Discovering conformation selective anti-EGFR nanobodies

Abstract

The epidermal growth factor receptor (EGFR) is an essential receptor tyrosine kinase that governs several cell-signaling pathways involved in growth and differentiation. Oncogenic EGFR mutations and overexpression are common in many cancer types and it is predicted more than 5% of all malignancies in the United States have mutant EGFR, with especially high prevalence in lung cancer and glioblastoma. Current anti-EGFR treatments lack longevity: over time, all cancers become resistant to anti-EGFR therapeutics with a median time of one year to resistance. In this work, I describe a novel approach to developing anti-EGFR therapies, targeting extracellular conformations that are upregulated in cancers. Chapter 1 of this dissertation provides an overview of EGFR activation in physiologic and pathogenic contexts, with an emphasis on targeting conformations that are stabilized by oncogenic mutations. In chapter two, I characterize ectodomain mutations that stabilize intermediate or preactive forms of EGFR on the cell surface. Chapter 3 covers the bulk of my dissertation work and describes the selection and characterization of a panel of anti-EGFR nanobodies, many of which show conformational selectivity. The asymmetric binding preferences of the nanobodies provide insight into conformations stabilized by common glioblastoma mutations, suggesting the location of the mutation underlies the distinct conformation that is stabilized. In Chapter 4, I discuss future directions for this work, and how these nanobodies can be further developed as research tools, diagnostics, and therapeutics. Additionally, I suggest alternative approaches for discovering nanobodies targeting conformations stabilized by oncogenic EGFR. Collectively, the work described in this dissertation enhances the current mechanistic understanding of EGFR activation and paves the way for improved anticancer agents.