Application of Taraxasterol in Specific Enhancement of DC Vaccine

Abstract

The application discloses application of taraxasterol in targeted enhancement of DC tumor vaccines and belongs to the technical field of cellular immunity. The application provides application of taraxasterol (Tara) in preparation of a reagent for enhancing the targeting property and safety of a tumor vaccine based on dendritic cells (DCs), and the Tara-assisted DC tumor vaccine can improve the intervention and treatment effect of the DC tumor vaccine, improve the inflammatory reaction to the organism, improve the targeting property and safety of the DC tumor vaccine treatment, and provides a scientific and valuable implementation scheme for a clinical test stage of the DC tumor vaccine anti-tumor treatment, and has obvious application prospect.

Description

Technical Field

[0001] This invention belongs to the field of cellular immunotherapy, specifically relating to the application of taraxasterol in targeted enhancement of DC tumor vaccines. Background Technology

[0002] Dendritic cell (DC)-based biotherapy has attracted considerable attention. Theoretically, DCs are the most effective APCs for stimulating adaptive immunity. By loading DCs with tumor antigens in vitro and then reinfusing them, peptide-loaded DCs activate specific T cells through the interaction of MHC / antigen peptide-TCR, thereby initiating an anti-tumor immune response to prevent or eliminate tumors. This method is safe with few side effects and offers unparalleled targeting for small and metastatic lesions compared to traditional methods. However, in practice, peptide-loaded DCs alone often fail to elicit a sufficiently strong anti-tumor immune response, resulting in treatment outcomes that are less than ideal.

[0003] Lung cancer is a malignant tumor that seriously threatens human health, ranking among the top three in both incidence and mortality among malignant tumors. Its pathogenesis is complex, and traditional radiotherapy and chemotherapy have unsatisfactory clinical efficacy, are prone to recurrence, and have low survival rates in advanced stages. There is an urgent need for research and development of new treatment options and strategies. Recent studies have shown that immunotherapy is a new and highly promising approach to cancer treatment, and when combined with chemotherapy drugs, it can significantly improve the tumor remission rate and 5-year survival rate. Extensive data show that DC-based lung cancer vaccines are currently a hot research topic and represent a novel and highly promising treatment method following surgery and radiotherapy / chemotherapy. However, a drawback is that lung cancer antigens are weak and have poor specificity, leading to poor efficacy of DC-based vaccines. Furthermore, while current methods for enhancing DC-based vaccines can improve their effectiveness, they can also cause severe inflammatory damage to the body, resulting in poor anti-tumor effects induced by the DC-based vaccines. Summary of the Invention

[0004] The purpose of this invention is to provide the application of taraxasterol in the targeted enhancement of DC tumor vaccines. By utilizing taraxasterol (Tara) to assist DC tumor vaccines in enhancing their anti-tumor effects, the killing effect of DC tumor vaccines on lung cancer cells can be significantly improved.

[0005] This invention provides the application of taraxasterol in the preparation of reagents that enhance the targeting and safety of dendritic cell-based tumor seedlings.

[0006] Preferably, the dendritic cells include umbilical cord blood, peripheral blood, and myeloid-derived dendritic cells.

[0007] Preferably, the tumors targeted by the dendritic cell-based tumor vaccine include lung cancer, melanoma, and pancreatic cancer.

[0008] The present invention also provides a highly targeted and safe antitumor drug, including taraxasterol and dendritic cell-based tumor vaccines.

[0009] Preferably, the method for preparing the tumor vaccine based on dendritic cells includes sensitizing dendritic cells with tumor antigens to obtain the tumor vaccine.

[0010] Preferably, when the antitumor drug is for non-small cell lung cancer, the method for preparing the non-small cell lung cancer vaccine based on dendritic cells includes using tumor cell lysate of non-small cell lung cancer as an antigen, co-incubating the antigen with myeloid-derived dendritic cells, and preparing the non-small cell lung cancer vaccine.

[0011] Preferably, the dosage of taraxasterol in the antitumor drug is 5-60 mg / kg, based on the dosage for mice.

[0012] Beneficial effects: This invention provides the application of taraxasterol (Tara) in the preparation of reagents that enhance the targeting and safety of dendritic cell-based tumor vaccines. By using Tara to assist DC vaccines, the therapeutic effect of DC vaccines can be improved, while also reducing the inflammatory response in the body, thus enhancing the targeting and safety of DC vaccine treatment. This provides a scientifically valuable implementation plan for the clinical trial stage of DC vaccine anti-tumor therapy and has significant application prospects. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the in vitro DC tumor seed induction process in this invention;

[0014] Figure 2 Figure showing the results of Tara-assisted DC tumor vaccine in vivo effectively enhancing the anti-non-small cell lung cancer effect;

[0015] Figure 3 This is a graph showing the safety test results of Tara in vivo;

[0016] Figure 4 The figure shows the safety assessment results of the in vivo Tara-assisted DC tumor vaccine antitumor effect;

[0017] Figure 5 The results of Tara-assisted DC tumor vaccine improving anti-tumor effects on inflammatory damage in the body are shown in the figure. Detailed Implementation

[0018] This invention provides the application of taraxasterol in the preparation of reagents that enhance the targeting and safety of dendritic cell-based tumor seedlings.

[0019] The taraxasterol described in this invention is a taraxer extract, which can be extracted in-house or purchased, requiring only a purity of 95% or higher. The dendritic cells (DCs) described in this invention preferably include those derived from myeloid, umbilical cord blood, and peripheral blood; the tumors targeted by the dendritic cell-based tumor vaccine preferably include lung cancer, melanoma, and pancreatic cancer. This invention uses a non-small cell lung cancer tumor vaccine based on myeloid dendritic cells as an example to illustrate that using Tara-assisted DC tumor vaccines can enhance the therapeutic effect of DC tumor vaccines, while simultaneously improving the body's inflammatory response and enhancing the targeting and safety of DC tumor vaccine treatment.

[0020] In this invention, Tara can enhance the anti-tumor effect mechanism of DC tumor vaccines. Tara targets PIK3R1 and AKT2 molecules in tumor cells, affecting the PI3K / AKT / mTOR / ECM pathway, reducing the production of type I collagen, and causing tumor cells to resist CD8+. + It is associated with increased cytotoxic sensitivity of T cells (CTLs).

[0021] The present invention also provides a highly targeted and safe antitumor drug, including taraxasterol and dendritic cell-based tumor vaccines.

[0022] The method for preparing a tumor vaccine based on dendritic cells according to the present invention preferably includes sensitizing dendritic cells with a tumor antigen component to obtain the tumor vaccine. For example, in the method for preparing a non-small cell lung cancer vaccine based on dendritic cells in an embodiment of the present invention, it preferably includes using tumor cell lysate of non-small cell lung cancer as an antigen, co-incubating the antigen with myeloid-derived dendritic cells to prepare the non-small cell lung cancer vaccine. Based on mouse dosage, the amount of taraxasterol in the antitumor drug of the present invention is preferably 5-60 mg / kg, more preferably 10 mg / kg.

[0023] To further illustrate the present invention, the application of taraxasterol provided by the present invention in targeted enhancement of DC tumor vaccines is described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] according to Figure 1The procedure described was as follows: following the article (Esmaily M, Masjedi A, Hallaj S, NabiAfjadi M, Malakotikhah F, Ghani S, Ahmadi A, Sojoodi M, Hassannia H, Atyabi F, Namdar A, Azizi G, Ghalamfarsa G, Jadidi-Niaragh F. Blockade of CTLA-4 increases anti-tumor response inducing potential of dendritic cell vaccine. J Control Release. 2020 Oct10; 326:63-74. doi:10.1016 / j.jconrel.2020.06.017.Epub 2020Jun21.PMID:32580042.), mature myeloid-derived dendritic cells (BMDCs) were cultured and induced in vitro. Mouse-derived lung cancer line LLC cells were subjected to repeated freeze-thaw cycles 5 times, following the procedure described in the article (Mahaweni). NM, Kaijen-Lambers ME, Dekkers J, Aerts JG, Hegmans JP. Tumor-derived exosomes as antigen delivery carriers indendritic cell-based immunotherapy for malignant mesothelioma. J Extracell Vesicles. 2013 Oct 24; 2. doi:10.3402 / jev.v2i0.22492.PMID:24223258; PMCID:PMC3823268.) The method disclosed in this study was used to prepare tumor antigens. These antigens were then co-incubated with dendritic cell-derived exosomes (BMDCs) to prepare a dendritic cell anti-LLC tumor vaccine (DCs-Vac). The results showed that DCs-Vac had a stronger antigen presentation capacity than BMDCs. Figure 1 ).

[0026] By using lung cancer cell line LLC cells (8×10 5 A lung cancer xenograft animal model was established in C57BL / 6WT mice via subcutaneous injection. After successful tumor bearing (7 days), mice were randomly divided into four groups: PBS group, Tara monotherapy group, DCs-Vac monotherapy group, and Tara and DCs-Vac combined therapy group. In the Tara group and the combined therapy group, mice were intraperitoneally injected with 10 mg / kg Tara every 3 days for a total of 6 times. In the combined therapy group, 12-24 hours after Tara injection, 1×10⁻⁶ mg / kg Tara was adopted via tail vein transfer.6 DCs-Vac (3 times, once every 7 days) were used to observe tumor growth. Mice were sacrificed on day 18 after the start of Tara injection. According to the article (Lei L#, Chen C#, Zhao J, Wang H, Guo M, Zhou Y, Luo J, Zhang J, Xu L*. Targeted expression ofmiR-7operatedby TTF-1promoter inhibited the growth of human lungcancer through NDUFA4 pathway. Mol ther-nucl acids, 2017, 6: 183-197.; Chang X, Zhao J, ZhouY, Guo M, YanY, WangY, Zhao X, Yang J, Chen C, Tang L, Qin M, Xu L*.MiR-7deficiency promotes Th1 polarization of CD4+T cells and enhances the antitumor effect in adoptive cell therapy for lung cancer. Immunol Res.2023Sep27.doi:10.1007 / s12026-023-09423-y.; Zhao J,Guo M, Yan Y, Wang Y, Zhao X, Yang J, Chen J, Chen C, Tang L, Zeng W, Liu Y, Qin M, Zhou Y, Xu L*. The miR-7 / EGFR axis controls the epithelial cell immunomodulation and regeneration and orchestrates the pathology in inflammatory bowel disease. J Adv Res. 2023 Apr23:S2090-1232(23)00117-0.) The method disclosed in this study observed changes in tumor weight, pathological structure, and lung tissue metastasis in model mice, and evaluated the anti-tumor effect of Tara-assisted DCs-Vac; after Tara treatment, changes in tissue weight index, pathological structure, biochemical indicators, and serum inflammatory cytokines in important organs such as the liver, lungs, and kidneys of mice were detected, and the safety effect of Tara intervention on the body was evaluated.

[0027] The results are as follows Figure 2As shown, compared with the PBS group, both Tara and DCs-Vac single intervention groups effectively inhibited tumor growth, but the combined intervention group (D+T) mice showed the slowest tumor growth. Figure 2 The tumor in the middle B group was the smallest in size and weight (only 1 / 10 of the tumor volume and weight in the PBS group). Figure 2 (C). H&E staining showed that, compared with the PBS group, the nucleoplasm of tumor cells in each intervention group was more porous and fragmented, with varying degrees of hemorrhage, necrosis, and fibrosis in the tumor area; however, the D+T group showed the most obvious nuclear fragmentation and the largest area of ​​necrosis. Figure 2 (D); and the D+T group had at least 10% lung metastases. Figure 2 The intervention showed the best effect in the middle (E). The above data prove that Tara combined with DC tumor vaccine can effectively inhibit the growth of NSCLC tumors in mice.

[0028] The results are as follows Figure 3 As shown in A, Tara executes as follows Figure 2 The tumor intervention method was consistent with that used in the Tara intervention group. Results showed that, compared with the normal group (Ctrl), there were no significant changes in the weight indices of organs such as the heart, liver, kidneys, brain, and lungs in the Tara intervention group. Figure 3 In the middle B and C), serum ALP, ALT, AST, LDH, HDL, TP, and TC levels showed no significant changes, while LDL and TG expression decreased. Figure 3 (D, E, and F). The above data demonstrate that Tara's in vivo intervention has good safety and a slight lipid-lowering function.

[0029] The results are as follows Figure 4 AD and H&E staining showed that, compared with the PBS group, the Tara single intervention group showed no significant changes in the histopathological changes of major organs such as the brain, heart, liver, and kidneys; however, the DC-Vac single intervention group showed varying degrees of inflammatory damage in the histopathological changes of major organs such as the brain, heart, liver, and kidneys; and compared with the DC-Vac single intervention group, the combined intervention group (D+T) showed significant improvement in the inflammatory damage of these tissues. Figure 4 (AD). Simultaneously, real-time quantitative PCR (... Figure 5 (B) and ELISA method ( Figure 5 The detection of C and D showed that, compared with the DC-Vac single intervention group, the expression levels of pro-inflammatory cytokines such as IFN-γ and TNF-α in the tumor tissue and serum of mice in the combined intervention group (D+T) were significantly reduced. Figure 5 (AD). The above data demonstrate that Tara combined with DC tumor vaccine can effectively inhibit the growth of NSCLC tumors in mice and significantly improve tissue inflammatory damage caused by DC tumor vaccine.

[0030] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of taraxasterol in the preparation of drugs that enhance the safety of dendritic cell-based tumor vaccines, characterized in that, The dendritic cells are myeloid-derived dendritic cells; The tumor targeted by the dendritic cell-based tumor vaccine is non-small cell lung cancer.

2. An antitumor drug, characterized in that, Including taraxasterol and dendritic cell-based tumor seedlings; The tumor targeted by the dendritic cell-based tumor vaccine is non-small cell lung cancer.

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