Elevated levels of EGFR have been detected in a variety of human cancers. Several reports have demonstrated that the Type III EGF receptor deletion-mutant (EGFRvIII) is frequently detected in various human cancers, including breast cancer. We have generated a monoclonal antibody against EGFRvIII, termed 4-5H. Fluorescence-activated cell sorting (FACS), immunoblotting, and immunohistochemistry were used to characterize mAb4-5H in comparison with mAbs 528 (anti-EGFR) and DH8.3/Ab-18 (anti-EGFRvIII). Both EGFRvIII mAbs 4-5H and DH8.3/Ab-18 demonstrate virtually no reactivity to wild-type EGFR expressing cell lines but strong staining in EGFRvIII expressing cells by immunohistochemistry. However, FACS analysis reveals that affinity of mAb DH8.3/Ab-18 to EGFRvIII is much weaker than mAb 4-5H and that of mAb528. Immunohistochemistry analysis of human tumor specimens revealed that mAb4-5H showed staining in a subset of breast carcinoma cells and brain tumor, but no staining in the adjacent normal tissues. We also show that EGFRvIII is phosphorylated in breast carcinoma, despite the absence of gene amplification of EGFR occurs in breast cancer. This study provides the first evidence that EGFRvIII is phosphorylated, and hence activated, in breast cancer.

[Proc Amer Assoc Cancer Res, Volume 45, 2004]