靶向促凋亡分子Immuno-caspase6的优化及对HER2阳性胃癌细胞杀伤作用的研究

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.

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。

靶向药物是肿瘤生物治疗的研究热点，我们前期研究构建一系列免疫促凋亡分子，并证实其在细胞内的转位途径，发现促进活性分子在细胞内的转位是提高其杀伤活性的有效手段。本课题在前期研究的基础上，对原有分子进行改建和优化，将流感病毒血凝素HA2引入靶向促凋亡分子，构建了新型促凋亡分子AFHC6。真核表达并纯化目的蛋白，将目的蛋白加到HER2阳性的胃癌细胞SGC7901培养上清，进行活性Caspase-6和早期内吞体的共定位，结果显示，融合HA2可以有效促进靶向促凋亡分子内化后的转位，缺少转位结构域和HA2的AC6大部分聚集在早期内吞体，效应分子无法转位到胞浆发挥作用。效应分子转位到胞浆后主要通过切割下游底物核膜蛋白Lamin A发挥作用，诱导肿瘤发生凋亡。将纯化后的目的蛋白加到HER2阳性肿瘤细胞的培养上清，MTT实验显示AFHC6较对照组可以有效抑制肿瘤细胞增殖，FCM显示其可以引起肿瘤细胞的凋亡，且目的蛋白对肿瘤细胞的促凋亡作用呈现剂量依赖性。建立荷瘤裸鼠模型，通过纯化蛋白尾静脉注射的方法对荷瘤裸鼠进行治疗，AFHC6较对照组可以抑制肿瘤增殖，延长裸鼠生存期。TUNEL实验显示AFHC6组的肿瘤组织内细胞发生了大量凋亡。而应用AFHC6治疗对裸鼠重要脏器没有明显影响。本研究进一步证实免疫促凋亡分子在细胞内的加工过程直接影响其对肿瘤细胞的杀伤活性，为HER2 阳性胃癌的免疫治疗提供新的候选分子和理论基础。