全人源EGFR单抗的筛选和鉴定

Screening and identification of human EGFR monoclonal antibodies

目的:制备全人源表皮生长因子受体(epidermal growth factor receptor,EGFR)单克隆抗体,为基于人EGFR靶点的肿瘤免疫调节治疗提供基础制剂。方法:采集110例胸部肿瘤(肺癌102例,乳腺癌8例)患者外周血,构建噬菌体单链抗体库。经噬菌体展示后筛选阳性克隆,测序并构建全长轻重链表达载体质粒,共转染HEK293。通过亲和层析获得纯化抗体,抗原抗体免疫反应测定抗体特异性和敏感性,生物膜干涉(biolayer interferometry,BLI)技术测定抗体亲和力和流式细胞技术(fluorescence activating cell sorter,FACS)检测对肿瘤细胞系EGFR结合活性。结果:成功构建肺癌单链抗体噬菌体库,抗体库库容≥108,筛选获得两株单抗,分别为BTHG17-1,BTHG17-2,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)测定结合抗原敏感性为10 ng/mL,亲和力在10-8 mol/L水平,两抗体结合相同表位,可特异性结合野生型EGFR和突变体EGFRvⅢ蛋白,具有结合肺癌和神经胶质瘤细胞活性。结论:获得较高亲和力全人源EGFR单链抗体(single-chain variable fragment,scFv),具有EGFR(包括EGFRVⅢ)特异性,为EGFR靶点封阻和免疫导向治疗研究提供一种新抗体工具。

Objective To prepare full human monoclonal antibody against epidermal growth factor receptor(EGFR)by phage antibody library and to provide a tool for tumor immunomodulatory therapies based on targeting EGFR.Methods The peripheral blood of 110 patients with thoracic tumors(102 of lung cancer and 8 of breast cancer)were collected to construct a phage single-chain antibody library.After phage display,positive clones were screened,sequenced.The full-length light and heavy chain expression vector plasmids were constructed,then co-transfected with HEK293.The purified antibodies were obtained by affinity chromatography,and the specificity and sensitivity of antibody were determined by antigen antibody immune reaction.The antibody affinity was determined by biolayer interferometry(BLI)technology and the binding activity of EGFR in tumor cell line was detected using fluorescence activating cell sorter(FACS).Results The phage library of single-chain variable fragment(scFv)against lung cancer was successfully constructed.The capacity of the library was≥108.Two monoclonal antibodies,BTHG17-1 and BTHG17-2,were obtained.The sensitivity of binding antigen was 10 ng/mL and affinity was around 10-8 mol/L.They specifically binded to both EGFRvⅢmutants and wild EGFR,and had the activity of binding lung cancer and glioma cells.Conclusion The moderate affinity of human monoclonal antibody has well specificity to EGFR(including EGFRvⅢ),which provides a new agent for immunotherapeutic researches for targeting EGFR.