Elucidation of the critical epitope of an anti-EGFR monoclonal antibody EMab-134

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.


Introduction
Epidermal growth factor receptor (EGFR) is a type-1 transmembrane glycoprotein, which is involved in cell growth and differentiation [1]. EGFR belongs to the human EGFR (HER) family of receptor tyrosine kinases [2][3][4] and forms homo-or heterodimers with other members of the HER family, such as HER2 [5] and HER3 [6]. EGFR overexpression is observed in many cancer types, including head and neck, lung, colorectal, breast, pancreatic, kidney, ovary, bladder, and prostate cancers [7].
In our previous study, we immunized mice with EGFR-expressed glioblastoma cells or purified recombinant EGFR to produce EMab-134 clone (IgG 1 , kappa), which reacted with endogenous EGFR of oral cancers in flow cytometry, Western blotting, and immunohistochemistry [11]. In immunohistochemical analysis, EMab-134 stained 36 of 38 (94.7%) oral cancer specimens. In this study, we evaluated the binding epitope of EMab-134 using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemistry.

Immunohistochemical analyses
This study examined one patient with oral cancer who underwent surgery at Tokyo Medical and Dental University [11]. The Tokyo Medical and Dental University Institutional Review Board reviewed and approved the use of human cancer tissues. Written informed consent was obtained for the use of human cancer tissue samples. Histological Sections (4-μm thick) were directly autoclaved in EnVision FLEX Target Retrieval Solution High pH (Agilent Technologies Inc.) for 20 min. After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific Inc.), sections were incubated with EMab-134 (5 μg/ml) or EMab-134 (5 μg/ml) plus peptides (5 μg/ml) for 1 h at room temperature, treated using an Envision+ kit (Agilent Technologies Inc.) for 30 min. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for 2 min, and counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Industries Ltd., Osaka, Japan).

Determination of binding-affinity by ELISA
EGFRec and a 375-394-amino acid (aa) peptide were immobilized at 1 μg/ml and 10 μg/ml, respectively. The plates were incubated with serially diluted antibodies (5 pg/ml -50 μg/ml) followed by 1:2000 diluted peroxidase-conjugated anti-mouse IgG (Agilent Technologies Inc.). The dissociation constants (K D ) were obtained by fitting the binding isotherms using the built-in one-site binding models in Prism software.

Results and discussion
We previously developed EMab-134, a novel anti-EGFR mAb, which exhibited high specificity and sensitivity against human EGFR [11]. EMab-134 was found to be useful not only for flow cytometry and Western blotting but also for immunohistochemical analyses with paraffin-embedded tissues. Therefore, the determination of the binding epitope of EMab-134 was deemed critical for developing a molecular targeted therapy against EGFR.
In this study, three deletion mutants of EGFR were expressed transiently in CHO-K1 cells, including dN152 [corresponding to 152-1210 aa], dN313 (corresponding to 313-1210 aa), and dN482 (corresponding to 482-1210 aa) (data not shown). All deletion mutants of EGFR contained an N-terminal PA tag [13] and were analyzed using flow cytometry for EMab-134 epitope mapping. NZ-1, an anti-PA tag mAb, detected all deletion mutants of EGFR. In contrast, EMab-134 did not react with dN482 (data not shown). These results indicated that the N-terminus of EMab-134 epitope existed between aa 313 and 482.
In conclusion, our results indicated that the critical epitope of EMab-134 is the 377-RGDSFTHTPP −386 sequence. Our findings can be used for the production of more functional anti-EGFR mAbs, which would be advantageous for eliciting antitumor effects against EGFRexpressing cancers.