针对HER2阳性肿瘤的靶向治疗，本课题组十年前提出了靶向促凋亡策略，所构建的靶向促凋亡分子由抗HER2单链抗体、内吞体膜转位结构域、凋亡效应分子三部分组成，证实其具有特异的抗肿瘤作用。然而，内吞体膜转位是限速步骤，包括Furin酶切割、凋亡效应分子释放入胞浆两个环节。对第一环节，我们筛选获得了最短、最有效的Furin酶切割序列Fdt；对第二环节，体外预实验显示流感病毒膜融合序列HA2可明显促进荧光素标记分子从内吞体释放。据此，本项目拟构建第二代HER2靶向促凋亡分子，解决内吞体膜转位的瓶颈问题。先对HA2功能结构域进行系列截短和突变分析，筛选内吞体释放效率高的最短HA2肽段，并与Fdt联用，构建二代分子e23sFv-Fdt-HA2-tBid，从细胞和动物水平观察其内吞体膜转位效率、抗肿瘤活性是否提高，验证以改善内吞体膜转位为突破的思路的有效性，进一步丰富靶向促凋亡策略的理论内涵和实用价值。

For the targeted therapy of HER2-positive tumors, we have well established a unique immunoproapoptotic strategy for ten years. A series of immunoproapoptotic molecules we generated consist of an anti-HER2 single-chain antibody, endosomal translocation domains and apoptotic effectors in turn, and their specific antitumor activities have been confirmed in vitro and in vivo. However, endosomal translocation is rate-limiting and involves two sequential steps of Furin cleavage of the immunoproapoptotic molecules and subsequent cytoplasmic release of the cleaved apoptotic effectors from endosomes. For the Furin cleavage step, we have screened to get the shortest and most efficient Furin-cleavable motif named as Fdt. For the cytoplasmic release step, our preliminary in vitro data showed incubation of the HA2 sequence of influenza Hemagglutinin led to endosomal disruption and accumulated cytosolic distribution of the indicator labeled with FITC. Based on the above progress, this project aims to construct the second generation of the HER2-targeted immunoproapoptotic molecules in order to overcome low endosomal translocation efficiency. We plan to design several truncated and mutated HA2 peptides according to its structure-function relationship and to screen out the shortest and most efficient endosome-disruptable motif. Both Fdt and the optimized HA2 motif would be used to construct the second-generation immunoproapoptotic molecule designated as e23sFv-Fdt-HA2-tBid. Cell-based assays and tumor-bearing mouse model would be applied to evaluate the putatively greater potential of e23sFv-Fdt-HA2-tBid in endosome translocation capability as well as antitumor effect. This project would testify the feasibility of improving immunoproapoptotic molecules with the limited endosome translocation as a major concern, which can enrich the tumor-targeted immunoproapoptotic strategy theoretically and practically.

本课题组多年前建立了具有自主知识产权的靶向促凋亡策略，由识别HER2的单链抗体、内吞体膜转位结构域、凋亡效应分子三部分组成靶向促凋亡分子，证实其在体内外对HER2阳性肿瘤具有特异的抗肿瘤活性。但这一策略的瓶颈问题是内吞体膜转位效率低。针对此问题，本项目旨在构建第二代HER2靶向促凋亡分子，从Furin酶切、效应分子释放入胞浆两个环节提高膜转位效率。首先，将前期筛选得到的最短、最有效的Furin酶切割序列Fdt引入第二代靶向促凋亡分子。同时，依据流感病毒膜融合序列HA2的功能结构域进行一系列截短分析，筛选内吞体释放效率高的最短HA2肽段，构建入二代分子e23sFv-Fdt-HA2-tBid。然后，通过原核和真核两套表达系统，纯化获得具有功能活性的e23sFv-Fdt-HA2-tBid融合蛋白，从细胞和动物水平证实其内吞体膜转位效率提高，抗肿瘤活性增强。上述结果已完成预定计划的研究内容，尤其在表达纯化技术路线上超额完成任务，除完成原计划的原核表达外，还摸索并完成了真核表达，为后期功能实验的优化提供了坚实的技术保障。前期相关的阶段性研究结果已在Cancer Lett和Mol Med Rep发表2篇英文论文，此外还发表4篇中文核心期刊论文，主体研究结果正在整理投稿1篇英文论文。