Research Of Screening Of Anti-EGFR Human Fab And The In Vitro Anti-cancer Efficacy Of Its Conjugation With Paclitaxel

Purpose: To generate a human Fab fragment against epidermal growthfactor receptor (EGFR); conjugate it to paclitaxel (Taxol) as animmuno-chemotherapy agent; and investigate its in vitro anti-tumorefficacy on A431 epidermoid carcinoma cells.Experimental Design: A431 cells (EGFR-positive), NIH 3T3 cells(EGFR-negative) and purified EGFR were used for subtractive panningon a human na(?)ve Fab phage library to generate a human anti-EGFR Fabfragment that binds the EGFR extracellular domain in nativeconformation and subsequently internalizes it into the cytosol. The Fabwas then conjugated with the chemotherapeutic Taxol, and cellproliferation and apoptosis (TUNEL) assays were conducted to determinethe effect of this Fab-drug conjugate on A431 cells.Results: A human Fab fragment that specifically recognizes the nativeEGFR extracellular domain and has internalization properties wasgenerated and characterized by immunoprecipitation, fluorescencestaining, flow cytometry, and Hum-Zap assay. The binding affinity topurified EGFR was 30 nM. The Fab-Taxol conjugate inhibited A431 cellproliferation at low concentrations and in a dose-responsive manner; more than 70% inhibition was observed at 52 pM. Furthermore, almost100% of cells underwent apoptosis after treatment with Fab-Taxol at 26pM for 48 hours.Conclusions: Our findings suggest that this Fab-Taxol conjugate couldbe a potential immuno-chemotherapeutic drug for clinical treatment ofEGFR-overexpressing tumors.