表皮生长因子受体特异性单链抗体的筛选及初步鉴定

Selection and initial characterization of an epidermal growth factor receptor scFv

目的从大型人源抗体库Griffin 1中直接筛选出靶向EGFR的单链抗体。方法以稳定转染的CHO-EGFR-GFP1细胞和未转染的CHO-K1细胞分别作为EGFR阳性和阴性细胞,采用负筛选的方法进行筛选;通过比较每轮投入及洗脱出噬菌体的效价比以及细胞ELISA检测多克隆p-scFv与阳性、阴性细胞结合情况对筛选过程进行监测;采用菌落PCR挑选含有全长scFv片断的菌落,进一步用细胞ELISA检测单克隆p-scFv与EGFR阳性及阴性细胞结合特异性;挑选EGFR特异性单克隆p-scFv采用DNA测序法确定克隆多样性。结果经过5轮筛选,细胞裂解液中洗脱出噬菌体效价有500倍以上增长;细胞ELISA结果显示多克隆p-scFv与CHO-EGFR-GFP1细胞和CHO-K1细胞均有明显结合,但有部分特异性结合EGFR;菌落PCR显示约45%克隆中含有完整的1kb scFv片断;经细胞ELISA、DNA测序检测共获得1株EGFR特异性单链抗体,命名为F4-scFv。结论F4-scFv可与筛选中所用EGFR阳性细胞特异性结合,为进一步研制以此单链抗体为靶向分子的抗肿瘤药物奠定了基础。

Objective To Select an epidermal growth factor receptor （EGFR） scFv from a large nonimmune phage display library. Methods CHO-EGFR-GFP1, a transfected cell line expressing EGFR-GFP fusion protein on membrane, and the untransfected cell line CHO-K1 were used as EGFR positive cells and negative cells respectively in the subtractive selection procedure. The efficiency of selection was monitored by comparing the number of phage recovered from the acid elution and cell lysate in each round, and by testing EGFR binding specificity of polyclonal phage-scFv （p-scFv） on CHO-EGFR-GFP1 and CHO-K1 cell with cell ELISA. Bacterial PCR was used to select clones containing a 1 kb insert. Cell ELISA was used to determine EGFR binding specificity of monoclonal p-scFv on EGFR positive and negative cell. The number of individual EGFR-binding clones was determined with nucleotide sequencing. Results 500-fold enrichments were observed by tittering phages in the cell lysate after five rounds of selection. A signal significantly greater than background binding was observed from the third to the fifth round of selection on CHO-EGFR-GFP1 and CHO-K1 cell, but a part of polyclonal scFv bound EGFR specially. About 45% of the selected clones contained a full-sized insert of 1 kb. One unique human anti- EGFR scFv （F4-scFv） was isolated by analyzing with cell ELISA and DNA sequencing. Conclusion F4-scFv binds specifically to EGFR positive cell line used in the selection, this work will be a fundament for further developing of anti-tumor drugs using this scFv as target molecule.