Activity and binding mechanism of cetuximab (Erbitux®) to the type III EGF deletion-mutant receptor

1238 The epidermal growth factor receptor (EGFR) plays an important role in the growth and survival of many human tumors of epithelial origin. EGFR variant III (EGFRvIII) is a truncated form of EGFR in which residues 6-273 have been deleted; this shortened receptor does not bind ligand, is constitutively active, and is reported to be co-expressed with EGFR on some human tumors including glioblastoma, breast, lung, and prostate. The deleted amino acid residues represent all of domain I and the amino-terminal 2/3 of domain II while domains III and IV of the extracellular region remain intact and should be structurally similar to those of the full-length receptor. Here we have tested the anti-EGFR monoclonal antibody ERBITUX (cetuximab) for its ability to bind EGFRvIII. A crystal structure of the complex of cetuximab bound to wild type EGFR extracellular domain reveals that the antibody binds exclusively to domain III of the receptor. A model of the EGFRvIII protein suggests that cetuximab binding should be unaffected by the deletion and be fully active against this mutant receptor. Chinese hamster ovary (CHO), 32D (non-tumorigenic murine hematopoetic cells), and U87-MG (EGFR positive Glioblastoma cells) stable transfectants were generated that express EGFRvIII. Analysis of receptor phosphorylation showed that EGFRvIII was constitutively phosphorylated in transfected cells. Flow cytometry and immunoprecipitation analysis of EGFRvIII transfectants showed specific binding of cetuximab to EGFRvIII. Binding of cetuximab to EGFRvIII on the cell surface led to the internalization of the cetuximab-EGFRvIII complex. This internalization led to a reduction in phosphorylated EGFRvIII in transfected cells and inhibition of cell proliferation. In addition, cetuximab induced an ADCC reaction in tumor cells expressing EGFRvIII. These results indicate the binding epitope for cetuximab is outside the region of the EGFRvIII deletion, consistent with the model structure. These data suggest that cetuximab may be a potential candidate for the treatment of tumors that also express EGFRvIII.