基于红细胞膜包被聚合物纳米粒的“功能鸡尾酒”癌症免疫治疗研究

Cancer immunotherapy is mainly focused on manipulating patient's own immune system to recognize and destroy cancer cells. The most widely used immunotherapeutic approach uses the ex vivo DC-based cancer vaccines, which remains insufficient in clinical patients (limited to <7% only). Our previous research mainly focused on single peptide and toll like receptor 4 agonist-MPLA co-delivery nanoparticles, however, the protective immunity developed in a mouse model can't completely result in tumor rejection. This may be attributed to that tumor cells could down regulate the antigen expression at their surface that makes them invisible to CTLs. In this project, we will co-deliver multi-MHC I/II peptide and MPLA in the same particles through a novel erythrocyte membrane-enveloped redox-sensitive polymeric nanoparticles combined with IFNγ to develop peptide-cocktail effect for making tumor cells more difficult to escape from the surveillance of the immune system and up-regulate the antigen expressions after treatment with IFNγ. The nanoparticles will be applied in this functional cocktail delivery system, which based on that erythrocyte can recognize and present antigens by itself or dendritic cells (DCs), promote T cell response and target DCs naturally and in situ. With assistance of targeting antibody and IFNγ, the active targeting delivery of cocktail to DCs would be further enhanced. This cancer vaccine system will provide a novel antigen delivery system and a mechanism of inhibiting tumor immune escape.

癌症免疫疗法可激活患者自身免疫系统，识别和杀灭肿瘤细胞，避免对正常细胞的损害，抑制肿瘤转移，预防复发。现广泛采用的抗原冲击致敏树突状细胞（DC）的方法，处理工序复杂，临床效应低。本研究前期发现载有单一抗原多肽和佐剂MPLA的聚合物纳米粒，可诱导T细胞免疫反应，一定程度上抑制肿瘤生长，但难以实现肿瘤的彻底清除。这可能与肿瘤可通过各种机制诱导抗原调变、肿瘤免疫微环境等有关。本项目以黑色素瘤为模型，利用红细胞对DC的靶向性、可激活DC识别和呈递抗原、促进T细胞活化等特性，构建抗体偶联的红细胞膜包被还原敏感聚合物纳米粒，用于装载不同表型的MHC I类抗原表位肽、T细胞辅助肽和佐剂，实现混合多肽和佐剂的高效共包封和呈递，发挥协同抗肿瘤和免疫增强作用，并结合IFNγ抑制肿瘤抗原调变而发展"功能鸡尾酒"纳米免疫疗法。相关研究将为感染和癌症类疾病构建安全、个性化的免疫治疗提供理论基础和实验依据。

肿瘤特异性多肽抗原是机体免疫系统识别和攻击的靶点，但是单个多肽只有一个抗原表位，免疫应答谱系窄，易导致免疫逃逸。使用混合多肽可增加抗原表位数量，拓宽应答谱系，有效克服肿瘤的免疫逃逸，实现肿瘤高效治疗。然而，不同抗原多肽亲、疏水性差异较大，如何实现其高效共装载，并增强树突状细胞DC主动摄取抗原是目前急需解决的一个重要科学问题。本项目设计了一种混合多肽（hgp100、p15E及TRP2）与免疫佐剂MPLA共载的红细胞膜包被聚合物纳米粒，以增强抗肿瘤免疫应答。首先，利用二硫键偶联以实现亲水性多肽的包封和细胞内快速释放，结合红细胞促进抗原提呈和T细胞活化的能力，并通过引入DC靶向基团和加入佐剂增强机体的免疫应答。构建的多肽纳米疫苗显示了较好的贮库效应，可聚集在淋巴结，诱导DC熟化和增加细胞因子分泌；此纳米疫苗具有良好的黑色素瘤的预防、治疗及转移抑制效果。其次，通过不同多肽聚合物混合制备的混合多肽疫苗均可达到多肽等比例混合共载的要求，预防实验与治疗实验结果类似，且hgp100与TRP2联用抗肿瘤效果最好，而三种多肽联用结果超出预想，比两种多肽联用的某些组合效果差。最后，进一步设计了磷脂包被杂化纳米载体以促进不同亲疏水性多肽在同一纳米粒中的共装载、高包封及增强抗肿瘤应答，以实现多种多肽和免疫佐剂MPLA的共输送，HGP100、P15E及磷酸化TRP2多肽的包封率分别达到77.7±2.5%、66.2±1.4%和61.7±1.5%；可刺激DC的熟化及细胞因子的分泌；激活机体自身的免疫系统来产生特异性的抗肿瘤免疫应答；与单一多肽纳米粒相比，混合多肽纳米粒能显著提高纳米疫苗识别并杀伤黑色素瘤细胞，抑制肿瘤的生长和转移。相关研究为探索结合“多肽功能鸡尾酒疗法”与药物递送载体构筑靶向DC细胞的肿瘤疫苗提供了良好的理论依据与实践基础，研究成果已相继在国际学术期刊ACS Nano、Journal of Controlled Release、Small、Theranostics等发表该基金资助文章18篇，其中直接相关文章4篇，多次参加国际国内学术会议做口头报告。

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