(535c) Novel Bispecific Fibronectin Domain Constructs Downregulate EGFR

The epidermal growth factor receptor (EGFR) is a validated target in oncology. Yet the FDA-approved antibodies against EGFR exhibit only modest therapeutic efficacy. We hypothesize that these deficiencies result from incomplete inhibition of receptor activity that arises from autocrine ligand stimulation, untargeted EGFR variant III, and insufficient receptor downregulation. Thus, as a complement to ligand competition, we sought to downregulate EGFR with novel heterobivalent constructs capable of crosslinking multiple EGFRs. To facilitate construction of bispecific agents we used the small, single-domain fibronectin type III module, which is a validated scaffold for molecular recognition.

Numerous fibronectin domains of picomolar to nanomolar affinity for EGFR were engineered via yeast surface display and directed evolution. Collectively, these clones target multiple conformational epitopes that were identified at amino acid resolution. Protein fusions of all homo- and hetero-bivalent combinations, connected via a flexible peptide linker, were tested for their ability to downregulate EGFR. Several heterobivalent agents downregulate EGFR robustly (up to 80% decrease in EGFR expression) and relatively swiftly (half-time of downregulation from 0.2-1.2 h). The impacts of linker length, domain orientation, epitope, and receptor density were investigated. The mechanism of action was studied via microscopy and analyses of both receptor and cellular phosphorylation. The downregulating bispecifics inhibit proliferation and migration of both normal and autocrine cell lines and thereby provide potential as therapeutic agents.