The epidermal growth factor receptor (EGFR) is a type-1 transmembrane receptor tyrosine kinase, which activates the downstream signaling cascades in many tumors, such as oral and lung cancers. We previously developed EMab-134, a novel anti-EGFR monoclonal antibody (mAb), which reacts with endogenous EGFR-expressing cancer cell lines and normal cells independent of glycosylation in Western blotting, flow cytometry, and immunohistochemical analysis. EMab-134 showed very high sensitivity (94.7%) to oral squamous cell carcinomas in immunohistochemical analysis. In this study, we performed enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of EMab-134. A blocking peptide (375-394 amino acids of EGFR) neutralized the EMab-134 reaction against oral cancer cells in flow cytometry and immunohistochemistry. The minimum epitope of EMab-134 was found to be the 377-RGDSFTHTPP-386 sequence. Our findings can be applied for the production of more functional anti-EGFR mAbs that in turn can be used for antitumor treatments.
Keywords: BSA, bovine serum albumin; DAB, 3,3-diaminobenzidine tetrahydrochloride; DMEM, Dulbecco's Modified Eagle's Medium; EDTA, ethylenediaminetetraacetic acid; EGFR; Epitope mapping; FBS, fetal bovine serum; Monoclonal antibody; Oral cancer; PBS, phosphate-buffered saline; SCC, squamous cell carcinoma; mAb, monoclonal antibody.