抗肝癌单链抗体融合TNFα导向作用的初步实验研究

目的 获得有效果的抗肝癌基因工程单链免疫细胞因子（TNFα）。方法 将抗肝癌单克隆抗体（mAb)HAb25的单链抗体（scFv25)与人TNFα基因偶联，构建抗肝癌免疫细胞因子（scFv25-TNFα，亚克隆人原核GST融合表达载体pGEX4T-1中，在大杆菌中进行表达。对肝癌细胞SMMC-7721涂片进行间接免疫荧光染色，测定纯化后目的蛋白的活性，通过对荷肝癌（SMMC-7721）裸鼠进行的初步抑瘤实验，检测scFv25-TNFα的导向治疗效果。结果scFv25-TNFα具有与亲本抗体HAb25类似的抗原结合特异性。scFv25-TNFα对荷肝癌裸鼠体内直径2mm左右的肿瘤具有明确的导向杀伤作用，达到3/3完全缓解，明显强于TNFα对照组。该组有交待得只有2/3，且无完全缓解。结论 scFv250-TNFα为具有一定效果的抗肝癌双功能抗体。

A priliminary study ontargeting effect of scFv25 fusing to TNF

Aim To obtain genetically engineered cytokine with potentialities of clinical application. Methods Anti-human hepatocellular carcinoma (HCC) single chain gene fragment(scFv25) was linked with human TNFα gene to form a fusion gene of anti--HCC cytokine， then it was subcloned into prokaryotic GST fusion expression vector pGEX 4T-1 and expressed in the host E.coli. Indirect immunofluorescent staining was performed on HCC cell smearing slides in order to evaluate the activity of purified aim protein, then initial tumor regression trial was carried out in nude mice bearing HCC to evaluate the targeting therapeutic value of scFv25-TNFα . Results scFv25-TNFα had similar specificity as parental antibody HAb25 for SMMC-7721 antigen. The tumor regression trial to the 2 mm HCC xenografts in nude mice showed that scFv25-TNFα had certain targeting cytotoxicity capacity and the efficiency was 3/3 with complete remission. The cytotoxicity of scFv25-TNFα was better than that of TNFα , whose efficiency was only 2/3 and no complete remission was seen. Conclusion scFv25-TNFα is the anti-HCC cytokine, which have certain potentialities of clinical application.