抗肝癌人源化单链抗体融合肿瘤坏死因子a的导向作用

目的 获得有临床应用潜力的人源化抗肝癌基因工程双功能抗体。方法 将鼠源性抗肝癌单克隆抗体HAb25的人源化单链抗体（hscFv25）基因与人TNF（基因偶连构建抗肝癌双功能抗体（hscFv25-TNF a），亚克隆入原核GST融合表达载体pGEX 4T-1中，在大肠杆菌中进行表达并纯化目的蛋白。对肝癌SMMC-7721细胞涂片进行间接免疫荧光染色测定纯化后的基因重组双功能抗体蛋白活性。MTT实验测定hscFv25-TNF a对靶细胞SMMC-7721杀伤活性。荷肝癌（SMMC-7721）裸鼠体内的初步抑瘤实验检测hscFv25-TNF a的导向治疗效果。结果 hscFv25-TNF a具有与亲本抗体HAb25相同的抗原结合特异性。1h预处理的MTT实验显示，hscFv25-TNF a对靶细胞SMMC-7721具有明确的杀伤作用，其IC50值为7.1mg/L。且该杀伤作用可被亲本抗体HAb25所封闭，表明hscFv25-TNF a对靶细胞的杀伤作用是抗体hscFv25介导的选择性杀伤作用。hscFv25-TNF a对荷肝癌裸鼠体内直径3.0mm左右的肿瘤具有明确的导向杀伤作用，有效率达到3/3，1/3完全缓解，强于TNF a对照组，该组有效者只有2/3，同时无完全缓解。结论 hscFv25-TNF a是具有一定临床应用潜力的抗肝癌双功能抗体。

Targeting studies of humanized scFv25 fusing to TNF

OBJECTIVE: To obtain humanized engineering bifunctional antibody, which has potentialities for clinical application. METHODS: Humanized anti-human hepatocellular carcinoma (HCC) single chain fragment (hscFv25) was linked with human TNF-alpha gene to form anti-HCC bifunctional antibody, 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 the purified aim protein, then MTT trial to evaluate the cytotoxicity of hscFv25-TNFalpha to SMMC-7721, finally primary tumor regression trial in nude mice bearing HCC to evaluate the targeting therapeutic value of hscFv25-TNFalpha. RESULTS: The hscFv25-TNFalpha had the similar specificity to parental antibody HAb25 for SMMC-7721 antigen. One hour predisposed MTT trial in control with parental antibody HAb25 affirmed that hscFv25-TNFalpha was cytotoxic to targeted cell SMMC-7721 with the IC(50) to be 7.1 microg/ml. The cytotoxicity can be inhibited by parental antibody HAb25. This indicated that the cytotoxicity of hscFv25-TNFalpha to targeted cell is antibody-mediated selective cytotoxicity. The tumor regression trial to the 3mm HCC xenografts in nude mice showed that hscFv25-TNFalpha had assured targeting cytotoxicity and the efficiency was nearly up to 3/3 (1/3 complete remission, 2/3 partial remission). The cytotoxicity of the hscFv25-TNFalpha was better than that of TNFalpha, whose efficiency was only 2/3 and without complete remission. CONCLUSION: hscFv25-TNFalpha is an anti-HCC bi-functional antibody which has potentialities for clinical application.