Application of efodipidine in the preparation of dendritic cell immune adjuvant

Abstract

The present invention relates to the field of cell immunotherapy technology, and in particular to the use of efonidipine in the preparation of dendritic cell immune adjuvants. The present invention provides the use of efonidipine in the preparation of dendritic cell immune adjuvants. The present invention has discovered that efonidipine can promote the maturation, migration and antigen presentation function of DCs, significantly improving the homing ability of DCs and CD8+T cell activation. In the present invention, the activation of DCs by efonidipine includes increasing the expression of maturation indicators CD80, CD40, and CD86, the expression of migration indicator CCR7, and antigen presentation activation of CD8+T cells. The efonidipine used in the present invention is a drug approved by the FDA for the clinical treatment of cardiovascular diseases. The present invention has discovered its application in immunotherapy, realizing new uses of old drugs and having significant economic value.

Description

Technical Field

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

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

[0003] Dendritic cells (DCs), as professional antigen-presenting cells (APCs), play a central role in bridging innate and adaptive immunity and are the only APCs capable of activating naive T cells. Studies have shown that DC function may be directly affected in patients with severe infection or cancer, manifested by downregulated expression of costimulatory molecules and decreased secretion of Th1 cytokines, as well as interference with DC migration and homing to lymphoid tissues, and even a tendency for DC precursors to differentiate into tolerogenic DCs. These findings highlight the importance of restoring and reestablishing impaired DC immune function in the host.

[0004] With the maturity of in vitro DC culture technology, adoptive infusion of DC vaccines loaded with specific antigens has become a new strategy for restoring damaged DC function and stimulating antigen-specific T cell immune responses. In clinical trials, monocyte-derived DCs (Mo-DCs) are a commonly used DC vaccine subset. The preparation process involves isolation from the patient's peripheral blood mononuclear cells, induction with GM-CSF and IL-4 for 5 days to generate immature DCs (imDCs), followed by antigen loading and adjuvant stimulation to allow them to mature into mature DCs (mDCs). Antigen loading and adjuvant stimulation are key steps in DC vaccine preparation, respectively determining the specificity and potency of T cell activation by DCs. The commonly used DC vaccine adjuvant, CC, consists 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 crucial 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] To address the above issues, the present invention provides the use of efonidipine in the preparation of dendritic cell immune adjuvants. The present invention discovered that efonidipine can promote the maturation, migration, and antigen presentation function of DCs, significantly enhancing 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:

[0007] The present invention provides the use of efodipidine in preparing a dendritic cell immune adjuvant.

[0008] 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; and 6) an immune adjuvant that increases cytokine secretion.

[0009] Preferably, the promoting dendritic cell maturation comprises: promoting the expression of one or more molecules selected from CD80, CD86 and CD40.

[0010] Preferably, the promoting dendritic cell migration includes promoting the expression of CCR7.

[0011] Preferably, the enhancing of CD8+ T cell activation comprises: promoting the expression of one or more molecules selected from CD44, CD25, CD69 and CD107α.

[0012] Preferably, the cytokines include one or more of TNF-α, IL-1β, IL-6 and IL-12p40.

[0013] The present invention provides a dendritic cell immune adjuvant, the active ingredient of which includes efonidipine; the concentration (unit dose) of efonidipine in the dendritic cell immune adjuvant is 10μM~20μM.

[0014] Preferably, the concentration of efodipime in the dendritic cell immune adjuvant is 10-15 μM.

[0015] Preferably, the concentration of efodipime in the dendritic cell immune adjuvant is 10 μM.

[0016] Beneficial effects:

[0017] The present invention provides the use of efonidipine in the preparation of a dendritic cell immune adjuvant. The present invention discovered that efonidipine can promote the maturation, migration and antigen presentation function of DCs, significantly improving the homing ability of DCs and CD8+T cell activation. In the present invention, the activation of DCs by efonidipine includes increasing the expression of maturation indicators CD80, CD40, and CD86, increasing the expression of migration indicator CCR7, and activating CD8+T cells through antigen presentation. The efonidipine used in the present invention is a drug approved by the FDA for the clinical treatment of cardiovascular diseases. The present invention discovered its application in immunotherapy, realizing new uses of old drugs and having significant economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0019] Figure 1 The results of the detection of apoptosis ratio of dendritic cells after 24 hours of treatment in different treatment groups in Example 1 of the present invention are as follows;

[0020] 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;

[0021] Figure 3 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;

[0022] Figure 4 Flow cytometric detection results of chemokine CCR7 on the surface of dendritic cells treated with different treatment groups for 24 hours;

[0023] Figure 5 Results of the detection of CD8+ T cell activation promoted by dendritic cells treated with different treatment groups for 24 hours;

[0024] Figure 6 These are the test results of how dendritic cells treated with different treatment groups for 24 hours promoted CD8+ T cells to secrete effector factors IFN-γ and TNF-α. DETAILED DESCRIPTION

[0025] The present invention provides the use of efodipidine in preparing a dendritic cell immune adjuvant.

[0026] 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 secretion of cytokines; the promoting dendritic cell maturation preferably includes: promoting the expression of one or more molecules among CD80, CD86 and CD40; the promoting dendritic cell migration preferably includes promoting the expression of CCR7; the enhancing CD8+T cell activation includes: promoting the expression of one or more molecules among CD44, CD25, CD69 and CD107α; the cytokines preferably include one or more of TNF-α, IL-1β, IL-6 and IL-12p40.

[0027] The present invention has discovered the role of efonidipine in immune activation, which can promote the maturation, migration, and antigen presentation of DCs, significantly enhancing their homing ability and CD8+ T cell activation. The activation of DCs by efonidipine in this invention includes increasing the expression of maturation markers CD80, CD40, and CD86, increasing the expression of the migration marker CCR7, and activating CD8+ T cells through antigen presentation, providing technical support for DC vaccine-based immunotherapy and the development of novel adjuvants.

[0028] Based on the above advantages, the present invention provides a dendritic cell immune adjuvant comprising efonidipine as an active ingredient; the concentration of efonidipine in the dendritic cell immune adjuvant is 10 μM to 20 μM. In the present invention, the concentration of efonidipine in the dendritic cell immune adjuvant is preferably 10 to 15 μM, more preferably 10 μM; the dosage form of the dendritic cell immune adjuvant preferably includes an injection. The concentration described in the present invention represents a unit dose of the dendritic cell immune adjuvant and is an effective concentration that does not affect dendritic cell apoptosis.

[0029] To further illustrate the present invention, the application of efodiprine provided by the present invention in preparing a dendritic cell immune adjuvant is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0030] 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); naive CD8+ T cell sorting kit (Miltenyi Biotec).

[0031] 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.

[0032] 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.

[0033] Bone marrow cells were prepared as follows:

[0034] (1) Remove 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 PBS that has been autoclaved and pre-cooled at 4°C, centrifuge at 400g for 5 minutes, and remove the supernatant;

[0035] (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;

[0036] (3) Resuspend the cells in complete culture medium and count them for later use.

[0037] Example 1

[0038] Experiment on the effect of different concentrations of efodipime on the apoptosis rate of dendritic cells treated with efodipime for 24 hours

[0039] (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in 6-well culture plates at 2 ml / well. The cells were cultured in a 37°C incubator containing 5% carbon dioxide for 7 days. The cell culture medium was replaced on the 3rd and 5th days of culture. On the 7th day, immature dendritic cells with low immunogenicity were obtained.

[0040] (2) The above immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml. The dendritic cells were treated with 10μM and 20μM efodipimerine at final concentrations for 24 hours. In the positive control group, the dendritic cells were treated with 100ng / mL LPS for 24 hours.

[0041] (3) Detect cell viability using the 7-AAD apoptosis kit.

[0042] The results are as follows Figure 1 As shown in the table, ns indicates no significant difference. The results show that compared with the blank control group, 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 the 20μM efodipine treatment group was slightly higher than that of the 10μM efodipine group.

[0043] Therefore, dendritic cells were treated with 10 μM efodipime in subsequent experiments.

[0044] Example 2 Experiment on the Effect of Efonidipine Treatment on the Secretion of Inflammatory Factors in Dendritic Cells

[0045] (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in 6-well culture plates at 2 ml / well. The cells were cultured in a 37°C incubator containing 5% carbon dioxide for 7 days. The cell culture medium was replaced on the 3rd and 5th days of culture. On the 7th day, immature dendritic cells with low immunogenicity were obtained.

[0046] (2) The immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml. 10 μM efodipime 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.

[0047] (3) Cell activity was detected using mouse TNF-α, IL-1β, IL-6, and interleukin 12 p40 subunit (IL-12 p40) ELISA kits.

[0048] 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 after 24 hours of treatment with 10 μM efodipine, the secretion of TNF-α, IL-1β, IL-6, and IL-12p40 in dendritic cells was increased compared to the blank control group. Specifically, 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.

[0049] Example 3 Experiment on the effect of 24-hour treatment of dendritic cells with efodipidine on the expression of co-stimulatory molecules CD80, CD86, and CD40 in dendritic cells

[0050] (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in 6-well culture plates at 2 ml / well. The cells were cultured in a 37°C incubator containing 5% carbon dioxide for 7 days. The cell culture medium was replaced on the 3rd and 5th days of culture. On the 7th day, immature dendritic cells with low immunogenicity were obtained.

[0051] (2) The immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml. 10 μM efodipime 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.

[0052] (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. CD11c+ cells in living cells are dendritic cells. The mean fluorescence intensity of CD80, CD86, and CD40 in dendritic cells can reflect the expression levels of CD80, CD86, and CD40, respectively.

[0053] 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 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.

[0054] 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.

[0055] Example 4 Experiment on the Effect of 24-Hour Treatment of Dendritic Cells with Efonidipine on the Expression of Dendritic Cell Chemokine Receptor Molecule CCR7

[0056] (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in 6-well culture plates at 2 ml / well. The cells were cultured in a 37°C incubator containing 5% carbon dioxide for 7 days. The cell culture medium was replaced on the 3rd and 5th days of culture. On the 7th day, immature dendritic cells with low immunogenicity were obtained.

[0057] (2) The above immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml, and 10 μM efodipime was added to treat the dendritic cells for 24 hours. In the positive control group, 10 μM LPS was added to treat the dendritic cells for 24 hours.

[0058] (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 in living cells are dendritic cells. The average fluorescence intensity of CCR7 in dendritic cells can respectively reflect the expression level of CCR7.

[0059] Test results such as Figure 4 As shown, ** is p The results showed that the average positive rate of CCR7 in the dendritic cells of the control group was 29.2%, while the average positive rate of CCR7 in the dendritic cells treated with 10 μM efodipine was 38.9%.

[0060] The above experimental results show that efodipidine can upregulate the expression of chemokine receptor molecule CCR7 on the surface of dendritic cells.

[0061] 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

[0062] (1) Mouse bone marrow cells were resuspended in complete culture medium at a concentration of 1.5×106 / ml and plated in 6-well culture plates at 2 ml / well. The cells were cultured in a 37°C incubator containing 5% carbon dioxide for 7 days. The cell culture medium was replaced on the 3rd and 5th days of culture. On the 7th day, immature dendritic cells with low immunogenicity were obtained.

[0063] (2) The above-mentioned immature dendritic cells were resuspended in fresh complete culture medium at a concentration of 1.5×106 / ml, and 10μM efodipime was added to treat the dendritic cells for 24 hours. The positive control group was added with cocktail adjuvants (final concentration of 10ng / mL IL-1β, final concentration of 10ng / mL IL-6, final concentration of 15ng / mL TNF-α, final concentration of 1μg / mL PGE2) and treated with dendritic cells for 24 hours.

[0064] (3) After the 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.

[0065] (4) Purify the naive CD8+ T cells of OT-I mice using a naive CD8+ T cell sorting kit, resuspend them in T cell culture medium, and count them for later use.

[0066] (5) The dendritic cells from the different treatment groups obtained in the above experimental steps were co-cultured with naive CD8+ T cells. 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).

[0067] (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 were considered living cells, and TCRβ+CD8+ cells in living cells were considered CD8+ T cells. The expression of CD25+, CD44+, CD69+, and CD107α+ in CD8+ T cells was used as activation index. The ratio of IFN-γ+ cells in CD8+ T cells can reflect the level of IFN-γ secretion, and the ratio of TNF-α+ cells in CD8+ T cells can reflect the level of TNF-α+ secretion.

[0068] Test results such as Figure 5 As shown, * is 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 CD44+, CD25+, CD69+, and CD107α+ cells 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.

[0069] The above experimental results show that 10μM efodipime treatment for 24 hours can significantly enhance the ability of dendritic cells to promote CD8+T cell proliferation.

[0070] The results of flow cytometry detection 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 efodipine for 24 hours was 1.1×106 and 6.02×105, respectively.

[0071] The above experimental results show that 10μM efodipime 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.

[0072] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. Use of efodipime in in vitro cultured dendritic cells, wherein the use is selected from one or more of the following: 1) Promote dendritic cell maturation; 2) Promote dendritic cell migration; 3) Promote dendritic cell antigen presentation function; 4) Enhance dendritic cell homing ability; 5) Enhance dendritic cell-mediated CD8+ T cell activation; 6) Increase dendritic cell cytokine secretion; The cytokines are one or more of TNF-α, IL-1β, IL-6, and IL-12p40. The concentration of efonidipine used was 10 μM.

2. The use according to claim 1, characterized in that The promoting the maturation of dendritic cells includes promoting the expression of one or more molecules among CD80, CD86 and CD40.

3. The use according to claim 1, characterized in that The promoting of dendritic cell migration includes promoting the expression of CCR7.

4. The use according to claim 1, characterized in that The dendritic cell-mediated enhancement of CD8+T cell activation includes: promoting the expression of one or more molecules among CD44, CD25, CD69 and CD107α.

Images