抗EGFRvⅢ/EGFR单抗9B9的制备和鉴定

Preparation and identification of a 9B9 monoclonal antibody specifically targeting EGFRvⅢ/EGFR

目的:制备抗EGFRvⅢ/EGFR单克隆抗体,并探讨其对人肝癌细胞株Huh7-EGFRvⅢ和表皮癌细胞系A431裸鼠移植瘤生长的抑制作用。方法:将稳转细胞株3T3-EGFRvⅢ免疫BALB/c小鼠,免疫小鼠脾细胞与小鼠骨髓瘤细胞SP2/0进行融合,ELISA法筛选出EGFRvⅢ阳性的克隆株并命名为9B9。Western blotting和免疫荧光鉴定9B9抗体与EGFRvⅢ/EGFR抗原结合的特性。建立裸鼠人肝细胞癌和表皮细胞癌移植瘤模型,分别腹腔注射PBS、西妥昔单抗和9B9抗体,比较它们对各移植瘤模型的抑瘤效果。结果:通过杂交瘤制备单克隆抗体技术筛选获得一株单抗9B9,Western blotting和免疫荧光结果显示其既能识别EGFRvⅢ又能识别EGFR。9B9抗体和西妥昔单抗对人肝癌细胞Huh7-EGFRvⅢ移植瘤的抑瘤率分别46%和42%,对人表皮细胞癌移植瘤的抑瘤率分别为86%和85%。结论:制备获得的单克隆抗体9B9可显著抑制裸鼠人肝细胞癌以及表皮细胞癌移植瘤的生长,具有与西妥昔单抗相似的抑瘤效果。

Objective：To prepare and identify monoclonal antibody specifically targeting epidermal growth factor receptor（EGFR） and（or） epidermal growth factor receptor vⅢ（EGFRvⅢ）,and to investigate its inhibitory effects on human hepatocellular carcinoma Huh7-EGFRvⅢ cell-and epidermal carcinoma A431 cell-implanted tumors in nude mice.Methods： BALB/c mice were immunized with 3T3 cells stably transfected with EGFRvⅢ（3T3-EGFRvⅢ）.Immunized spleen cells were fused with myeloma SP2/0 cells,and anti-EGFRvⅢ monoclonal antibody positive hybridoma cells（named 9B9 antibody and 9B9 cells,respectively） were selected and identified by ELISA.The specific interaction between 9B9 antibody and EGFRvⅢ/EGFR antigen was detected by Western blotting and immunofluorescence assay.Huh7-EGFRvⅢ cell-（human hepatocellular carcinoma Huh7 cells stably transfected with EGFRvⅢ） and epidermal cell carcinoma A431 cell-bearing mouse models were established and were divided into PBS group,Cetuximab group and 9B9 antibody group.Then,anti-tumor effect of 9B9 antibody was examined and compared with those of PBS and Cetuximab.Results： A monoclonal antibody,named 9B9 antibody,was obtained by hybridoma technique and it reacted with both EGFRvⅢ antigen and EGFR antigen as detected by Western blotting and immunofluorescence.The inhibitory rates of Cetuximab and 9B9 antibody against Huh7-EGFRvⅢ cells-implanted tumors were 42% and 46%,respectively,and those against A431 cells-implanted tumors were 85% and 86%,respectively.Conclusion： 9B9 monoclonal antibody can effectively inhibit the growth of human hepatocellular carcinoma cell-and epidermal cell carcinoma cell-implanted tumors,and the effects resemble that of Cetuximab.