Mechanisms of Extracellular Oncogenic Dysregulation and Antibody Targeting of the Epidermal Growth Factor Receptor

Regulation of the Epidermal Growth Factor Receptor (EGFR) by its growth factor ligands is critical in many biological processes, including development and tissue maintenance and growth. Aberrant overexpression or activation by mutation of EGFR is associated with many human tumors. In these contexts, constitutive signaling can lead to cellular transformation and oncogenesis, thereby driving the cancer. The EGFR is the target of several existing or developing cancer therapies or immunotherapies, including monoclonal antibodies that prevent its activation. Activating mutations in cytoplasmic tyrosine kinase domain have been identified in many cancers, and have been the focus of mechanistic work. In this dissertation, I focus on the mode of oncogenic dysregulation by novel extracellular mechanisms. Extracellular oncogenic variants of EGFR include point mutations and alternative splice variants of EGFR. I find through biochemical analysis of the activating missense mutations in the extracellular region of EGFR that the soluble extracelluar region of EGFR (sEGFR) harboring these mutations bind ligands with elevated affinities. The dimerization energetics of these sEGFR mutants is not measurably altered, which suggests that additional interactions from the membrane and/or the intracellular region are important to this novel mode of extracellular oncogenic dysregulation of EGFR. I present preliminary progress towards the application of hydrogen/deuterium exchange coupled to mass spectrometry to analyze such allosteric (dys)regulation of the EGFR. In a second focus, I studied mechanisms of antibody targeting of EGFR. There are several monoclonal therapeutic antibodies that are in clinical development or use that target the EGFR/ErbB/HER family of receptor tyrosine kinases, including cetuximab/Erbitux™, panitumumab/Vectibix™, and necitumumab/Portrazza™, which all target EGFR, as well as trastuzumab/Herceptin™ and pertuzumab/Perjeta™, which target ErbB2/HER2. Unfortunately, as observed for most targeted therapies for cancer, resistance to these antibody therapies limits the duration of their effective treatment. Recent exome sequencing analyses of KRAS-WT colorectal cancer patients resistant to cetuximab treatment has identified epitope mutations as a mechanism of resistance. Whereas these mutated receptors bind cetuximab with dramatically decreased affinities, I report that they retain high affinity binding for necitumumab, a humanized IgG1 anti-EGFR antibody that shares the same epitope as cetuximab and panitumumab, and was recently FDA approved for squamous non-small cell lung carcinoma. I determined an X-ray crystal structure of the Fab fragment of necitumumab with the most commonly found resistance mutation—S492R (or S468R using the numbering scheme that starts at the beginning of the mature EGFR protein). This structure reveals a relatively hydrophobic cavity in the paratope of necitumumab that can accommodate the arginine at position S492/468 in the EGFR epitope. Further I find that other cetuximab and panitumumab resistance variants of EGFR are also permissive for necitumumab binding, suggesting significant plasticity in binding of necitumumab to EGFR. A survey of structures of therapeutic antibodies bound to their targets suggests that paratope shape may be an important property to consider in the selection of monoclonal antibodies in therapeutic strategies. Another mechanism of oncogenic dysregulation is the gene rearrangement of EGFR that results in EGFR variant III (EGFRvIII), an important target of many classes of immunotherapies for glioblastoma multiforme (GBM). I show in small angle X-ray scattering analyses of the ectodomain of EGFRvIII some evidence of structural flexibility in domain II that may be important for its documented transactivation of other receptor tyrosine kinases. I also report an X-ray crystal structure of the ectodomain of EGFRvIII in complex with the antigen binding or VHH domain of a camelid heavy-chain only antibody (HCAb), that has ~25-fold specificity for EGFRvIII compared to wild type EGFR. The structure reveals that the VHH gains specificity for EGFRvIII by targeting an epitope on domain IV that is sterically occluded in wild type EGFR by the intramolecular ‘tether’. This structure provides the direct evidence of dynamic uncoupling of the ‘tether’. My work corroborates the utility of the ‘tether’ as a source of antibody specificity for oncogenic EGFR, and is the first structural view of specific antibody targeting of an oncogenic EGFR variant

Structural Evaluation of EGFR Inhibition Mechanisms for Nanobodies/VHH Domains

SummaryThe epidermal growth factor receptor (EGFR) is implicated in human cancers and is the target of several classes of therapeutic agents, including antibody-based drugs. Here, we describe X-ray crystal structures of the extracellular region of EGFR in complex with three inhibitory nanobodies, the variable domains of heavy chain only antibodies (VHH). VHH domains, the smallest natural antigen-binding modules, are readily engineered for diagnostic and therapeutic applications. All three VHH domains prevent ligand-induced EGFR activation, but use two distinct mechanisms. 7D12 sterically blocks ligand binding to EGFR in a manner similar to that of cetuximab. EgA1 and 9G8 bind an epitope near the EGFR domain II/III junction, preventing receptor conformational changes required for high-affinity ligand binding and dimerization. This epitope is accessible to the convex VHH paratope but inaccessible to the flatter paratope of monoclonal antibodies. Appreciating the modes of binding and inhibition of these VHH domains will aid in developing them for tumor imaging and/or cancer therapy

Complex Relationship between Ligand Binding and Dimerization in the Epidermal Growth Factor Receptor

The epidermal growth factor receptor (EGFR) plays pivotal roles in development and is mutated or overexpressed in several cancers. Despite recent advances, the complex allosteric regulation of EGFR remains incompletely understood. Through efforts to understand why the negative cooperativity observed for intact EGFR is lost in studies of its isolated extracellular region (ECR), we uncovered unexpected relationships between ligand binding and receptor dimerization. The two processes appear to compete. Surprisingly, dimerization does not enhance ligand binding (although ligand binding promotes dimerization). We further show that simply forcing EGFR ECRs into preformed dimers without ligand yields ill-defined, heterogeneous structures. Finally, we demonstrate that extracellular EGFR-activating mutations in glioblastoma enhance ligand-binding affinity without directly promoting EGFR dimerization, suggesting that these oncogenic mutations alter the allosteric linkage between dimerization and ligand binding. Our findings have important implications for understanding how EGFR and its relatives are activated by specific ligands and pathological mutations

EGFR mutations cause a lethal syndrome of epithelial dysfunction with progeroid features

The epidermal growth factor receptor (EGFR) is part of a large family of receptors required for communicating extracellular signals through internal tyrosine kinases. Epidermal growth factor (EGF) signaling is required for tissue development, whereas constitutive activation of this signaling pathway is associated with oncogenic transformation. We identified homozygous c.1283G>A (p.Gly428Asp) mutations in the extracellular domain of EGFR in two siblings. The children were born prematurely, had abnormalities in skin and hair, suffered multisystem organ failure, and died in the neonatal period from intestinal perforation. EGF failed to induce mutated receptor phosphorylation in patient-derived fibroblasts and activation of downstream targets was suppressed. The heterologously expressed extracellular domain was impaired in stability and the binding of EGF. Cells from the affected patient undergo early senescence with accelerated expression of β-galactosidase and shortened telomeres at all passages when compared to controls. A comparison of homozygous inherited regions from a separate report of a patient from the same ethnic background and EGFR genotype confirms the pathogenicity of EGFR mutations in congenital disease