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J Immunol Methods. 2001 Feb 1;248(1-2):17-30.

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

Author information

1
Department of Anesthesia, University of California, San Francisco, Room 3C-38, San Francisco General Hospital, 1001 Potrero Avenue, San Francisco, CA 94110, USA. marksj@anesthesia.ucsf.edu

Abstract

The first step in developing a targeted cancer therapeutic is generating a ligand that binds to a receptor which is either tumor specific or sufficiently overexpressed in tumors to provide targeting specificity. For this work, we generated human monoclonal antibodies to the EGF receptor (EGFR), an antigen overexpressed on many solid tumors. Single chain Fv (scFv) antibody fragments were directly selected by panning a phage display library on tumor cells (A431) overexpressing EGFR or Chinese hamster ovary cells (CHO/EGFR cells) transfected with the EGFR gene and recovering endocytosed phage from 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 EGFR negative cells. Phage antibodies and multivalent immunoliposomes constructed from scFv were endocytosed by EGFR expressing cells as shown by confocal microscopy. Native scFv primarily stained the cell surface, with less staining intracellularly. The results demonstrate how phage antibodies binding native cell surface receptors can be directly selected on overexpressing cell lines or transfected cells. Use of a transfected cell line allows selection of antibodies to native receptors without the need for protein expression and purification, significantly speeding the generation of targeting antibodies to genomic sequences. Depending upon the format used, the antibodies can be used to deliver molecules to the cell surface or intracellularly.

PMID:
11223066
[Indexed for MEDLINE]

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