Tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles and preparation method and application thereof

Abstract

The present invention relates to a tumor vaccine based on nanovesicles fused with tumor cell and immune cell membranes, and a preparation method and application thereof. The present invention adopts nanovesicles fused with dendritic cells and tumor cell membranes overexpressing CD40L as tumor antigen carriers, has good tissue compatibility and immune organ targeting, CD40L can promote dendritic cells to better exert immune functions; nanovesicles formed based on cell membranes have low research and development costs and simple procedures, and improve clinical conversion application rate; using cell membranes as materials for nanovesicles reduces their cytotoxicity and improves safety; the tumor vaccine based on nanovesicles fused with tumor cell and immune cell membranes of the present invention can also be used to prepare tumor therapeutic drugs.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment drugs, and in particular to a tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles, and a preparation method and application thereof. Background Art

[0002] Tumor vaccines are mainly composed of tumor antigens, appropriate adjuvants, effective delivery vectors, and effective administration methods. According to the different delivery forms of antigens, tumor vaccines can be divided into three categories, including nucleic acids that can encode antigens, proteins of tumor antigens, and engineered cells that can load or express cancer antigens. Tumor vaccines can promote the recognition of tumor antigens through different methods, thereby enhancing anti-tumor immunity, inhibiting tumor growth or even eliminating tumors. At present, tumor vaccines mainly rely on the following methods: designing nucleic acids containing tumor-related antigens and transferring them into cells through transfection methods such as electroporation to make them express tumor antigens to enhance tumor immunity; vaccines based on peptides or proteins are screened or computer-designed proteins or peptides related to tumor immunity or that can affect the biological characteristics of tumors, so that they enter the tumor and play a role, thereby achieving the purpose of killing tumors; engineered cell vaccines operate on autologous or allogeneic immune cells in vitro (such as by co-incubation with antigens for stimulation, or antigen loading, etc.), and then infuse them back into the body for tumor treatment).

[0003] The main limitations of current tumor vaccines are as follows: (1) low immunogenicity of the vaccine;

[0004] (2) The vaccine delivery system affects the effectiveness of tumor vaccines; (3) The synthesis cost is high and the synthesis time is long. (4) The safety is uneven

[0005] The main limitations of current vaccines are as follows:

[0006] (1) Low immunogenicity of the vaccine: Low expression of tumor antigens or disappearance of antigenic epitopes are the reasons for low immunogenicity of the vaccine. (2) The vaccine delivery system affects the effect of tumor vaccines. (3) High R&D cost and long synthesis time: The technology designed in the synthesis path of other methods is complex, and the cycle is long and the cost is high. (4) Safety varies: Some tumor vaccines designed using whole cells, if not fully inactivated, will promote the occurrence of tumors when entering the body. Relatively speaking, there is no need to worry about this when using cell membranes to synthesize tumor vaccines. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles and a preparation method and application thereof in response to the above problems and requirements.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] The method for preparing a tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles comprises the following steps:

[0010] Step 1, respectively treating tumor cells and dendritic cells overexpressing CD40L to obtain tumor cell membranes and dendritic cell membranes;

[0011] Step 2: Mix the tumor cell membrane and the dendritic cell membrane at a mass ratio of (4-5):1, and use an extruder equipped with a filter membrane to perform more than 10 extrusion cycles to obtain a tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles.

[0012] Furthermore, step 1 specifically includes the following contents:

[0013] Tumor cells and dendritic cells overexpressing CD40L were lysed on ice using a cell membrane protein and cytoplasm protein extraction kit, and then centrifuged at 800g for 10 minutes to extract the supernatant, which was then centrifuged at 15000g for 30 minutes to obtain the cell membrane.

[0014] Furthermore, step 2 specifically includes the following contents: quantifying the tumor cell membrane and the dendritic cell membrane using the BCA method, mixing them at a mass ratio of 5:1 between the tumor cell membrane and the dendritic cell membrane, and performing 15 extrusion cycles using an extruder equipped with 5μm, 1μm, and 400nm polycarbonate filter membranes to obtain a tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles.

[0015] The tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles is prepared by the above method.

[0016] The application of tumor vaccines based on tumor cell and immune cell membrane fusion nanovesicles in the preparation of tumor therapeutic drugs.

[0017] After adopting the above technical solution, the present invention has the following advantages compared with the prior art:

[0018] (1) Nanovesicles that fuse dendritic cells and tumor cell membranes that overexpress CD40L are used as tumor antigen carriers, which have good tissue compatibility and immune organ targeting. CD40L can promote dendritic cells to better exert their immune function.

[0019] (2) Nanovesicles formed based on cell membranes have low R&D costs and simple processes, which can improve the clinical translation application rate.

[0020] (3) Using cell membrane as the material of nanovesicles reduces its cytotoxicity and improves its safety.

[0021] The present invention is described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Figure 2 is related to the activation effect of CDLVDV on immune cells in vitro; (A) shows the experimental design, (B) and (C) show the flow cytometric analysis of CD80 and CD86 on BMDC after culturing with different nanoparticles for 24 hours, and the percentage of CD80+ and CD86+ BMDC cells (n=3), (D) shows the secretion of IL-12 in the supernatant determined by ELISA (n=3), (E) and (F) show the flow cytometric analysis of the stimulated BMDC cells and spleen T cells. Expression of CD4 on T cells after 24 hours of co-culture of lymphocytes. The bar graph shows the percentage of CD4+T cells (n=3). (G) and (H) are flow cytometric analysis of CD8+T cells after 24 hours of co-culture of stimulated BMDC cells and spleen T lymphocytes. The data are expressed as mean ± standard deviation. The statistical significance was calculated by one-way ANOVA. "ns" indicates not significant; (*) p<0.05, (**) p<0.01, (***) p<0.001;

[0023] Figure 2 Figures related to the lymph node accumulation and immune stimulatory effects of CDLVDV in vivo; (A) shows the fluorescent images of the inguinal lymph nodes of C57BL / 6 mice 12 hours and 24 hours after injection of PBS, DiO-labeled CDLV or DiO-labeled CDLVDV; (B) shows the statistical analysis of the fluorescent signals of the draining inguinal lymph nodes; (C) and (D) show the detection and analysis of DiO+ cells in the CD11c+ cells in the draining lymph nodes by flow cytometry; (E) and (F) show the maturation of DCs in the lymph nodes analyzed by flow cytometry; the bar graphs show the expression of CD86+ and CD8 in CD11c+ cells. 0+ cells (n=5), (G) shows the results of the spleen index of mice under different intervention measures, (H) and (I) show the flow cytometric analysis of CD4 expression on T cells in the spleen, and the bar graph shows the percentage of CD4+T cells in CD3+ cells (n=5), (J) and (K) show the flow cytometric analysis of CD8+ on T cells in the spleen, and the bar graph shows the percentage of CD8+T cells in CD3+ cells (n=5), data are expressed as mean ± standard deviation, "ns" means not significant; (*) p<0.05, (**) p<0.01, (***) p<0.001;

[0024] Figure 3Figures related to the anti-tumor effect of CDLVDV in vivo; (A) shows the growth curve of subcutaneous tumors, and the tumor volume was measured every 3 days; (B) shows the tumor weight measurement; (C) shows the Kaplan-Meier survival curve of mice in each group; (D) and (E) show the flow cytometric analysis of CD4 T cells in tumors, and the bar graphs show the percentage of CD4+T cells (n=5); (F) and (G) show the flow cytometric analysis of CD8 expression on T cells in tumor tissues, and the bar graphs show the percentage of CD8+T cells (n=5); data are expressed as mean ± standard deviation, and statistical significance was calculated by one-way analysis of variance, "ns" indicates not significant; (*) p<0.05, (**) p<0.01, (***) p<0.001;

[0025] Figure 4 This is a photo of a naked tumor after obtaining a subcutaneous tumor from a mouse;

[0026] Figure 5 Synergistic effect of CDLVDV combined with PD-L1 antibody Synergistic therapeutic effect of CDLVDV and PD-L1 antibody (A) Body weight curve of mice in each group, (B) Tumor weight measurement, (C) Subcutaneous tumor growth curve, tumor volume was measured every three days, (D) and (E) Flow cytometric analysis of CD4+T cells in tumors, the bar graph shows the percentage of CD4+T cells (n=5), (F) and (G) Flow cytometric analysis of CD8 expression on T cells in tumor tissues, the bar graph shows the percentage of CD8+T cells (n=5), data are expressed as mean ± SD, statistical significance was calculated by one-way ANOVA, "ns" indicates not significant; (*) p<0.05, (**) p<0.01, (***) p<0.001;

[0027] Figure 6 This is a photo of a nude tumor after obtaining a subcutaneous tumor from a mouse. DETAILED DESCRIPTION

[0028] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0029] 1. Synthesis and identification of CDLVDV

[0030] LLC cells overexpressing CD40L were constructed and characterized by qPCR and western blot. After obtaining cell membrane fragments, LLC cells overexpressing CD40L and DC membranes were labeled with DiR and DiO, and then CDLVDVs were prepared by extrusion and centrifugation. The hybridization of nanoparticles was confirmed by fluorescence microscopy. Then, we confirmed its structure by TEM. DLS analysis showed that the particle size distribution of TMPs was mainly around 200nm.

[0031] 2. CDLVDV can target DC in vitro

[0032] We investigated the targeting ability of CDLVDV on DC2.4. We investigated the uptake efficiency of DC2.4 cells by fluorescence imaging and flow cytometry. DC2.4 cells were stimulated with DiI-labeled CDLV and CDLVDV for 4 h. Fluorescence microscopy results showed that the cells took up the nanoparticles. Flow cytometric analysis showed that the fluorescence signal of cells in the CDLVDV group was significantly higher than that in the CDLV group, indicating that homologous targeting of DC cell membranes played a role in the uptake process and membrane hybridization also inherited its characteristics.

[0033] 3. CDLVDV stimulates DC maturation in vitro and indirectly induces T cell activation

[0034] DCs are one of the most critical APCs in the immune response, responsible for recognizing and processing antigens. The interaction between CD40 and CD40L on the surface of DCs can enhance the anti-tumor function of DCs. Therefore, we first used BMDCs as the research object to study the role of CDLVDV. The overall design of the experiment is as follows Figure 1 As shown in A. We determined the immunostimulatory effect of CDLVDV on BMDC by flow cytometry and ELISA. First, we prepared CDLVDV with different ratios of DC and LLC cells to determine the optimal ratio for mature DC. The determined ratio was 1:5 (DCs: LLC). Flow cytometry results showed that BMDC stimulated by CDLVDV expressed higher levels of CD86 and CD80 than other groups, indicating that it had a stronger ability to mature BMDC ( Figure 1 B and C). In addition, the CD80- and CD86-positive cells in the CDLVDV group were significantly higher than those in the CDLV group, indicating that hybridization can also enhance the effect.

[0035] Then, we detected the IL-12 level in the DC supernatant. The IL-12 level in the CDLVDV group was higher than that in the other groups ( Figure 1D). The results showed that with the hybridization of the LLC membrane overexpressing CD40L with the DC membrane, the immunostimulatory capacity of the vesicles was upregulated and BMDCs were affected by CDLVDV. Then, to evaluate the effect of stimulated DCs on T cells, we co-cultured DCs and T cells. T cells extracted from the spleen were co-cultured with DCs stimulated with different vesicles. After 48 hours, the proportions of various T cells were detected by flow cytometry. The results showed that the CDLVDV group had the highest proportion of CD4+ and CD8+ T cells, which was higher than that of other groups. At the same time, the proportions of CD4+ and CD8+ T cells in the mixed vesicle group were higher than those in the pure vesicle group. This proves that CDLVDV can activate T cells by interacting with DCs.

[0036] 4. CDLVDV accumulates in lymph nodes and spleen and exhibits potential immunostimulatory effects in vivo

[0037] C57BL6 / J mice were subcutaneously injected with equal amounts of Dil-labeled CDLVDV and CDLV. 24 hours later, in vivo imaging and fluorescence signal results showed that CDLVDV was significantly accumulated in the inguinal lymph nodes ( Figure 2 A and B). Flow cytometry results showed that the CDLVDV group also had a higher efficiency in taking up CD11c+ cells than the CDLV group ( Figure 2 C and D). In addition, we also found that the lymph nodes in the CDLVDV group were larger than those in the other groups. We also performed another batch of experiments to examine the effects of CDLVDV on lymph nodes. C57BL6 / J mice were treated with PBS, DV, LV, CDLV, LVDV, and CDLVDV. Inguinal lymph nodes were isolated and further analyzed by flow cytometry. The results showed that the proportion of CD86+ and CD80+ in CD11c+ cells in the lymph nodes increased, indicating that CDLVDV can stimulate the maturation of DCs in vivo ( Figure 2 E and F).

[0038] The spleen is the most important immune organ responsible for recognizing and absorbing tumor antigens. To detect the biodistribution of CDLVDV in the spleen, mice were injected with DiR-labeled particles via the tail vein. In vivo imaging was performed to measure the biodistribution. The results showed that 24 hours after tail vein injection of CDLVDV, CDLVDV accumulated mainly in the liver and spleen. We then evaluated the immunostimulatory ability of CDLVDV on the spleen. The spleen index showed that the CDLVDV group was the largest, indicating its potential immunostimulatory effect ( Figure 2 G). In addition, the CD4+T cells and CD8+T cells in the spleen of the CDLVDV group increased, which was significantly higher than that of the other groups ( Figure 2 H to K). Thus, we confirmed the immunostimulatory effects of CDLVDV in vivo.

[0039] 5.CDLVDV exerts anti-tumor effects in vivo by increasing T cell infiltration

[0040] To verify the anti-tumor effect of CDLVDV, we intervened tumor-bearing mice by tail vein injection. The intervention was performed 7 days after tumor inoculation, every three days, and samples were collected for testing on the 19th day. During the observation period, we recorded the tumor volume and measured the tumor weight at the end of the experiment. The results showed that the tumor weight and volume of the CDLVDV group were the smallest among all groups, indicating that CDLVDV can inhibit tumor growth ( Figure 3 A and B, Figure 4 ). At the same time, we found that the tumor volume of each group increased every day, but the CDLVDV group always maintained the lowest, which proved that CDLVDV can play a role in the early stage of the tumor. Comparison between the CDLV group and the CDLVDV group showed that the CDLV group had an anti-tumor effect caused by CD40L to a certain extent, but the fusion vesicles could better activate the immune response. During another 40-day treatment period, mice that died or had a tumor volume of more than 2000 cubic millimeters were excluded. The survival rate of the CDLVDV group was higher than that of other groups, indicating that CDLVDV can prolong the survival time of tumor mice ( Figure 3 C). Flow cytometry results of tumor tissues showed that the CD4+ and CD8+ T cells in the CDLVDV group were the highest among all groups. In addition, compared with single-component vesicles, the fusion product had a better effect ( Figure 3 D to G in the middle).

[0041] Synergistic therapeutic effect of 6CDLVDV and PD-L1 antibody

[0042] We have confirmed that CDLVDV's anti-tumor immunity enhances anti-tumor immunity through T cell infiltration. PDL1 antibody is already a first-line drug in tumor immunotherapy. Therefore, we want to confirm whether our products can be used together to enhance the anti-tumor efficacy of PDL1.

[0043] As ICB drugs are applied in clinical practice, immunotherapy has become a promising method for treating cancer. The application of PD-1 or PD-L1 inhibitors can activate T cells and enhance the anti-tumor effect of T cells. Given that CDLVDV can activate T cells by interacting with DCs, we tested the efficacy of CDLVDV combined with PD-L1 antibody. Figure 5 The safety of these treatments is further confirmed by the weight curves shown in A, where there was no weight loss during the treatment period. Similar to previous experiments, at the end of the intervention, the weight of the bare tumor was measured, as shown in Figure 5 B. In addition, the changes in subcutaneous tumor volume in the four groups are shown in Figure 5C. It is clear that the CDLVDV+ICB group had a better inhibitory effect on tumor growth, while ICB or CDLVDV alone had a poorer inhibitory effect on tumors. This confirms that CDLVDV and ICB have a synergistic effect on tumor regression, just like PD-L1 antibodies. Notably, flow cytometry also showed that the percentage of CD4+T cells and CD8+T cells in the tumors of the CDLVDV+ICB group was significantly higher than that of the CDLVDV and ICB group, further demonstrating the improvement in the efficiency of immunotherapy ( Figure 5 D to G in the middle), Figure 6 This shows that the therapeutic effect of CDLVDV combined with ICB (PD-L1 antibody) is better.

[0044] The above is an example of the best implementation of the present invention, and the parts not described in detail are common knowledge of ordinary technicians in the field. The protection scope of the present invention shall be based on the content of the claims, and any equivalent transformation based on the technical enlightenment of the present invention is also within the protection scope of the present invention.

Claims

1. A method for preparing a tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles, characterized in that: The following steps are involved: Step 1, treating LLC tumor cells and dendritic cells overexpressing CD40L respectively to obtain tumor cell membranes and dendritic cell membranes; Step 2 Specific The method includes the following steps: quantifying the tumor cell membrane and dendritic cell membrane using the BCA method, mixing the tumor cell membrane and dendritic cell membrane at a mass ratio of 5:1, and performing 15 extrusion cycles using an extruder equipped with 5μm, 1μm, and 400nm polycarbonate filter membranes to obtain a tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles.

2. The method for preparing a tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles according to claim 1, characterized in that: Step 1 specifically includes the following: Tumor cells and dendritic cells overexpressing CD40L were lysed on ice using a cell membrane protein and cytoplasm protein extraction kit, and then centrifuged at 800g for 10 minutes to extract the supernatant, which was then centrifuged at 15000g for 30 minutes to obtain the cell membrane.

3. A tumor vaccine based on tumor cell and immune cell membrane fusion nanovesicles, characterized in that: Prepared by the method according to any one of claims 1 to 2.

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