Epidermal growth factor receptor downregulation by small heterodimeric binding proteins

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Protein Engineering Design and Selection (Impact Factor: 2.54). 12/2011; 25(2):47-57. DOI: 10.1093/protein/gzr056
Source: PubMed


No single engineered protein has been shown previously to robustly downregulate epidermal growth factor receptor (EGFR), a
validated cancer target. A panel of fibronectin-based domains was engineered to bind with picomolar to nanomolar affinity
to multiple epitopes of EGFR. Monovalent and homo- and hetero-bivalent dimers of these domains were tested for EGFR downregulation.
Selected orientations of non-competitive heterodimers decrease EGFR levels by up to 80% in multiple cell types, without activating
receptor signaling. These heterodimers inhibit autophosphorylation, proliferation and migration, and are synergistic with
the monoclonal antibody cetuximab in these activities. These small (25 kDa) heterodimers represent a novel modality for modulating
surface receptor levels.

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    • "Research Article not inhibit EGFR internalization completely, suggesting the existence of other as yet undefined mechanisms (Goh et al., 2010; Huang et al., 2007; Wang et al., 2007). Recently, different antibody combinations or multitopic constructs showed a reduction in surface-expressed EGFR as a result of the kinaseindependent internalization of EGFR, suggesting the involvement of EGFR clustering in EGFR internalization (Boersma et al., 2011; Friedman et al., 2005; Hackel et al., 2012; Spangler et al., 2010). "
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    ABSTRACT: EGFR signaling is attenuated by endocytosis and degradation of receptor/ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple posttranslational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.
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    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.
    Preview · Article · Jun 2012 · Journal of Molecular Biology
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    ABSTRACT: Genes encoding membrane proteins have been estimated to comprise as much as 30% of the human genome. Among these membrane, proteins are a large number of signaling receptors, transporters, ion channels and enzymes that are vital to cellular regulation, metabolism and homeostasis. While many membrane proteins are considered high-priority targets for drug design, there is a dearth of structural and biochemical information on them. This lack of information stems from the inherent insolubility and instability of transmembrane domains, which prevents easy obtainment of high-resolution crystals to specifically study structure-function relationships. In part, this lack of structures has greatly impeded our understanding in the field of membrane proteins. One method that can be used to enhance our understanding is directed evolution, a molecular biology method that mimics natural selection to engineer proteins that have specific phenotypes. It is a powerful technique that has considerable success with globular proteins, notably the engineering of protein therapeutics. With respect to transmembrane protein targets, this tool may be underutilized. Another powerful tool to investigate membrane protein structure-function relationships is computational modeling. This review will discuss these protein engineering methods and their tremendous potential in the study of membrane proteins.
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