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J. Biol. Chem. 279 (29): 30375-30384

© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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*

Terrance G. Johns{ddagger}§¶, Timothy E. Adams§||, Jennifer R. Cochran§**, Nathan E Hall{ddagger}{ddagger}, Peter A. Hoyne||, Mark J. Olsen**, Yong-Sung Kim**, Julie Rothacker{ddagger}{ddagger}, Edouard C. Nice{ddagger}{ddagger}, Francesca Walker{ddagger}{ddagger}, Gerd Ritter§§, Achim A. Jungbluth§§, Lloyd J. Old§§, Colin W. Ward||, Antony W. Burgess{ddagger}{ddagger}, K. Dane Wittrup**, , and Andrew M. Scott{ddagger}

{ddagger}Tumour Targeting Program, Ludwig Institute for Cancer Research, Austin Hospital, Heidelberg 3084, Australia, ||CSIRO Health Sciences and Nutrition, Parkville 3052, Australia, **Chemical Engineering & Bioengineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, {ddagger}{ddagger}Epithelial Biochemistry Laboratory, Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville 3050, Australia, and §§Ludwig Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

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 (~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 (~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.

Received for publication February 4, 2004. Revision received April 8, 2004.

* This work was supported in part by National Institutes of Health Grant CA96504, the Ludwig Trust, and the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

To whom correspondence should be addressed: Oncogenic Signalling Laboratory, Ludwig Institute for Cancer Research, Austin Hospital, Level 6, Harold Stokes Building, Studley Road, Heidelberg 3084, Australia. Tel.: 613-9496-3068; Fax: 613-9496-5892; E-mail: terry.johns{at}

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