Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library

The first step in developing a targeted cancer therapeutic is generating a ligand that binds to a receptor which is either tumor specific or sufficien tly overexpressed in tumors to provide targeting specificity. For this work , we generated human monoclonal antibodies to the EGF receptor (EGFR), an a ntigen overexpressed on many solid tumors, single chain Fv (scFv) antibody fragments were directly selected by panning a phage display library on tumo r cells (A431) overexpressing EGFR or Chinese hamster ovary cells (CHO/EGFR cells) transfected with the EGFR gene and recovering endocytosed phage fro m within the cell. Three unique scFvs were isolated, two from selections on A431 cells and two from selections on CHO/EGFR cells. All three scFv bound native receptor as expressed on a panel of tumor cells and did not bind EG FR negative cells. Phage antibodies and multivalent immunoliposomes constru cted from scFv were endocytosed by EGFR expressing cells as shown by confoc al microscopy. Native scFv primarily stained the cell surface, with less st aining intracellularly. The results demonstrate how phage antibodies bindin g native cell surface receptors can be directly selected on overexpressing cell lines or transfected cells. Use of a transfected cell line allows sele ction of antibodies to native receptors without the need for protein expres sion and purification, significantly speeding the generation of targeting a ntibodies to genomic sequences. Depending upon the format used, the antibod ies can be used to deliver molecules to the cell surface or intracellularly . (C) 2001 Elsevier Science BN. All rights reserved.