分级响应的PD-L1抗体/siRNA外泌体组合物介导肿瘤多机制联合治疗

The key scientific issue for siRNA drug development is how to achieve safe and efficient siRNA delivery in vivo. Exosome is one of novel drug delivery system that has been proved to successfully deliver multiple types of therapeutics, including siRNA, miRNA, protein and small molecules. Block of PD-1 / PD-L1 pathway is a powerful and amazing tumor immunotherapy strategy. Dlin-MC3-DMA, a kind of ionizable lipid, is a quasi FDA-approved nucleic acid pharmaceutical excipients. This project aims to develop a proteinase/acid successive-responsive immune siRNA exosome (pariExosome) for combined treatment of cancer.. In this project, Dlin-MC3-DMA and anti-PLK1 siRNA will be first loaded into M1 macrophage-derived exosomes by electroporation. Furthermore, a oligopeptide linker that can be cleaved by matrix metalloproteinase 2, a proteinase highly-expressed in tumor tissue and involved in tumor progression, was designed and utilized to modify anti-PD-L1 antibody on the surface of the exosomes, by click chemistry. The prepared nanosystem is termed pariExosome. pariExosome possesses versatile properties, including active tumor targeting, tumor immunotherapy, proteinase-responsive antibody dissociation, acid-responsive siRNA release from endosome/lysosome, etc. A series of studies will performed to evaluate the anti-tumor effect of pariExosome by using two colon cancer models established with CT26 and MC38 cell lines. In a word, this work will explore a novel synergistic cancer treatment strategy that employs tumor-targeted siRNA delivery and immunotherapy, which may provide new theories, new techniques and new solutions for a wider range of cancer treatments.

siRNA体内递送是其制药应用的核心科学问题，外泌体是一种新型药物传输系统，拮抗PD-1/PD-L1是代表性的肿瘤免疫治疗策略，MC3是FDA即将批准的酸响应性核酸药物辅料。本项目拟研究PD-L1抗体可逆修饰的酶/酸分级响应性siRNA外泌体（pariExosome）多途径联合治疗肿瘤的机制与效应。. 首先通过电穿孔将MC3、抗PLK1 siRNA装载到M1型巨噬细胞来源的外泌体；设计利用肿瘤高表达的基质金属蛋白酶2可降解的多肽连接子，通过点击化学技术将PD-L1抗体修饰在外泌体表面得到pariExosome，其将具备肿瘤靶向性、肿瘤免疫治疗、酶响应抗体解离、酸响应siRNA释放与长效抑制等多重特征。通过在CT26/MC38结肠癌模型上开展分子、细胞、动物各层次研究，阐明该策略的可行性与作用机制，以建立全新的抗体-siRNA组合物研究平台，为更广泛的疾病治疗提供新理论、新技术、新方案。

嵌合抗原受体（Chimeric Antigen Receptor, CAR）-T细胞疗法虽然在血液系统恶性肿瘤中显示出良好的治疗效果与应用前景，但在实体瘤中的应用仍面临严峻的挑战。本项目中，承担团队构建了一个类似CAR-M的RNAisome （Chimeric Antigen Receptor macrophages like RNAisome，CAR-MiR ）药物递送平台。 CAR-MiR药物递送平台中的关键原件是由 M1型巨噬细胞分泌的EV M1 EV 与 siPLK1形成的RNAisome。为了提高肿瘤相关的抗原特异性，在CAR-MiR引入 PD-L1抗体从而研究免疫检查点阻断的抗肿瘤活性。MMP-2底物肽和光敏剂Chlorine6（Ce6）被引入研究对CAR-MiR递送体系时空可控性与 siRNA逃逸效率的影响。研究结果证实了M1 EV的趋炎性和 aPD-L1抗体的靶向性，在肿瘤部位富集的过程中，肿瘤微环境中的 MMP2酶促进 aPD-L1从CAR-MiR中脱落，阻断PD-1/PD-L1通路，并激活 CD8+T细胞群；CAR-MiR经过激光的照射会产生大量的 ROS，ROS与M1 EV会促进M2巨噬细胞向M1巨噬细胞复极化；ROS会促进 siRNA的胞内释放 ，从而导致肿瘤细胞内PLK1基因表达的抑制。. 整体上，该项目的实施，证实了CAR-MiR可以明显抑制肿瘤的生长，延长小鼠的生存期，为 siRNA的递送研究提供了一种创新的策略，将PD-L1 抗体免疫治疗与siRNA 药物的靶点广谱性结合起来，建立新的抗体-siRNA 联用、以及肿瘤治疗研究平台，为更广泛的疾病治疗提供新理论、新技术、新方案。

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