肝细胞癌人源抗c-Met抗体Fab的筛选及鉴定

Screening and identification of human anti-c-Met Fab from a phage antibody library

目的从大容量人源Fab抗体库中筛选全人源抗c-Met抗体，并对抗体与肝癌细胞的结合活性进行初步鉴定。方法利用Met-Fc融合蛋白对大容量人源Fab抗体库进行固相筛选，经过5轮固相筛选，随机挑选30个克隆经酶联免疫吸附法差减鉴定，阳性克隆进行可溶性表达；用c-Met表达阳性的人肝癌细胞株鉴定抗c-Met抗体Fab的结合活性。结果Western blot、免疫荧光结果显示，c-Met分子表达于SMMC721、BEL7402人肝癌细胞膜上；从大容量人源Fab抗体库中筛选出1株抗c-Met抗体Fab（AM2-26），经免疫共沉淀、荧光激活细胞分类术、免疫荧光分析，结果显示AM2-26与人肝癌细胞表面c-Met分子有较好的结合活性。结论从人源Fab抗体库中筛选的AM2-26能够与肝癌细胞表面c-Met分子特异性结合，为研制用于肝癌生物治疗的靶向药物，提供了候选分子。

Objective To screen anti-c-Met Fab from a phage antibody library and identify its binding activity. Methods The expression of c-Met of HCC lines was identified by Western blot and immunofluorescence. Antibodies against c-Met were screened with immobilized antigen. After five rounds of panning, 30 randomly selected clones were identified by phage ELISA to select specific clones with high affinity. The positive clones were selected for Fab soluble expression in TOP10F and the binding activities were analysed in HCC lines. Results c-Met expressed in HCC membrane was confirmed by Western blot and immunofluorescence. A Fab fragment named AM2-26 with fine activity to c-Met was selected. AM2-26 binding specificity was confirmed by IP, FACS and immunofluorescence. Conclusion The anti-c-Met Fab binding to c-Met in HCC provides a promising candidate for the biotherapy of hepatoma.