基于抗体技术的靶向药物是肿瘤生物治疗的研究热点。我们前期将抗HER2单链抗体、白喉毒素的弗林蛋白酶识别序列和重组Caspase6相融合构建了靶向促凋亡分子（e23sFv-Fdt-Caspase6），证实了其可以在体内外有效杀伤HER2阳性肿瘤细胞。然而该分子内化进入靶细胞后，活性Caspase6从内吞体转位至胞浆的效率不高，严重限制了其对靶细胞的杀伤。为提高重组蛋白转位效率，本研究拟对原有分子进行改建和优化，引入流感病毒血凝素HA2以破坏内吞体膜结构稳定性并促进Caspase6的转位，提高对靶细胞的杀伤。本研究将构建多种融合HA2的靶向促凋亡分子，经原核系统表达纯化后，检测其亲和活性和内化活性，比较HA2在不同位置的重组蛋白的转位效率和在体内外对HER2阳性胃癌细胞的促凋亡能力，筛选出杀伤活性最高的分子。本研究将为HER2阳性胃癌的免疫治疗提供新的候选分子，为肿瘤靶向治疗提供新的理论基础。

Targeted drug based on antibody of tumor biological treatment has been a hotspot of cancer research. We previously generated a targeted pro-apoptotic molecule (e23sFv-Fdt-caspase6) , which consists of the anti-HER2 single-chain antibody, Furin cleavage sequence of the diphtheria toxin and recombinant Caspase-6, and demonstrated it potent selective killing activities on HER2 positive tumor cells in vitro and in vivo. Following the receptor-mediated endocytosis in HER2 positive gastric cancer cells, the efficiency of active Caspase6 translocating from endosomes to cytoplasm is low, which limits its killing activities severely. To improve the translocation efficiency of the recombinant molecules, the original molecule e23sFv-Fdt-caspase6 will be subjected to reconstruction and optimization by introducing the influenza virus hemagglutinin HA2 into the molecule. The influenza virus hemagglutinin HA2 was confirmed to destruct membrane structural stability of endosomes, which could promote translocation of active Caspase6 to perform its cytotoxicity on HER2 positive tumor cells. We intend to generate a series of targeted pro-apoptotic molecules (novel Immuno-caspase6) fused HA2 at different sites. The novel Immuno-caspase6 proteins will be bacterially expressed and purified, and the affinity and internalization activity of target proteins are to be evaluated in tumor cells with different HER2 overexpression level. In addition, to screen the most active molecule, the translocation efficiency and specific pro-apoptotic activity of novel Immuno-caspase6 will be compared with the parental protein. These studies will provide new candidate molecules to immunotherapy of HER2 positive gastric cancer, and enrich the theoretical basis of targeted tumor therapy.