title: Generation and functional characterization of intracellular antibodies interacting with the kinase domain of human EGF receptor.
abstract: Intracellular expression of single-chain antibodies (scFvs) represents a promising approach for selective interference with cellular proto-oncogenes such as the epidermal growth factor receptor (EGFR). Previously, we have shown that intrabodies targeted to the lumen of the endoplasmic reticulum prevent the transit of EGFR or the related ErbB2 molecule to the cell surface, thereby inactivating their transforming potential. While intramolecular disulfide bridges important for antibody stability are correctly formed during expression in the secretory pathway, scFvs expressed in the reducing environment of the cytosol are often inactive. To overcome this problem and to generate antibody fragments that interact with the intracellular domain of human EGFR in the cytoplasm, here we have chosen a two-step approach combining classical selection of scFvs by phage display with subsequent expression in yeast. After enrichment of EGFR-specific antibody fragments from a combinatorial library by biopanning, a yeast two-hybrid screen was performed using the intracellular domain of EGFR as bait. Screening of 1.5 x 10(5) preselected scFv plasmids under highly stringent conditions yielded 223 colonies that represented at least five independent scFv clones functional in the intracellular milieu of eukaryotic cells. Interaction of selected antibody fragments with the intracellular domain of EGFR was confirmed in GST pull-down and coimmunoprecipitation experiments. Upon cytoplasmic expression in human tumor cells, scFvs colocalized with EGFR at the plasma membrane demonstrating their functionality in vivo.
authors.affiliation: Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, D-co596 Frankfourt an Main, Germany.
authors.forename: Stephen
authors.initials: S
authors.lastname: Hyland
authors.forename: Roger R
authors.initials: RR
authors.lastname: Beerli
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Hynes
authors.forename: Winfried
authors.initials: W
authors.lastname: Wels
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D001435:Bacteriophages; D001483:Base Sequence; D019556:COS Cells / Q000378:metabolism; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
chemical_list: D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D066246:ErbB Receptors