Application of efonidipine in preparation of dendritic cell immunologic adjuvant

Abstract

The invention relates to the technical field of cellular immunotherapy, in particular to application of efonidipine in preparation of a dendritic cell immunologic adjuvant. The invention provides an application of efonidipine in preparation of a dendritic cell immunologic adjuvant. It is found that efonidipine can promote maturation, migration and antigen presentation functions of DCs, and the homing ability of DCs and CD8 + T cell activation are remarkably improved. The activation aspect of efonidipine on DCs comprises improvement of expression of maturation indexes CD80, CD40 and CD86, expression of a migration index CCR7, activation of CD8 + T cells by antigen presentation and the like. The efonidipine used in the invention is a drug approved by FDA for clinical treatment of cardiovascular diseases, the application of the efonidipine in immunotherapy is found, new use of old drugs is realized, and the efonidipine has great economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of cell immunotherapy, and in particular to the application of efodipidine in the preparation of dendritic cell immune adjuvant. Background Art

[0002] Patients with severe infections or tumors may suffer from antigen presentation dysfunction due to abnormal activation of the immune system, which in turn affects the efficacy of cytotoxic T cells (CTLs), which is one of the key factors leading to the failure of immune response. Therefore, restoring the patient's impaired immune presentation function through immune cell therapy is crucial to enhance the clearance ability of the immune system.

[0003] Dendritic cells (DCs), as professional antigen-presenting cells (APCs), play a central role in the connection between innate immunity and acquired immunity, and are the only APCs that can activate naive T cells. Studies have shown that the function of DCs in patients with severe infection or tumors may be directly affected, manifested by down-regulation of co-stimulatory molecule expression and reduced secretion of Th1 cytokines, as well as interference with the migration and homing of DCs to lymphoid tissues, and even causing DCs precursor cells to tend to differentiate into tolerant DCs. These findings emphasize the importance of restoring and reconstructing the damaged immune function of DCs in the host.

[0004] With the maturity of DCs in vitro culture technology, adoptive infusion of DC vaccines carrying specific antigens has become a new strategy to repair damaged DCs function and stimulate antigen-specific T cell immune response. In clinical trials, monocyte-derived DCs (Mo-DCs) are a commonly used DC vaccine subset. The preparation process includes separation from the patient's peripheral blood monocytes, induction with GM-CSF and IL-4 for 5 days to obtain immature DCs (imDCs), followed by antigen loading and adjuvant stimulation to develop into mature DCs (mDCs). Antigen loading and adjuvant stimulation are key links in the preparation of DC vaccines, which determine the specificity and response intensity of DCs activating T cells, respectively. The commonly used DC vaccine adjuvant CC is composed of IL-1β, IL-6, TNF-α and PGE2, which can upregulate the expression of costimulatory molecules CD40, CD80, and CD86 on the surface of DCs and promote the secretion of Th1 cytokines. However, long-term use has found that CC adjuvants have functional defects, such as the stimulated DCs can only reach a partial mature state and are unable to synthesize cytokines such as IL-12 that are essential for T cell activation. In addition, the ability of CC-stimulated DC vaccines to home to lymphoid tissues is extremely low, at only 3% to 5%. Summary of the invention

[0005] In order to solve the above problems, the present invention provides the use of efonidipine in the preparation of dendritic cell immune adjuvants. The present invention finds that efonidipine can promote the maturation, migration and antigen presentation function of DCs, significantly improve the homing ability of DCs and CD8+T cell activation, and can be used to prepare dendritic cell immune adjuvants.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides the use of efodipidine in preparing dendritic cell immune adjuvant.

[0007] Preferably, the dendritic cell immune adjuvant includes one or more of 1) to 6): 1) an immune adjuvant that promotes dendritic cell maturation; 2) an immune adjuvant that promotes dendritic cell migration; 3) an immune adjuvant that promotes the antigen presentation function of dendritic cells; 4) an immune adjuvant that enhances the homing ability of dendritic cells; 5) an immune adjuvant that enhances CD8+T cell activation; 6) an immune adjuvant that increases the secretion of cytokines.

[0008] Preferably, the promoting maturation of dendritic cells comprises: promoting the expression of one or more molecules of CD80, CD86 and CD40.

[0009] Preferably, the promoting dendritic cell migration includes promoting the expression of CCR7.

[0010] Preferably, the enhancing of CD8+T cell activation comprises: promoting the expression of one or more molecules of CD44, CD25, CD69 and CD107α.

[0011] Preferably, the cytokines include one or more of TNF-α, IL-1β, IL-6 and IL-12p40.

[0012] The present invention provides a dendritic cell immune adjuvant, the effective component of which includes efonidipine; the concentration (unit dose) of efonidipine in the dendritic cell immune adjuvant is 10 μM-20 μM.

[0013] Preferably, the concentration of efodipidine in the dendritic cell immune adjuvant is 10-15 μM.

[0014] Preferably, the concentration of efodipidine in the dendritic cell immune adjuvant is 10 μM.

[0015] Beneficial effects: The present invention provides the use of efonidipine in the preparation of dendritic cell immune adjuvants. The present invention finds that efonidipine can promote the maturation, migration and antigen presentation function of DCs, and significantly improves the homing ability of DCs and CD8+T cell activation. In the present invention, the activation of DCs by efonidipine includes improving the expression of maturation indicators CD80, CD40, CD86, improving the expression of migration indicator CCR7, antigen presentation activation of CD8+T cells, etc. The efonidipine used in the present invention is a drug approved by the FDA for clinical treatment of cardiovascular diseases. The present invention finds its application in immunotherapy, realizes new uses of old drugs, and has great economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 The results of the detection of cell apoptosis ratios of dendritic cells treated in different treatment groups for 24 hours in Example 1 of the present invention; Figure 2 ELISA results of inflammatory factors TNF-α, IL-6, IL-1β, and IL-12p40 after dendritic cells were treated with different treatment groups; Figure 3 The flow cytometric detection results of costimulatory molecules CD80, CD86, and CD40 on the surface of dendritic cells treated with different treatment groups for 24 hours; Figure 4 Flow cytometric detection results of chemokine CCR7 on the surface of dendritic cells treated with different treatment groups for 24 hours; Figure 5 Results of the detection of CD8+T cell activation promoted by dendritic cells treated with different treatment groups for 24 hours; Figure 6 These are the test results of dendritic cells treated with different treatment groups for 24 hours promoting CD8+T cells to secrete effector factors IFN-γ and TNF-α. DETAILED DESCRIPTION

[0018] The present invention provides the use of efodipidine in preparing dendritic cell immune adjuvant.

[0019] In the present invention, the dendritic cell immune adjuvant preferably includes one or more of 1) to 6): 1) an immune adjuvant that promotes dendritic cell maturation; 2) an immune adjuvant that promotes dendritic cell migration; 3) an immune adjuvant that promotes the antigen presentation function of dendritic cells; 4) an immune adjuvant that enhances the homing ability of dendritic cells; 5) an immune adjuvant that enhances CD8+T cell activation; 6) an immune adjuvant that increases the amount of cytokine secretion; the promotion of dendritic cell maturation preferably includes: promoting the expression of one or more molecules of CD80, CD86 and CD40; the promotion of dendritic cell migration preferably includes promoting the expression of CCR7; the enhancement of CD8+T cell activation includes: promoting the expression of one or more molecules of CD44, CD25, CD69 and CD107α; the cytokines preferably include one or more of TNF-α, IL-1β, IL-6 and IL-12p40.

[0020] The present invention has discovered the role of efonidipine in immune activation, which can promote the maturation, migration and antigen presentation function of DCs, and significantly improve the homing ability of DCs and CD8+T cell activation. In the present invention, efonidipine activates DCs by increasing the expression of maturation indicators CD80, CD40, and CD86, increasing the expression of migration indicator CCR7, activating CD8+T cells by antigen presentation, etc., providing technical support for immunotherapy based on DC vaccines and the development of new adjuvants.

[0021] Based on the above advantages, the present invention provides a dendritic cell immune adjuvant, the active ingredient of which includes efonidipine; the concentration of efonidipine in the dendritic cell immune adjuvant is 10μM~20μM. In the present invention, the concentration of efonidipine in the dendritic cell immune adjuvant is preferably 10~15μM, more preferably 10μM; the dosage form of the dendritic cell immune adjuvant preferably includes an injection. The concentration described in the present invention is a unit dose of the dendritic cell immune adjuvant, which is an effective concentration that does not affect the apoptosis of dendritic cells.

[0022] In order to further illustrate the present invention, the application of efonidipine provided by the present invention in preparing dendritic cell immune adjuvant is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0023] The materials and sources used in the following examples are as follows: Efonidipine (Selleck); dimethyl sulfoxide (DMSO, Sigma); RPMI 1640 medium (Gibco); fetal bovine serum (Gibco); penicillin-streptomycin (Gibco); glutamine (Gibco); granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech); IL-4 (Peprotech); lipopolysaccharide (LPS, purchased from Sigma); anti-CD11c, anti-CD80, anti-CD86, anti-CCR7, anti-CXCR4, anti-CCR5, anti-CD8, anti-TCRb, anti-CD25, anti-CD69, anti-CD44, anti-CD107α, anti-IFNγ, anti-TNF-α and other flow cytometry antibodies purchased from Biolegend; CCK-8 kit (Biyuntian); initial CD8+T cell sorting kit (Miltenyi Biotec).

[0024] The complete culture medium used in the following examples is RPMI 1640 culture medium containing GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, and glutamine, and the final concentrations of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, and glutamine are 15 ng / ml, 10 ng / ml, 10%, 100 U / ml, 100 μg / ml, and 2 mM, respectively.

[0025] The sequence of the polypeptide OVA257-264 used in the following examples is SIINFEKL (shown in SEQ ID NO. 1), which was purchased from InvivoGen.

[0026] The bone marrow cells were prepared as follows: (1) Take the femur and tibia of the mouse, remove the muscle, disinfect and clean, flush the bone marrow cells into a 50 ml centrifuge tube with high-pressure sterilized PBS precooled at 4°C, centrifuge at 400g for 5 minutes, and remove the supernatant; (2) Resuspend the cells in PBS, filter the resuspended cells with a 40 μm filter to remove impurities such as broken bones, centrifuge at 400 g for 5 minutes, and remove the supernatant; (3) Resuspend the cells in complete culture medium and count them for later use.

[0027] Example 1 Experiment on the effect of different concentrations of efodipidine on the apoptosis ratio of dendritic cells treated with efodipidine for 24 hours (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in a 6-well culture plate at 2 ml / well. The plates were cultured in an incubator at 37°C containing 5% carbon dioxide for 7 days. The medium was replaced on days 3 and 5 of culture. Immature dendritic cells with low immunogenicity were obtained on day 7.

[0028] (2) The above immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml. Efonidipine at a final concentration of 10 μM and 20 μM was added to treat the dendritic cells for 24 hours. In the positive control group, 100 ng / mL LPS was added to treat the dendritic cells for 24 hours.

[0029] (3) Detect cell activity using 7-AAD apoptosis kit.

[0030] The results are as follows Figure 1 As shown, ns indicates no significant difference. The results show that compared with the blank control group cells, the apoptosis rate of dendritic cells treated with 10μM and 20μM efodipine for 24 hours did not change significantly, and the average apoptosis rate of cells in the 20μM efodipine treatment group was slightly higher than that of 10μM efodipine.

[0031] Therefore, dendritic cells were treated with 10 μM efodipidine in subsequent experiments.

[0032] Example 2 Experiment on the effect of efodipidine treatment on the secretion of inflammatory factors in dendritic cells (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in a 6-well culture plate at 2 ml / well. The plates were cultured in an incubator at 37°C containing 5% carbon dioxide for 7 days. The medium was replaced on days 3 and 5 of culture. Immature dendritic cells with low immunogenicity were obtained on day 7.

[0033] (2) The above immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml, and 10 μM efodipidine was added to treat the dendritic cells for 24 hours. In the positive control group, 100 ng / mL LPS was added to treat the dendritic cells for 24 hours.

[0034] (3) Cell activity was detected using mouse TNF-α, IL-1β, IL-6, and interleukin 12 p40 subunit (IL-12 p40) ELISA kits.

[0035] The experimental results are as follows Figure 2 Compared with the control group, ns means no significant difference, * means p <0.05, *** p<0.001. The results showed that the secretion of TNF-α, IL-1β, IL-6, and IL-12p40 in dendritic cells after 24 hours of treatment with 10μM efodipidine was upregulated compared with the blank control group. Among them, the secretion of TNF-α was 1.45 times that of the control group, and the secretion of IL-6 was 1.32 times that of the control group.

[0036] Example 3 Experiment on the effect of efodipidine treatment on dendritic cells for 24 hours on the expression of co-stimulatory molecules CD80, CD86 and CD40 in dendritic cells (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in a 6-well culture plate at 2 ml / well. The plates were cultured in an incubator at 37°C containing 5% carbon dioxide for 7 days. The medium was replaced on days 3 and 5 of culture. Immature dendritic cells with low immunogenicity were obtained on day 7.

[0037] (2) The above immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml, and 10 μM efodipidine was added to treat the dendritic cells for 24 hours. In the positive control group, 100 ng / mL LPS was added to treat the dendritic cells for 24 hours.

[0038] (3) After collecting the cells, the expression levels of co-stimulatory molecules CD80, CD86, CD40, and MHC II on the surface of dendritic cells were detected by flow cytometry surface staining. CD11c+ cells in living cells are dendritic cells, and the average fluorescence intensity of CD80, CD86, and CD40 in dendritic cells can reflect the expression levels of CD80, CD86, and CD40, respectively.

[0039] Test results such as Figure 3 Compared with the control group, ns means no significant difference, ** means p <0.01, *** p <0.001, **** p The results showed that the average positive ratios of CD80, CD86, and CD40 in the control group dendritic cells were 35.2%, 36.1%, and 26.3%, respectively, while the average positive ratios of CD80, CD86, and CD40 in the dendritic cells treated with 10 μM efodipidine were 47.5%, 50.1%, and 41%, respectively.

[0040] The above experimental results show that efodipidine can upregulate the expression of co-stimulatory molecules CD80, CD86, and CD40 on the surface of dendritic cells.

[0041] Example 4 Experiment on the effect of efodipidine treatment on the expression of chemokine receptor molecule CCR7 on dendritic cells for 24 hours (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in a 6-well culture plate at 2 ml / well. The plates were cultured in an incubator at 37°C containing 5% carbon dioxide for 7 days. The medium was replaced on days 3 and 5 of culture. Immature dendritic cells with low immunogenicity were obtained on day 7.

[0042] (2) The above immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml, and 10 μM efodipidine was added to treat the dendritic cells for 24 hours. For the positive control group, 10 μM LPS was added to treat the dendritic cells for 24 hours.

[0043] (3) After collecting the cells, the expression level of the chemokine receptor molecule CCR7 on the surface of dendritic cells was detected by flow cytometry surface staining. FVD- cells are living cells, and CD11c+ cells among the living cells are dendritic cells. The average fluorescence intensity of CCR7 in dendritic cells can respectively reflect the expression level of CCR7.

[0044] Test results such as Figure 4 As shown, ** is p The results showed that the average positive ratio of CCR7 in the control group dendritic cells was 29.2%, and the average positive ratio of CCR7 in the dendritic cells treated with 10 μM efodipidine was 38.9%.

[0045] The above experimental results show that efodipidine can upregulate the expression of chemokine receptor molecule CCR7 on the surface of dendritic cells.

[0046] Example 5 Experiment on the effect of dendritic cell-mediated CD8+ T cell activation and secretion of effector factors IFN-γ and TNF-α after 24-hour treatment of dendritic cells with efodipidine (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in a 6-well culture plate at 2 ml / well. The plates were cultured in an incubator at 37°C containing 5% carbon dioxide for 7 days. The medium was replaced on days 3 and 5 of culture. Immature dendritic cells with low immunogenicity were obtained on day 7.

[0047] (2) The above immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml, and 10 μM efodipidine was added to treat the dendritic cells for 24 hours. The positive control group was added with a cocktail adjuvant (a final concentration of 10 ng / mL IL-1β, a final concentration of 10 ng / mL IL-6, a final concentration of 15 ng / mL TNF-α, and a final concentration of 1 μg / mL PGE2) and treated with the dendritic cells for 24 hours.

[0048] (3) After stimulation of the two groups of cells, live cells were obtained by Ficoll centrifugation, resuspended in T cell culture medium, and counted for later use.

[0049] (4) Purify the naive CD8+ T cells of OT-I mice using a naive CD8+ T cell isolation kit, resuspend them in T cell culture medium, and count them for later use.

[0050] (5) The dendritic cells of the different treatment groups obtained in the above experimental steps were co-cultured with naive CD8+ T cells, respectively. Each co-culture system was 500 μl and contained 5×105 dendritic cells, 2.5×106 naive CD8+ T cells and 10 ng / ml OVA257-264 (SIINFEKL, shown in SEQ ID NO.1).

[0051] (6) After 24 hours of co-culture, the proliferation of CD8+T cells was detected by flow cytometry surface staining. After 4 days of co-culture, the levels of IFN-γ and TNF-α secreted by CD8+T cells were detected by flow cytometry intracellular staining. FVD- cells are living cells, TCRβ+CD8+ cells in living cells are CD8+T cells, and CD25+, CD44+, CD69+, and CD107α+ in CD8+T cells are activation indicators. The proportion of IFN-γ+ cells in CD8+T cells can reflect the level of IFN-γ secretion, and the proportion of TNF-α+ cells in CD8+T cells can reflect the level of TNF-α+ secretion.

[0052] Test results such as Figure 5 As shown, * p <0.05, ** p <0.01, *** p <0.001, **** p The results showed that compared with the control group of dendritic cells, the number of activated cells of CD44+, CD25+, CD69+, and CD107α+ in the CD8+T cells co-cultured with dendritic cells treated with 10 μM efodipidine for 24 hours was 3.4×106, 2.3×106, 7×105, and 1.8×106, respectively.

[0053] The above experimental results show that 10 μM efodipidine treatment for 24 hours can significantly enhance the ability of dendritic cells to promote CD8+T cell proliferation.

[0054] The flow cytometry results of CD8+T cells secreting effector factors IFN-γ and TNF-α are as follows Figure 6 As shown, *** is p <0.001, **** pThe results showed that compared with the control group of dendritic cells, the number of IFN-γ+ and TNF-α+ cells in the CD8+T cells co-cultured with dendritic cells treated with 10 μM efodipidine for 24 hours was 1.1×106 and 6.02×105, respectively.

[0055] The above experimental results show that 10μM efodipidine treatment for 24 hours can significantly enhance the ability of dendritic cells to promote CD8+T cells to secrete effector factors IFN-γ and TNF-α.

[0056] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of efodipidine in the preparation of dendritic cell immune adjuvant.

2. The use according to claim 1, characterized in that: The dendritic cell immune adjuvant includes one or more of 1) to 6): 1) an immune adjuvant that promotes dendritic cell maturation; 2) an immune adjuvant that promotes dendritic cell migration; 3) an immune adjuvant that promotes the antigen presentation function of dendritic cells; 4) an immune adjuvant that enhances the homing ability of dendritic cells; 5) an immune adjuvant that enhances CD8+T cell activation; 6) an immune adjuvant that increases the secretion of cytokines.

3. The use according to claim 2, characterized in that: The promoting the maturation of dendritic cells includes: promoting the expression of one or more molecules among CD80, CD86 and CD40.

4. The use according to claim 2, characterized in that: The promoting of dendritic cell migration includes promoting the expression of CCR7.

5. The use according to claim 2, characterized in that: The enhancing of CD8+T cell activation includes: promoting the expression of one or more molecules among CD44, CD25, CD69 and CD107α.

6. The use according to claim 2, characterized in that: The cytokines include one or more of TNF-α, IL-1β, IL-6 and IL-12p40.

7. A dendritic cell immune adjuvant, characterized in that: The effective ingredients include efonidipine; the concentration of efonidipine in the dendritic cell immune adjuvant is 10 μM-20 μM.

8. The dendritic cell immune adjuvant according to claim 7, characterized in that: The concentration of efodipidine in the dendritic cell immune adjuvant is 10-15 μM.

9. The dendritic cell immune adjuvant according to claim 7 or 8, characterized in that: The concentration of efodipidine in the dendritic cell immune adjuvant is 10 μM.

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