30 minutes.

[0076] (3) DC2.4 nuclear staining

[0077] Remove the fixative, wash the confocal dish twice with PBS buffer, then add 500 μL DAPI staining solution to cover the sample. Leave at room temperature for 3 minutes. Aspirate the DAPI staining solution, and wash the confocal dish twice with PBS buffer, 3 minutes each time.

[0078] Then, confocal microscopy was used to observe the uptake of DC2.4 into vesicles from dendritic cells of different groups. Figure 6 As shown, it was found that DC2.4 was able to effectively take up vesicles from dendritic cells of each group compared with the PBS group.

[0079] Example 10

[0080] Effect of different concentrations of tumor antigen peptide gp100 on the maturation of mouse bone marrow-derived dendritic cells

[0081] In order to screen out the optimal concentration of melanoma antigen peptides that promote the maturation of bone marrow-derived dendritic cells, flow cytometry was used to detect the effects of different concentrations of tumor antigen peptide gp100 (IMDQVPFSV-C (DSPE-PEG1000-Mal)) on mouse bone marrow-derived dendritic cells. The detection indicators were the proportion of bone marrow-derived dendritic cell maturation markers CD11c, CD80, and CD86 positive cells.

[0082] Dendritic cells were extracted from mouse bone marrow and cultured at 1 × 10 6Cells were inoculated in 6-well plates with RPMI1640 complete medium (RPMI-1640 + 10% FBS + 1% penicillin-streptomycin double antibody + 10ng / mL IL-4 + 20ng / mL GM-CSF). Different concentrations of tumor antigen peptide gp100 (0μg / mL, 0.5μg / mL, 1μg / mL, 2μg / mL, 5μg / mL) and negative and positive control group drugs (PBS, 1μg / mL LPS) were added to each group of cells, so that the culture system of each well was 2mL. After 24h of co-incubation, the bone marrow-derived dendritic cells in the well plate were gently scraped off with a cell spatula, centrifuged at 4°C using a low-temperature centrifuge, and the supernatant was discarded and the cells were resuspended in 100μL PBS, and 1μL of CD11c, CD80 and CD86 flow cytometry antibodies were added, mixed thoroughly by vortexing, and co-incubated at 4°C in the dark for 30min. After the incubation, centrifuge at 4°C in a low-temperature centrifuge, discard the supernatant and resuspend in pre-cooled PBS, transfer the cell suspension into a flow tube, and make the volume of each tube of cell suspension 700 μL. Flow cytometry (FCM) was used to detect the expression of bone marrow-derived dendritic cell surface markers CD11c, CD80 and CD86 to screen the optimal concentration of melanoma antigen peptides that promote the maturation of bone marrow-derived dendritic cells. The results are shown in Figure 7 According to the flow cytometry results, the CD11c + CD80 + With CD86 + The results showed that 2μg / mL of tumor antigen peptide gp100 can achieve high-level activation of bone marrow-derived dendritic cells and enhance the antigen presentation ability of dendritic cells. (The above experiments were set up in three parallel experiments, and the results were expressed as mean ± standard deviation (Mean ± SD), *P < 0.05, ****P < 0.0001).

[0083] Embodiment 11

[0084] Evaluation of the differentiation effects of different groups of vaccines on spleen-derived lymphocytes in mice

[0085] In order to detect the differentiation effect of the vaccine on 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 is the proportion of lymphocyte differentiation markers CD3, CD4, CD8 positive cells), specifically:

[0086] According to the vaccine preparation method of Example 4, the engineered bionic 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, respectively.

[0087] Lymphocytes were extracted from mouse spleen and the cells were divided into 1×10 6 Each well was inoculated in a 6-well plate, and the cells were cultured in RPMI 1640 complete medium (RPMI-1640 + 10% FBS + 1% penicillin-streptomycin double antibody). Different vaccine groups (g-diDCVac, gp100 vaccine, DCNV vaccine, diDCNV vaccine and g-DCNV vaccine obtained in Example 4) and negative and positive control group drugs (PBS, 10ng / mL IL-2) were added to each group of cells, so that the culture system per well was 2mL. After incubation for 48h, the lymphocytes in the well plate were gently scraped off using a pipette, centrifuged at 4°C using a low-temperature centrifuge, and the cells were resuspended in 100μL PBS after discarding the supernatant, and 1μL of CD3, CD4, and CD8 flow antibodies were added, and they were fully mixed by vortexing, and incubated for 30min in the dark at 4°C. After the incubation, the 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 into a flow cytometer so that the volume of each cell suspension was 700 μL. The expression of lymphocyte surface markers CD3, CD4, and CD8 was detected by flow cytometry (FCM) to evaluate the activation of lymphocytes by different vaccine groups. The results are shown in Figure 8 According to the flow cytometry results, the CD3 + CD4 T lymphocytes + With CD8 + The results showed that CD4 + / CD8 + The proportion of T lymphocytes in the g-diDCVac group was the smallest, indicating that CD8 + T lymphocytes accounted for the largest proportion, further indicating that the g-diDCVac group could significantly induce lymphocytes to CD8 + T lymphocyte differentiation and efficient activation of lymphocyte immune killing function (the above experiments were set up three times in parallel, and the results were expressed as mean ± standard deviation (Mean ± SD), *P < 0.05, ***P < 0.001, ****P < 0.0001).

[0088] Example 12

[0089] Evaluation of the anti-tumor efficacy of different groups of vaccines in in vivo treatment experiments on melanoma mice

[0090] (1) Healthy 5-week-old C57BL / 6 female mice were selected and a mouse melanoma model was established by subcutaneously injecting B16 cells into the right back of the mice. 1.0×10 6 B16 cells, recorded as Day 0. The mice were randomly divided into PBS, DCNV vaccine group, gp100 vaccine group, di-DCNV vaccine group, g-DCNV vaccine group, and g-diDCVac vaccine group (obtained in Example 4), 5 mice in each group, and the vaccines of the corresponding groups were injected on days 3, 6, 9, 12, and 15, wherein the preparation methods of DCNV vaccine, gp100 vaccine, di-DCNV vaccine, and g-DCNV vaccine were the same as those in Example 11. The concentration of gp100 antigen peptide in the vaccine containing gp100 antigen peptide was 5 μg / mouse, the concentration of vesicles in each group of vaccines was 200 μg / mL, the injection dose was 200 μL / mouse, each mouse weighed 14-19 g, and the mice were killed by cervical dislocation on the 24th day.

[0091] (2) Every 3 days, the mice were weighed and the long and short diameters of the tumors were measured to calculate the tumor volume. Fig. 9 As shown, the tumors in the g-diDCVac group were significantly smaller than those in the other groups, indicating that g-diDCVac has a good anti-tumor effect on mouse melanoma (*P<0.05, ****P<0.0001).

[0092] (3) Tumor tissues of mice in each group were taken for TUNEL and Ki67 immunohistochemical staining. Ki67 is a nuclear proliferation antigen that reflects cell proliferation. TUNEL is a method for detecting cell apoptosis. Fig.10 As shown (pictures taken under a microscope), the results of tumor tissue immunohistochemistry TUNEL staining are basically consistent with the results of tumor proliferation antigen Ki67, further illustrating that the melanoma vaccine of the present invention can effectively inhibit tumor growth and promote tumor tissue apoptosis, and has a good anti-tumor effect on mouse melanoma.

[0093] (4) The hearts, livers, spleens, lungs, and kidneys of mice in each group were sliced ​​and stained with H&E. The results were as follows: Fig.11 As shown (pictures taken under a microscope), it is proved that the melanoma vaccine of the present invention has good safety in the process of treating melanoma mice.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An engineered bionic dendritic cell vesicle, characterized in that: The interior of the engineered bionic dendritic cell vesicle carries an agonist of an interferon gene-stimulated receptor, and the outer membrane surface of the engineered bionic dendritic cell vesicle is coupled with a melanoma antigen peptide gp100, wherein the amino acid sequence of the melanoma antigen peptide gp100 is IMDQVPFSV.

2. The engineered biomimetic dendritic cell vesicle according to claim 1, characterized in that: The agonist of the receptor for stimulation of interferon genes includes diABZI.

3. The engineered biomimetic dendritic cell vesicle according to claim 1, characterized in that: The particle size of the engineered bionic dendritic cell vesicle is 50 to 300 nm.

4. The engineered biomimetic dendritic cell vesicle according to claim 1, characterized in that: The melanoma antigen peptide gp100 is gp100 whose C-terminus is modified by distearoylphosphatidylethanolamine and PEG1000.

5. The method for preparing the engineered bionic dendritic cell vesicles according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: mixing dendritic cells with a buffer solution containing an agonist to obtain a cell resuspension; extruding the cell resuspension to obtain bionic dendritic cell vesicles; and mixing and stirring the bionic dendritic cell vesicles with a buffer solution containing a melanoma antigen peptide gp100 to obtain engineered bionic dendritic cell vesicles.

6. The preparation method according to claim 5, characterized in that: The density of dendritic cells in the cell resuspension is 2.0×10 6 ~5.0×10 6 / mL, and the concentration of the agonist in the cell resuspension is greater than 0 and below 2.54 μg / mL.

7. The preparation method according to claim 5, characterized in that: After the biomimetic dendritic cell vesicles are mixed with a buffer solution containing the melanoma antigen peptide gp100, the concentration of the melanoma antigen peptide gp100 is greater than 0 and less than 5 μg / mL.

8. Use of the engineered biomimetic dendritic cell vesicles according to any one of claims 1 to 4 or the preparation method according to any one of claims 5 to 7 in the preparation of a melanoma vaccine.

9. A melanoma vaccine, characterized in that: The melanoma vaccine comprises the engineered bionic dendritic cell vesicles according to any one of claims 1 to 4, and the concentration of the engineered bionic dendritic cell vesicles in the melanoma vaccine is 200 to 400 μg / mL.

10. The melanoma vaccine according to claim 9, characterized in that The melanoma vaccine also includes trehalose, and the mass concentration of the trehalose in the melanoma vaccine is 2% to 5%.

Images