抗肝癌单链抗体融合TNFα导向作用的初步实验研究

A priliminary study on targeting effect of scFv_(25) fusing to TNFα against hepatocellular carcinoma

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ｐＧＥＸ ４Ｔ－１中，在大肠杆菌中进行表达。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，该组有效者只有２／３，且无完全缓解。结论ｓｃＦｖ２５－ＴＮＦα为具有一定效果的抗肝癌双功能抗体。

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.