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J. Biol. Chem., Vol. 282, Issue 5, 2840-2850, February 2, 2007

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The Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor AG1478 Increases the Formation of Inactive Untethered EGFR Dimers

IMPLICATIONS FOR COMBINATION THERAPY WITH MONOCLONAL ANTIBODY 806^*

Hui K. Gan, Francesca Walker, Antony W. Burgess, Angela Rigopoulos, Andrew M. Scott^¶, and Terrance G. Johns¹

From the Oncogenic Signalling Laboratory and ^¶Tumour Targeting Program, Ludwig Institute of Cancer Research, Austin Hospital, Level 6, Harold Stokes Building, Studley Road, Heidelberg, Victoria 3084 and the Epithelial Biochemistry Laboratory, Royal Melbourne Hospital, Parkville, Victoria 3052, Australia

The epidermal growth factor receptor (EGFR) has at least two fundamental conformations: an inactive tethered conformation and an active untethered, ligand-bound "back-toback" dimer, which may be part of an oligomeric complex. Monoclonal antibody (mAb) 806 is an EGFR-specific antibody that only binds a transitional form of the receptor after it untethers but before forming the back-to-back, ligated, active oligomer. We have shown that AG1478, a tyrosine kinase inhibitor of the EGFR, synergistically inhibits the growth of tumors overexpressing EGFR when used in combination with mAb 806 but the mechanism for this was not elucidated (Johns, T. G., Luwor, R. B., Murone, C., Walker, F., Weinstock, J., Vitali, A. A., Perera, R. M., Jungbluth, A. A., Stockert, E., Old, L. J., Nice, E. C., Burgess, A. W., and Scott, A. M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15871–15876). We now show that AG1478 increases binding of mAb 806 to the cell surface through two distinct mechanisms: an immediate effect on the conformation of EGFR and a longer term increase in cell surface under-glycosylated EGFR, an event known to increase mAb 806 reactivity. Cross-linking studies demonstrated the presence of spontaneously occurring mAb 806-reactive dimers on the surface of cells overexpressing EGFR, which are rapidly increased by AG1478. Because they react with mAb 806, these dimers must exist in a conformation distinct from the ligated back-to-back dimer. Indeed, we detected similar dimers in 293T cells expressing the EGFR lacking the small dimerization/activation arm essential to the formation of the back-to-back dimer. Thus, some of the EGFR on the cell surface of cancer cells must exist as an untethered dimer that adopts a previously unreported conformation that is inactive. This information was used to optimize the therapeutic synergy between mAb 806 and AG1478 in a xenograft model.

Received for publication, May 30, 2006 , and in revised form, November 8, 2006.

^* This work was partly supported by the National Health and Medical Research Council of Australia (Program Grant 280912). 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.

¹ To whom correspondence should be addressed. Tel.: 613-9496-3068; Fax: 613-9496-5892; E-mail: terry.johns{at}ludwig.edu.au.

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