[Show abstract][Hide abstract] ABSTRACT: Dysregulation of epidermal growth factor receptor (EGFR) is a hallmark of many epithelial cancers, rendering this receptor an attractive target for cancer therapy. Much effort has been focused on the development of EGFR-directed antibody-based therapeutics, culminating in the clinical approval of the drugs cetuximab and panitumumab. Unfortunately, the clinical efficacy of these drugs has been disappointingly low, and a particular challenge to targeting EGFR with antibody therapeutics has been resistance, resulting from mutations in the downstream raf and ras effector proteins. Recent work demonstrating antibody cocktail-induced synergistic downregulation of EGFR motivated our design of cetuximab-based antibody-fibronectin domain fusion proteins that exploit downregulation-based EGFR inhibition by simultaneously targeting multiple receptor epitopes. We establish that, among our engineered multiepitopic formats, trans-triepitopic antibody fusions demonstrate optimal efficacy, inducing rapid EGFR clustering and internalization and consequently ablating downstream signaling. The combined effects of EGFR downregulation, ligand competition, and immune effector function conspire to inhibit tumor growth in xenograft models of cetuximab-resistant BRAF and KRAS mutant cancers. Our designed triepitopic constructs have the potential to enhance the efficacy and expand the scope of EGFR-directed therapies, and our multiepitopic may be readily applied to other receptor targets to formulate a new class of antibody-based therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Due to its common dysregulation in epithelial-based cancers and extensive characterization of its role in tumor growth, epidermal growth factor receptor (EGFR) is a highly validated target for anticancer therapies. There has been particular interest in the development of monoclonal antibodies (mAbs) targeting EGFR, resulting in two approved mAb-based drugs and several others in clinical trials. It has recently been reported that treatment with combinations of noncompetitive mAbs can induce receptor clustering, leading to synergistic receptor down-regulation. We elucidate three key aspects of this phenomenon. First, we show that highly potent combinations consisting of two noncompetitive mAbs that target EGFR domain 3 reduce surface receptor levels by up to 80% with a halftime of 0.5-5 h in both normal and transformed human cell lines to an extent inversely proportional to receptor density. Second, we find the mechanism underlying down-regulation to be consistent with recycling inhibition. Third, in contrast to the agonism associated with ligand-induced down-regulation, we demonstrate that mAb-induced down-regulation does not activate EGFR or its downstream effectors and it leads to synergistic reduction in migration and proliferation of cells that secrete autocrine ligand. These new insights will aid in ongoing rational design of EGFR-targeted antibody therapeutics.
Proceedings of the National Academy of Sciences 07/2010; 107(30):13252-7. DOI:10.1073/pnas.0913476107 · 9.67 Impact Factor