Combined use of multiple monoclonal antibodies targeting epidermal growth factor receptor for improved cancer therapeutics

Epidermal growth factor receptor (EGFR) overexpression and its deregulated signaling is one of the hallmarks of multiple neoplasms. Although the use of monoclonal antibodies (mAbs) binding to the ectodomain of EGFR has shown promising results in inhibiting tumor progression, clinical failures in the anticipated outcome resulted in exploring new modalities for improved treatment.During the initial phase of this thesis, we observed that combined use of two EGFR targeting mAbs---mAb425 (EMD55900) and C225 (Cetuximab/Erbitux), synergistically inhibited the growth and EGF dependent signaling of EGFR overexpressing breast cancer cells. This observation has led to an effort to identify the mechanisms that govern the synergistic behavior.Binding competition and mutagenesis studies showed that mAb425 and C225 had distinct binding epitopes. mAb425/C225 combination inhibited EGF binding more efficiently than individual mAbs. EGF disrupted mAb-EGFR complexes containing individual mAbs but not the complexes with mAb combination. Further flow cytometry and surface plasmon resonance analysis suggested that both the mAbs were able to recognize most of the conformational states of EGFR. Overall, these results indicate that simultaneous binding of two non-competing mAbs, mAb425 and C225 stabilizes the inactive form of the receptor which EGF cannot recognize thus achieving efficient inhibition of EGF binding.We also observed that co-incubation with mAb425 and C225 resulted in enhanced EGFR downregulation, compared to individual mAbs. The use of Fab fragments showed no significant receptor internalization. Monovalent Fab fragments failed to inhibit cell growth and EGF dependent signal transduction, suggesting that EGFR crosslinking by bivalent IgG molecules is required for inhibition. Live cell confocal imaging showed that mAb combination significantly reduced receptor recycling. Collectively these results suggest that EGFR oligomerization by mAb425/C225 combination is vital for synergistic receptor downregulation.We believe that the mechanisms of synergy presented may be further extended to other mAb combinations. Such combinations could synergize by adopting the efficient ligand blocking, faster EGFR downregulation, or a combination of both these mechanisms. We hope that the information gained from these studies may be used as a model to guide researchers to engineer novel mAbs and accelerate their transition from bench to clinic.