Screening of scFv-displaying phages recognizing distinct extracellular domains of EGF receptor by target-guided proximity labeling method.

摘要

We recently constructed the scFv-displaying phage library with extremely high repertoire and have successfully utilized for screening scFv antibodies against various proteins, polysaccharides and glyco-lipids. Here, we developed a new screening strategy to isolate scFv antibodies against cell surface EGF receptor (EGFR). For this, we applied two slightly different methods of "target-guided proximity labeling," such as Proximity selection (ProxiMol) method and a new sulfo-SBED labeling method with the aide of monoclonal anti-human EGFR antibody B4G7 as a guide molecule. ProxiMol method relies on the Biotin-labeling of scFv-displaying phages that bound to the target in a vicinity of 100A from the guide molecule, whereas sulfo-SBED method transfers Biotin to scFv-displaying phages, which bound to the target in a distance of 20 A. After two rounds of panning on the EGFR-overexpressing A431 cells starting from approx. 1 x 10(1)(2) pfu, 47 each of Biotin-labeled scFv-displaying phages were recovered using Streptoavidin-coated magnetic beads, and among them total 11 scFv-phages were found to be definitely positive for binding to A431 cell surface by ELISA assay. Restriction mapping and sequencing analysis of these scFv-phage DNAs revealed that they encode 4 different scFv-nucleotide sequences in total. Immuno-fluorescent microscopy provided evidence that these 4 scFv antibodies bind specifically to EGFR on the A431 cells, showing slightly different staining patterns. Thus, "target-guided proximity labeling" methods were powerful for isolating scFv-displaying phages that recognize distinct extracellular domains of the target receptor. This novel screening strategy could be applicable to many other cell surface antigens and receptors.