Dendritic cell vaccine for preventing and treating EGFR (epidermal growth factor receptor) sensitive gene mutation type lung adenocarcinoma and preparation method of dendritic cell vaccine

Abstract

The invention discloses a dendritic cell vaccine for preventing and treating EGFR (epidermal growth factor receptor) sensitive gene mutation type lung adenocarcinoma and a preparation method of the dendritic cell vaccine. The preparation method comprises the following steps: irradiating tumor cells with X rays with the total dose of 9-18Gy in a segmentation mode, and inducing the tumor cells to generate exosomes; and loading the exosome to the dendritic cells to obtain the dendritic cell vaccine. The exosome generated by the X-ray induced tumor cells is adopted to promote the expression of surface costimulatory molecules and the secretion of cell factors in the dendritic cell maturation process, and the ability of T lymphocyte proliferation and tumor cell killing is improved. According to the dendritic cell vaccine, the curative effect of osimertinib on EGFR sensitive gene mutation type lung adenocarcinoma can be improved, and a new method is provided for improving the survival rate of EGFR sensitive gene mutation type lung adenocarcinoma patients.

Description

Technical Field

[0001] This invention belongs to the fields of molecular immunology and biomedical technology, specifically relating to a dendritic cell vaccine for the prevention and treatment of EGFR-sensitive gene-mutant lung adenocarcinoma and its preparation method. Background Technology

[0002] Targeted therapy has significantly improved the survival rate of patients with epidermal growth factor receptor (EGFR) sensitive gene mutations, but long-term response is still uncommon for most patients and drug resistance is inevitable. The combination therapy strategy of targeted therapy with other treatments is an important direction of current research.

[0003] Dendritic cell vaccines, as a promising immunotherapy approach, are an important alternative to targeted drug combination therapy strategies. Tumor antigens are key components in inducing effective anti-tumor immune responses in dendritic cell vaccines. The antigen loading methods of dendritic cell vaccines are diverse and play a crucial role in inducing effective anti-tumor effects. Tumor cell lysates, peptides, total proteins, cDNA, total RNA, mRNA, and inactivated whole cells can all serve as tumor antigens to activate dendritic cells, obtain specific tumor antigens, and then reinfuse them to treat tumors. In recent years, tumor-derived exosomes have become an important source of tumor antigens for the preparation of dendritic cell vaccines. They can deliver various bioactive molecules, such as proteins, miRNAs, and mRNAs, between cells. These substances can be carried by tumor-derived exosomes to dendritic cells and may become tumor antigens that trigger tumor-specific immune responses. Therefore, stimulating tumor cells to produce exosomes is a key step in the successful construction of dendritic cell vaccines. However, the composition of exosomes is affected by various factors, and how to effectively stimulate tumor cell exosomes to secrete immunogenic substances remains a challenge for researchers and requires continuous exploration. Summary of the Invention

[0004] In order to effectively promote the secretion of immunogenic substances by tumor cell exosomes and improve the survival rate of patients with EGFR-sensitive gene mutations, the present invention provides the following technical solution.

[0005] In a first aspect, the present invention provides a method for preparing a dendritic cell vaccine for preventing and treating EGFR-sensitive gene-mutant lung adenocarcinoma, comprising the following steps: S1 uses X-rays with a total dose of 9-18 Gy and employs a fractionated irradiation mode to induce tumor cells to produce exosomes; S2, the exosomes are loaded into dendritic cells to obtain the dendritic cell vaccine.

[0006] Preferably, the dose of X-rays in step S1 is selected from any one of 9 Gy, 12 Gy, 16 Gy or 18 Gy.

[0007] Preferably, the number of times the segmented irradiation is 2 to 6, for example, 2, 3, 4, 5 or 6 times.

[0008] More preferably, the X-ray irradiation mode is selected from one of 3Gy×3f (f represents the number of irradiation fractions, the same below; for example, 3Gy×3f here means that the irradiation dose is 3Gy per irradiation and the number of irradiations is 3), 3Gy×4f, 4Gy×3f, 6Gy×2f, 8Gy×2f, 4Gy×4f, 6Gy×3f, 9Gy×2f, and 3Gy×6f.

[0009] Furthermore, the X-ray irradiation mode is 6Gy×3f, which has a significantly better irradiation effect than 3Gy×3f, 3Gy×4f, 4Gy×3f, 6Gy×2f, 8Gy×2f, 4Gy×4f, 9Gy×2f, and 3Gy×6f.

[0010] Preferably, the tumor cells in step S1 include, but are not limited to, breast cancer cells, colorectal cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells, and esophageal cancer cells.

[0011] Preferably, the dendritic cells in step S2 are derived from bone marrow, thymus, peripheral blood, or lymphatic organs, and more preferably from bone marrow.

[0012] In a second aspect, the present invention provides a dendritic cell vaccine prepared according to the preparation method described in the first aspect.

[0013] Preferably, the dendritic cell vaccine is in the form of a cell suspension.

[0014] Preferably, the dendritic cell vaccine further includes pharmaceutically acceptable excipients, such as pharmaceutically acceptable carriers, diluents, and adjuvants.

[0015] Preferably, the carrier comprises a viral carrier (adenovirus or lentivirus) or nanomaterials.

[0016] Preferably, the diluent is selected from one or more of physiological saline, PBS, glucose injection, or sterile isotonic buffer solution.

[0017] Preferably, the adjuvant is selected from any one or more of Freund's complete adjuvant (FCA), poly-ICLC, STING agonist, or imiquimod.

[0018] Preferably, the dendritic cell vaccine is administered via any one or more of the following routes: subcutaneous, intramuscular, intradermal, or intravenous.

[0019] More preferably, the single dose of the dendritic cell vaccine is 4-6 × 10⁻⁶. 6 Cells, for example: 4 × 10 6 Cells, 5×10 6 Cells, 6×10 6 cell.

[0020] Thirdly, the present invention provides a pharmaceutical composition for preventing and treating EGFR-sensitive gene-mutant lung adenocarcinoma, comprising the dendritic cell vaccine described in the second aspect.

[0021] Preferably, the pharmaceutical composition further includes a targeted drug against EGFR.

[0022] Preferably, the targeted drug includes gefitinib, erlotinib, icotinib, afatinib, osimertinib, or dabrafenib; more preferably, the targeted drug is osimertinib.

[0023] Fourthly, the present invention provides the use of the dendritic cell vaccine described in the second aspect or the pharmaceutical composition described in the third aspect in the preparation of a product for the prevention and treatment of EGFR-sensitive gene-mutant lung adenocarcinoma.

[0024] Preferably, the product comprises the dendritic cell vaccine described in the second aspect or the pharmaceutical composition described in the third aspect.

[0025] Preferably, the product further includes pharmaceutically permissible excipients (such as carriers, excipients, diluents, or adjuvants).

[0026] The beneficial effects of this invention are: 1. Exosomes produced by tumor cells by X-ray induction can promote the expression of surface co-stimulatory molecules and the secretion of cytokines (such as interferon and interleukin) during the maturation of dendritic cells, thereby enhancing the proliferation of T lymphocytes and their ability to kill tumor cells.

[0027] 2. This invention has found that the irradiation effect of using an X-ray irradiation mode of 6Gy×3f is significantly better than other low-dose fractionation methods (such as 3Gy×6f and 2Gy×9f) under the same total irradiation dose.

[0028] 3. The dendritic cell vaccine of the present invention can improve the efficacy of osimertinib against EGFR-sensitive gene-mutant lung adenocarcinoma, providing a new method for improving the survival rate of patients with EGFR-sensitive gene-mutant lung adenocarcinoma. Attached Figure Description

[0029] Figure 1 The image shows the morphology of DCs on day 9 of induced differentiation under a 40x microscope in Example 1; Figure 2 The image shows surface molecular markers of mouse dendritic cells (BMDCs) in Example 1; Figure 3 The image shown is a flow cytometry plot of BMDC co-stimulatory molecules expressed 24 hours after treatment with exosomes or LPS in Example 1. Figure 4 The figure shows the expression results of BMDC co-stimulatory molecules after 24 hours of treatment with exosomes or LPS in Example 1; Figure 5 The image shows the results of ELISA analysis of cytokines secreted by BMDCs in Example 1; Figure 6 The figure shown is a flow cytometry representation of CFSE-positive cells (T cell proliferation) under different treatment conditions in Example 2; Figure 7 The figure shown is a statistical graph of CFSE-positive cells (T cell proliferation) under different treatment conditions in Example 2; Figure 8 The image shows the results of ELISA analysis of cytokines secreted by T cells in Example 2; Figure 9 The figure shows the cytotoxicity of CTL cells stimulated by different DC groups in Example 2; Figure 10 The figure shows the flow cytometry gating strategy in Example 2 and the flow cytometry representation of CD45 anexinV+ under different treatment conditions. Figure 11 The figure shown is a statistical graph of the proportion of CD45 anexinV+ cells under different treatment conditions in Example 2; Figure 12 The diagram shown is the experimental flowchart for Example 3; Figure 13 The figure shows the tumor size of mice in different groups at different time points in Example 3; Figure 14 The figure shown is a graph of tumor volume changes in mice in each group in Example 3. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to embodiments and accompanying drawings. The advantages and features of the present invention will become clearer as the description unfolds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0031] It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0032] Example 1: Preparation of Dendritic Cell Vaccine 1.1 X-ray irradiation of tumor cells LLC cell lines were purchased from the National Biomedical Experimental Cell Resource Bank (BMCR) and cultured in DMEM medium (Gibco) supplemented with 10% FBS (Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin. LLC cells were transfected with an EGFR-19DEL overexpressing lentivirus constructed by GeneChem (Shanghai, China) to construct LLC cells expressing human EGFR-19del protein. Before irradiation, the medium was replaced with Hieff Serum-free medium (for exosomes) (Yisheng Bio, cat: 41210ES76). Cells grown to approximately 80% confluence were irradiated in various modes: 0 Gy, 3 Gy × 3 f, 6 Gy × 3 f, 9 Gy × 1 f, 18 Gy × 1 f, 3 Gy × 6 f, and 2 Gy × 9 f. The irradiation was performed using an RS2000 biological irradiator with parameters set at 160 kV, 25 mA, and a dose rate of 1.175 Gy / min.

[0033] 1.2 Exosome isolation Exosomes were isolated using ultracentrifugation. The supernatant from LLC-EGFR19Del cell culture was first centrifuged at 300×g for 10 minutes to remove cells and large particles, then centrifuged at 2000×g for 30 minutes to obtain a coarse exosome pellet. This pellet was then transferred to an ultracentrifuge tube and centrifuged at 10000×g for 70 minutes to remove detached microvesicles. After centrifugation, the supernatant was collected, balanced, and placed in an ultracentrifuge (Beckman Optima™). The pellet was centrifuged at 100000×g for 120 minutes at 4°C. The pellet was then resuspended in PBS, and the centrifugation was repeated once. After resuspending in sterile PBS, the pellet was filtered through a 0.22 μm filter and aliquoted at -80°C for storage.

[0034] 1.3 Exosome-induced DC activation Nine days after mouse bone marrow cells were induced to differentiate, non-adherent and semi-adherent cells were harvested by centrifugation at 300×g for 5 min and regarded as immature dendritic cells (iDCs). These cells were cultured for 24 hours with exosomes derived from irradiated tumor cells. After cell collection, the DC phenotype was detected by flow cytometry. The supernatant was collected to quantify the cytokines secreted by the DCs.

[0035] Depend on Figure 1 As can be seen under a microscope, dendritic protrusions are visible on the surface of the induced differentiated BMDC.

[0036] Depend on Figure 2 It can be seen that more than 85% of the cells are CD11c positive and express DC surface markers, including MHC-II, CD40, CD80 and CD86.

[0037] Depend on Figure 3 and Figure 4 As can be seen from the flow cytometry test, compared with the negative control without exosomes and the positive control with LPS, irradiated tumor cell-derived exosomes (RA-TEXs) promoted the maturation of iDCs to varying degrees, with 6 Gy × 3 f-TEXs showing the strongest stimulatory effect.

[0038] Depend on Figure 5 As can be seen from ELISA detection, irradiated tumor cell-derived exosomes (RA-TEXs) promote the maturation of iDCs to varying degrees. Among them, 6Gy×3f-TEXs have the strongest stimulating effect and can effectively stimulate iDCs to secrete cytokines INF-γ, IL-12 and IL-10.

[0039] Example 2: Effects of dendritic cell vaccine on tumor cells 2.1 T-cell killing effect of dendritic cell vaccine After euthanizing normal C57BL / 6 mice (Spyford Beijing Biotechnology Co., Ltd.), spleens were harvested, ground into a single-cell suspension, filtered, washed with lysine, and lymphocytes were isolated using a mouse spleen lymphocyte isolation kit (SolarBio, China). T lymphocyte concentration was detected by flow cytometry. The isolated T cells were then activated in a system containing CD3, CD28, and IL-2 (BioLegend, USA), stained with 5M CFSE (Thermo, USA) at 37°C for 10 minutes, and then washed by centrifugation with medium containing 10% FBS. Next, DCs treated with LPS and exosomes for 24 hours were co-incubated with CFSE-labeled T cells. After 3 days of co-culture, the supernatant was collected, and cytokine secretion by T cells was measured by ELISA. Cells were harvested, and CFSE-labeled T cell proliferation was detected by flow cytometry.

[0040] To assess the cytotoxic efficacy of T cells, T cells stimulated by exosome-treated dendritic cells (DCs) served as effector cells (E) and interacted with target cells (T cells, or tumor cells). These cells were co-incubated for 24 h at E / T ratios of 5:1, 10:1, and 20:1. The cytotoxic activity of CTLs against tumor cells was assessed by measuring the release of lactate hydrogenase (LDH) and Annexin V flow cytometry staining of tumor cells. Cell mortality was calculated using an LDH release kit (Beyotime, China) at 450 nm absorbance, and the proportion of necrotic cells was analyzed by Annexin V staining flow cytometry.

[0041] Depend on Figure 6 and Figure 7 It can be seen that iDCs did not induce a continuous CFSE decreasing peak pattern, indicating that T cells hardly proliferate in the absence of antigen peptide stimulation. LPS and C-TEXs stimulation resulted in less than 15% T cell proliferation. RA-TEXs-stimulated DCs, especially those stimulated with 6 Gy*3f-TEXs, could increase the T cell proliferation rate to 83.3%.

[0042] Figure 8 ELISA data showed the cytokine secretion results after co-incubation of DCs and T cells with different treatments. The highest secretion of TNF-α and IL-4 by T cells was observed in the 6 Gy × 3 f group.

[0043] Figure 9 The LDH killing assay results showed the killing effect of T cells on tumor cells at different effector-to-target ratios (5:1, 10:1, and 20:1). RA-TEXs-stimulated dendritic cells induced CTL-specific cytotoxicity against tumor cells. The tumor cells in the 6 Gy × 3 f-TEXs group released the most LDH, demonstrating that this method of stimulating dendritic cells is most effective in inducing T cell killing of tumor cells.

[0044] Figure 10 and Figure 11 Flow cytometry results demonstrated the tumor-killing effect of T cells induced by iDCs under different stimulation modalities. The 6 Gy × 3 f-TEXs group exhibited the strongest tumor-killing activity, with approximately 72.7% of tumor cells being Annexin V positive.

[0045] 2.2 The inhibitory effect of combined dendritic cell vaccine and osimertinib on tumors according to Figure 12 The experiment was conducted according to the procedure shown below. Four healthy adult C57BL / 6 female mice (Speedford Beijing Biotechnology Co., Ltd.) aged 6 weeks were used. LLC-EGFR19Del cells (2×10⁻⁶) were injected subcutaneously into the right scapula. 6 (cells / mouse). On day 7 post-vaccination, mice were randomly divided into a control group, a DC group, an osimertinib group, and a DC vaccine plus osimertinib combination group. The specific treatments for each group were as follows: (1) Control group: PBS 100 μL / d was injected via tail vein; (2) Osimertinib group: Mice were given osimertinib by gavage once a day, 50 mg (kg·d); (3) DC group: Mice were immunized with DCs stimulated by RA-EXO via tail vein injection, once every 3 days, 2×10 6 Cells / 100 μL·d, treatment lasted for two weeks; (4) DC vaccine combined with osimertinib group: mice were immunized by intravenous injection of RA-EXO-stimulated DCs (once every 3 days, 2×10). 6 (100 μL / animal), and simultaneously received osimertinib by gavage (50 mg / kg / day once daily) for two weeks.

[0046] The average tumor burden at the start of treatment was 100 mm. 3 (The average value of the group ranged from 89 to 110 mm) 3 The tumor size was measured in two dimensions every 3 days using calipers, and the tumor volume was calculated according to Formula 1 to plot the tumor growth curve. Mice were euthanized by cervical dislocation on day 21 after tumor inoculation, and the tumor was dissected and photographed for record-keeping.

[0047] Tumor volume (mm) 3 )=(width)2×(length)×1 / 2 (Formula 1) Depend on Figure 13 and Figure 14 It can be seen that both osimertinib and 6Gy×3f-TEXS-stimulated DCs led to a certain degree of tumor regression. Compared with other groups, the tumor volume was significantly reduced and tumor growth was inhibited in the DCs combined with osimertinib group.

[0048] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for preparing a dendritic cell vaccine for the prevention and treatment of EGFR-sensitive gene-mutant lung adenocarcinoma, characterized in that: The preparation method includes the following steps: S1, X-rays with a total dose of 9-18 Gy are irradiated in a segmented mode to induce tumor cells to produce exosomes; S2, the exosomes are loaded into dendritic cells to obtain the dendritic cell vaccine.

2. The preparation method according to claim 1, characterized in that: In step S1, the number of divisions is 2 to 6.

3. The preparation method according to claim 1, characterized in that: In step S1, the tumor includes breast cancer, colorectal cancer, lung cancer, liver cancer, stomach cancer, and esophageal cancer; and / or In step S2, the dendritic cells are derived from bone marrow.

4. The preparation method according to any one of claims 1-3, characterized in that: In step S1, tumor cells are irradiated using a fractionation mode, with each irradiation dose being 6 Gy, and the number of irradiations being 3.

5. A dendritic cell vaccine, characterized in that: The dendritic cell vaccine is prepared according to any one of claims 1-4.

6. The dendritic cell vaccine according to claim 5, characterized in that: The dendritic cell vaccine is in the form of a cell suspension.

7. The dendritic cell vaccine according to claim 5 or 6, characterized in that: The dendritic cell vaccine can be administered via one or more of the following routes: subcutaneous, intramuscular, intradermal, or intravenous. Preferably, the single dose of the dendritic cell vaccine is 4-6 × 10⁻⁶. 6 cell.

8. A pharmaceutical composition for preventing and treating EGFR-sensitive gene-mutant lung adenocarcinoma, characterized in that: The pharmaceutical composition comprises the dendritic cell vaccine according to any one of claims 5-7.

9. The pharmaceutical composition according to claim 8, characterized in that: The pharmaceutical composition also includes a targeted drug against EGFR; Preferably, the targeted drug includes gefitinib, erlotinib, icotinib, afatinib, osimertinib, or dabrafenib.

10. The use of the dendritic cell vaccine of any one of claims 5-7 or the pharmaceutical composition of any one of claims 8-9 in the preparation of a product for the prevention and treatment of EGFR-sensitive gene-mutant lung adenocarcinoma.

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