== The relationship between antibody-induced EGFR degradation and growth inhibition for each of the 58 mAbs and mixtures of two antibodies was investigated in the cell lines HN5 and A431NS (Fig. inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is usually superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain name III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain name. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation. Key words:EGFR, antibody synergy, functional screening, epitope binning, antibody combinations == Introduction == The epidermal growth factor receptor (EGFR/ErbB1) belongs to the ErbB family of Ethynylcytidine receptor tyrosine kinases, which also includes HER-2/ErbB2, HER-3/ErbB3 and HER-4/ErbB4.1EGFR is known to bind at least six ligands, including epidermal Ethynylcytidine growth factor (EGF) and transforming growth factor- (TGF).2Unliganded EGFR is thought to rest in a tethered inactive form where a putative dimerization arm localized in domain II (dII) is sequestered by interactions with domain IV Ethynylcytidine (dIV). For ligand binding to occur, domain name 1 (dI) and domain name III (dIII) must undergo conformational changes that bring them close together for formation of a ligand binding pocket. These ligand-mediated structural changes induce an untethered active receptor conformation where the dimerization domain is usually exposed and capable of promoting receptor dimerization with other monomers, shifting the monomer dimer equilibrium in favor of the dimeric receptor state.35Receptor homo- or hetero-dimerization with EGFR or other members of the ErbB family results in activation of intracellular tyrosine kinase domains, leading to autophosphorylation that controls signaling cascades important for cell growth, proliferation, survival and motility.6,7Owing to involvement in these cellular processes, EGFR signaling is frequently unbalanced in human malignancies, either due to increased ligand production, receptor overexpression, receptor mutations or cross-talk with other receptor systems.811Changes in EGFR status have been linked to the development and maintenance of a malignant phenotype and correlated to poor clinical prognosis.12For this reason EGFR is an attractive target for anti-cancer therapy.13Two monoclonal antibodies (mAbs), cetuximab (Erbitux) and panitumumab (Vectibix), are approved for the treatment of colorectal cancers14,15and head and neck cancers (cetuximab only14). One of KIAA0538 the main mechanisms of tumor growth inhibition by mAbs is usually thought to be disruption of ligand induced EGFR tyrosine kinase activation. The mAbs do this by sterically blocking access of the ligand to EGFR dIII. 16Despite the clinical success of cetuximab and panitumumab, recent in vitro results show that cancer cells that were initially sensitive to treatment escape by switching on alternative receptor tyrosine kinases (HER2, MET or IGF1R). However, importantly the Ethynylcytidine cells remain dependent on the presence of EGFR.1720 Previous studies have described how mixtures of antibodies targeting multiple distinct epitopes on HER-2 are more effective than single mAbs at inhibiting growth of HER-2 dependent cells in vitro and tumor growth in various animal models.2124Furthermore, in vitro studies have demonstrated that mixtures of antibodies with non-overlapping epitopes are more efficient at inducing EGFR downregulation compared with the individual antibodies.22,25A mixture of the mAbs 806 and 528 have also been shown to induce degradation of the 27 EGFR in a xenograft model of the U87-MG-27 EGFR cell line.26 Finally, antibody effector functions like antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) could contribute to the activity of tumor growth inhibitory anti-EGFR mAbs by inducing tumor cell killing. While single anti-EGFR mAbs have been demonstrated to activate ADCC through binding to FcIIIa receptor, only antibody mixtures have been shown to activate CDC in vitro.27,28 Based on these reports, we hypothesized that a systematic discovery and selection approach might lead to improved antibody mixtures for targeting EGFR. The result was the identification of Sym004, a drug candidate composed of an equimolar mixture of the two chimeric mAbs, 992 and 1024, recognizing non-overlapping epitopes on EGFR dIII. Sym004 was superior to reference.