title: Identification of the epitope for the epidermal growth factor receptor-specific monoclonal antibody 806 reveals that it preferentially recognizes an untethered form of the receptor.
abstract: The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial cancers, an observation often correlated with poor clinical outcome. Overexpression of the EGFR is commonly caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (de2-7 EGFR or EGFRvIII) bearing an internal deletion in its extracellular domain. Monoclonal antibody (mAb) 806 is a novel EGFR antibody with significant antitumor activity that recognizes both the de2-7 EGFR and a subset of the wild type (wt) EGFR when overexpressed but does not bind the wt EGFR expressed in normal tissues. Despite only binding to a low proportion of the wt EGFR expressed in A431 tumor cells (approximately 10%), mAb 806 displays robust antitumor activity against A431 xenografts grown in nude mice. To elucidate the mechanism leading to its unique specificity and mode of antitumor activity, we have determined the EGFR binding epitope of mAb 806. Analysis of mAb 806 binding to EGFR fragments expressed either on the surface of yeast or in an immunoblot format identified a disulfide-bonded loop (amino acids 287-302) that contains the mAb 806 epitope. Indeed, mAb 806 binds with apparent high affinity (approximately 30 nm) to a synthetic EGFR peptide corresponding to these amino acids. Analysis of EGFR structures indicates that the epitope is fully exposed only in the transitional form of the receptor that occurs because EGFR changes from the inactive tethered conformation to a ligand-bound active form. It would seem that mAb 806 binds this small proportion of transient receptors, preventing their activation, which in turn generates a strong antitumor effect. Finally, our observations suggest that the generation of antibodies to transitional forms of growth factor receptors may represent a novel way of reducing normal tissue targeting yet retaining antitumor activity.
authors.affiliation: Tumour Targeting Program, Ludwig Institute for Cancer Research, Austin Hospital, Heidelberg 3084, Australia. terry.johns@ludwig.edu.au
authors.forename: Terrance G
authors.initials: TG
authors.lastname: Johns
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Adams
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: Cochran
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Hall
authors.forename: Peter A
authors.initials: PA
authors.lastname: Hoyne
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Olsen
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
authors.forename: Julie
authors.initials: J
authors.lastname: Rothacker
authors.forename: Edouard C
authors.initials: EC
authors.lastname: Nice
authors.forename: Francesca
authors.initials: F
authors.lastname: Walker
authors.forename: Gerd
authors.initials: G
authors.lastname: Ritter
authors.forename: Achim A
authors.initials: AA
authors.lastname: Jungbluth
authors.forename: Lloyd J
authors.initials: LJ
authors.lastname: Old
authors.forename: Colin W
authors.initials: CW
authors.lastname: Ward
authors.forename: Antony W
authors.initials: AW
authors.lastname: Burgess
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scott
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D066246:ErbB Receptors / Q000737:chemistry* / Q000276:immunology; D005434:Flow Cytometry; D017353:Gene Deletion; D014644:Genetic Variation; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D000939:Epitopes; D008024:Ligands; D010455:Peptides; D066246:ErbB Receptors