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 (EGFRvIII) 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.


METHODS
Here we have tested the anti-EGFR monoclonal antibody cetuximab for its interaction with EGFRvIII. Chinese hamster ovary (CHO), 32D (non-tumorigenic murine hematopoetic cells), and U87-MG stable transfectants were generated to express EGFRvIII.


RESULTS
Analysis of receptor phosphorylation showed that the EGFRvIII was constitutively phosphorylated in transfected cells. Flow cytometry, direct binding, and immunoprecipitation analysis of EGFRvIII transfectants showed specific binding of cetuximab to EGFRvIII. Cetuximab bound to EGFRvIII 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 EGFRvIII on the cell surface led to at least 50% of the cetuximab-EGFRvIII complex internalized from cell surface of CHO-EGFRvIII after 3 hours. This internalization led to a reduction in phosphorylated EGFRvIII in transfected cells. Furthermore, incubation of cells expressing EGFRvIII with cetuximab resulted in 40-50% inhibition of cell proliferation.


CONCLUSIONS
These data suggest that cetuximab may be a potential candidate for the treatment of tumors that also express EGFRvIII.
Patel
Dipa
D
Departments of Experimental Therapeutics, ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA.
Lahiji
Armin
A
Patel
Sheetal
S
Franklin
Matthew
M
Jimenez
Xenia
X
Hicklin
Daniel J
DJ
Kang
Xiaoqiang
X
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism* / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized; D016466:CHO Cells; D045744:Cell Line, Tumor; D000068818:Cetuximab; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation; D010766:Phosphorylation; D015398:Signal Transduction; D013379:Substrate Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; C410723:epidermal growth factor receptor VIII; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D000068818:Cetuximab