智能磁性/荧光纳米探针高灵敏检测及同步无损分离循环PD-L1外泌体

Anti-PD-1 antibodies have shown remarkable promise in treating tumors by overcoming PD-L1/PD-1-mediated tumor immune escape. However, the patient response rate is less than 50%. Therefore, predictive biomarkers of the clinical response are urgently needed for precision immunotherapy. Our previous study has revealed that the level of circulating PD-L1 exosomes in peripheral blood positively correlates with the patient response to anti-PD-1 therapy, thus providing a rationale for the application of circulating PD-L1 exosome as a predictor for immunotherapy. The present project is designed to further realize: 1) the highly sensitive quantitative detection and simultaneous non-destructive isolation of circulating PD-L1 exosomes based on intelligent magnetic and fluorescent nanobioprobes, which are constructed with aptamer, magnetic nanospheres and quantum-dot nanospheres to achieve the combination of capture, detection and release; 2) the development of a sensitive liquid biopsy strategy based on the level of circulating PD-L1 exosomes to predict the patient response to anti-PD-1 therapy; 3) the investigation of individual differences of circulating PD-L1 exosomes in terms of their molecular composition, biological functions, and correlation with the patient response to anti-PD-1 therapy, in order to further improve the accuracy of circulating PD-L1 exosome as a predictive biomarker. Our research would facilitate the application of circulating PD-L1 exosome as a predictor for precision immunotherapy.

基于PD-1单抗的免疫治疗可通过逆转肿瘤免疫逃逸实现治疗甚至治愈肿瘤的目的，但该疗法仅对不到50%的患者有效。因此，寻找能够预测免疫治疗有效性的生物标志物对于实现精准免疫治疗至关重要。我们近期研究表明肿瘤患者外周血中循环PD-L1外泌体的浓度水平与免疫治疗反应性密切相关，可用于早期预测免疫治疗的有效性。本项目拟在上述工作基础上，以核酸适配体为识别元件，以磁性纳米球为分离载体，结合量子点纳米球的信号放大效应，构建集“捕获-检测-释放”为一体的智能磁性/荧光纳米探针，用于高灵敏检测及同步无损分离肿瘤患者外周血中循环PD-L1外泌体，以发展通过定量检测其浓度水平来预测免疫治疗有效性的液体活检策略，同时探究循环PD-L1外泌体的分子组成和功能在不同患者个体中存在的差异性及其与免疫治疗有效性的关联，以望进一步提高其用于免疫治疗有效性预测的精准度。本项目研究对于推动肿瘤精准免疫治疗的发展具有重要意义。

近年来，外泌体作为疾病早期诊断及预后评估的潜在生物标志物受到了广泛关注，如PD-L1外泌体的浓度水平与免疫治疗反应性密切相关，可用于早期预测免疫治疗的有效性。然而，难以同时高效分离并定量检测这些纳米级的外泌体是当前外泌体领域转化应用的技术瓶颈之一。本项目主要围绕磁性-荧光纳米标记技术展开，并将其应用于外泌体的快速高灵敏定量检测及同步无损分离中。我们将适配体高效偶联在磁性微球表面，借助适配体的“栓系”作用，实现DNA多联体在磁性微球表面的有序自组装，利用链霉亲和素与生物素作用，实现量子点在DNA多联体上的大量负载，构建集“捕获-检测-释放”为一体的智能磁性/荧光纳米探针。该智能探针能够在捕获外泌体的同时有效释放作为检测信号载体的DNA多联体，一步法实现了外泌体的快速高灵敏定量检测。同时，我们利用适配体的高效捕获及其互补链的竞争结合，巧妙地发展了一种同步无损分离PD-L1外泌体的新策略，完成了PD-L1外泌体携带生物信息表达谱研究及其关键致病分子的筛选，揭示了PD-L1外泌体更倾向于钙粘蛋白介导的细胞粘附以靶向抑制免疫细胞，而PD-L1阴性亚群更容易粘附富含胶原和纤维蛋白的细胞外基质。综上所述，本项目中所建立的一步法高灵敏定量检测外泌体的新方法，以及特定外泌体亚群的无损分离新策略，均具有较好的普适性，对推动外泌体作为液体活检标志物的应用具有重要意义，为揭示PD-L1外泌体在肿瘤发生发展中的作用机制及推动肿瘤精准免疫治疗的发展提供了重要思路。