噬菌体展示全人源抗GPC3的单链抗体的筛选及鉴定

Screening and characterization of human anti-GPC3 single chain Fv antibody fragment selected by phage display

使用全人源噬菌体单链抗体文库进行筛选,获得特异性靶向磷脂酰肌醇聚糖-3(GPC3)的单链抗体,并对单链抗体的生物学活性和抗原表位进行鉴定。经过4轮"结合-淘洗-洗脱-扩增"后,利用噬菌体ELISA和IMGT数据库分析抗体序列的靶向性和完整性。单链抗体的基因序列与表达载体p ET-22b进行双酶切并连接,重组载体转化大肠杆菌E.coli Rosetta(DE3),抗体表达后经Ni^(2+)柱亲和层析纯化,通过SDS-PAGE和Western blot对单链抗体分子质量进行鉴定。抗体对抗原的亲和力常数通过ELISA和SPR实验获得,流式细胞术分析其与细胞膜受体的结合能力。经筛选获得4个单链抗体(1F7、1D7、1D4和1B10)具有良好的靶向性和亲和力,其中1F7单链抗体的亲和力和结合能力最高。本课题抗GPC3单链抗体为双特性抗体和免疫毒素等新一类免疫治疗药物的开发奠定基础。

The aim of the experiments is to screen human single-chain variable fragment（sc Fv）targeting glypican 3 from phage display library,and analyze its biological activity.After several rounds of panning,the binders with high affinity were obtained through phage ELISA and IMGT analysis.The desired sc Fv gene was then ligated with p ET-22b vector yielding recombinant plasmids,which was then introduced into E.coli Rosetta（DE3）.Soluble sc Fv protein was expressed and further purified using Ni^（2＋）affinity chromatography.The purified proteins were identified by SDS-PAGE and Western blot.Subsequently,the affinity and cell based binding activity were measured using Surface Plasmon Resonance（SPR）and flow cytometry assay,separately.Four enriched sequences with relatively high binding affinity were found（1F7,1D7,1D4 and 1B10）.We also found that 1F7 sc Fv showed better targeting ability and higher affinity.The sc Fv could pave the way for new immunotherapies,such as bispecific anitbody,antibody-drug conjugate and chimeric antigen receptor T-cell immunotherapy cell,etc.