ATPS-86MUTATIONS IN THE EGF RECEPTOR EXTRACELLULAR DOMAIN REVEAL AN UNTETHERED TRANSITIONAL STATE WHICH MEDIATES mAb806 BINDING

The epidermal growth factor receptor (EGFR) is both amplified and frequently mutated in glioblastoma. While downstream inhibition of EGFR signaling through small molecule tyrosine kinase inhibitors has shown little clinical efficacy in glioblastoma, monoclonal antibody targeting is proving to be a promising alternative. The monoclonal antibody 806 (mAb806) specifically binds the most common EGFR mutant, EGFRvIII, along with a subset of amplified wild type EGFR, but not EGFR expressed at normal levels. The selectivity of mAb806 is thought to be due to its targeting a tumor-specific epitope of EGFR which is only accessible as the receptor transitions between an inactive tethered conformation to an active ligand-bound dimer. Here we used molecular dynamics simulations coupled with in vitro assays in glioblastoma cells to characterize the transitional conformation of EGFR in mAb806 binding. Focusing on the mutations R108K and G63R, molecular dynamics simulation revealed a spontaneous untethering of EGFR to a compact conformation, which is characterized by a backwards rotation of domain I-II exposing both the dimerization arm and the mAb806-epitope. This configuration is structurally equivalent to EGFRvIII, suggesting that several glioblastoma mutations converge to remove domain I from the dimerization interface. Next, we evaluated the effect of these point mutations on the ability of mAb806 to bind glioblastoma cells in vitro. Using site-directed mutagenesis coupled with lentiviral-transduction, we expressed EGFR-R108K or EGFR-G63R in U87 glioblastoma cells. Investigating mAb806 binding by FACS, we show that mAb806 binds the mutant cells five times greater than control cells, validating our simulation predictions. Future studies will examine the effect of mAb806 in an in vivo tumor model. Significantly, as these point mutations have been found in several GBM patients, our results indicate the possibility of a broader patient spectrum for the use of ABT-806, a humanized version of mAb806 currently in phase I clinical trials.