Combination antibody treatment down-regulates epidermal growth factor receptor by inhibiting endosomal recycling

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.ErbB | monoclonal antibody | tyrosine kinase | trafficking E pidermal growth factor receptor (EGFR) is a member of the ErbB family of single-pass transmembrane receptor tyrosine kinases (RTKs). Under normal conditions, EGFR activation is tightly regulated by its native ligands, epidermal growth factor (EGF) and transforming growth factor-α (TGFα), which bind to the receptor extracellular domain (1). Ligand binding induces conformational changes in EGFR that stabilize homo-or heterodimerization, leading to autophosphorylation of its intracellular domain. Phosphorylation of selected tyrosine residues activates signaling effectors in downstream pathways, including the mitogenactivated protein kinase (MAPK) and phosphoinositol kinase 3 (PI3K) pathways, eliciting responses such as growth, migration, differentiation, and apoptosis (2). The primary mechanisms of signal attenuation are receptor deactivation via phosphatase activity and receptor degradation following endocytosis (3). Ligand activation accelerates receptor endocytosis and simultaneously decreases the recycling to degradation ratio to terminate EGFR signaling (4).Dysregulation of EGFR leading to unregulated growth has been observed in a variety of cancers. Means of dysregulation include receptor overexpression, which occurs in one-third of all epithelial-based tumors (5), mutation, aberrant localization, autocrine ligand secretion, and obstruction of endocytosis (3). Due to its prevalence and altered expression in cancer patients, several therapeutic strategies have been employed to target EGFR, one of which involves the use of monoclonal antibodies (mAbs) that bind the receptor ectodomain. mAbs...

Heparanase-neutralizing antibodies attenuate lymphoma tumor growth and metastasis

“…4D). This is in agreement with the notion that combining two different mAbs increases the inhibitory outcome (31).…”

Inhibition of pancreatic carcinoma by homo- and heterocombinations of antibodies against EGF-receptor and its kin HER2/ErbB-2

“…The antitumor actions of homocombinations of mAbs to EGFR and HER2 have been associated with forced internalization and subsequent receptor degradation in lysosomes (14,17,21). Furthermore, this mechanism might entail collapse of large antigen-antibody lattices into endocytic vesicles, which use clathrinas well as caveolin-mediated trafficking and avoid a recycling pathway that governs trafficking of EGFR bound to a single mAb (18,23). Interestingly, the data we present in Fig.…”

“…However, another way to improve the efficacy of mAbs to surface receptors comprises combinations of two or more mAbs, each targeting a distinct receptor's epitope. For example, it has been reported that certain pairs of anti-EGFR antibodies can accelerate receptor endocytosis and degradation (17), probably through a mechanism involving inhibition of receptor recycling (18). Consistent with these observations, a mixture of two anti-EGFR mAbs, called Sym004, inhibited cancer cell growth (14).…”

“…Previous studies established similar effects on gastric (20,21) and breast cancer (11,14,30), including the aggressive triple negative subtype (23). Importantly, synergy requires engagement of two nonoverlapping antigenic sites (17,18), and occupation of the ligand-binding site of EGFR or the dimerization site of HER2 might enhance synergy (14,21). Unlike homocombinations of mAbs, which have been extensively studied and already reached clinical application (22), studies of antibody heterocombinations are relatively new.…”

Enhanced Targeting of the EGFR Network with MM-151, an Oligoclonal Anti-EGFR Antibody Therapeutic

“…Our results build on and contribute to the emerging field of oligoclonal inhibitors of ErbB and other cell-surface receptors (51)(52)(53), which notably includes the approved combination of trastuzumab (Herceptin) and pertuzumab (Perjeta) targeting ErbB2 in metastatic breast cancer (57) and the early-stage clinical development of Sym004 targeting EGFR (5, 6). Our observations of near-complete downregulation of EGFR and enhanced CDC activity by MM-151 are consistent with these studies and provide an exciting prospect to elicit additional EGFR inhibition and pathway-extrinsic cytotoxicity beyond that achievable by single monoclonal antibodies.…”