Monoclonal antibody cetuximab binds to and down-regulates constitutively activated epidermal growth factor receptor vIII on the cell surface

Background: 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 (EGFR nu III) is a truncated form of EGFR that does not bind ligand, is constitutively active, and is reported to be coexpressed with EGFR in some human tumors including breast, glioblastoma, lung, and prostate. Materials and Methods: Here we have tested the anti-EGFR monoclonal antibody cetuximab for its interaction with EGFR nu III. Chinese hamster ovary (CHO), 32D (non-tumorigenic murine hematopoetic cells), and U87-MG stable transfectants were generated to express EGFR nu III. Results: Analysis of receptor phosphorylahon showed that the EGFR nu III was constitutively phosphorylated in transfected cells. Flow cytometry, direct binding, and immunoprecipitation analysis of EGFR nu III transfectants showed specific binding of cetuximab to EGFR nu III. Cetuximab bound to EGFR nu III with a KD of 0.38 nM determined by Scatchard analysis and 1.1 nM determined by Biacore analysis respectively. In internalization studies, binding of cetuximab to the EGFR nu III on the cell surface led to at least 50% of the cetuximab-EGFR nu III complex internalized from cell surface of CHO-EGFR nu III after 3 hours. This internalization led to a reduction in phosphotylated EGFR nu III in transfected cells. Furthermore, incubation of cells expressing EGFR nu III with cetuximab resulted in 40-50% inhibition of cell proliferation. Conclusion: These data suggest that cetuximab may be a potential candidate for the treatment of tumors that also express EGFR nu III.