Biochemistry and Biophysics Reports 14 (2018) 54–57

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Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

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

TMika K. Kanekoa, Shinji Yamadaa, Shunsuke Itaia, Yao-Wen Changa, Takuro Nakamuraa,⁎Miyuki Yanakaa, Yukinari Katob,abDepartment of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapanA R T I C LE I N FO

A B S T R A C T

Keywords:EGFR
Epitope mapping
Monoclonal antibody
Oral cancer

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, andimmunohistochemical 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 inturn can be used for antitumor treatments.

1. Introduction

glioblastoma cells or puriﬁed recombinant EGFR to produce EMab-134
clone (IgG1, 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, andimmunohistochemistry.

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 [2–4] and forms homo- or heterodimers with othermembers of the HER family, such as HER2 [5] and HER3 [6]. EGFR
overexpression is observed in many cancer types, including head andneck, lung, colorectal, breast, pancreatic, kidney, ovary, bladder, andprostate cancers [7].
Monoclonal antibodies (mAbs) against EGFR have been developed
for cancer treatment; e.g., cetuximab (a mouse–human chimeric mAb;
IgG1) against head and neck and colorectal cancers; panitumumab (afully human mAb; IgG2) against colorectal cancers; and necitumumab(a fully human mAb; IgG1) against non-small cell lung cancers [8–10].
Anti-EGFR mAbs possess diverse functional mechanisms, such as
blocking ligand binding, blocking dimerization, EGFR endocytosis,antibody-dependent cellular cytotoxicity, and complement-dependent
cytotoxicity.
In our previous study, we immunized mice with EGFR-expressed

2. Materials and methods
2.1. Cell lines
LN229/EGFR was previously established [11,12]. HSC-3 (oralsquamous carcinoma cell line from tongue) was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).
LN229/EGFR and HSC-3 were cultured in Dulbecco's Modiﬁed Eagle'sMedium (DMEM; Nacalai Tesque, Inc., Kyoto, Japan), supplemented
with 10% heat-inactivated fetal bovine serum (Thermo Fisher ScientiﬁcInc., Waltham, MA), 100 units/ml penicillin, 100 μg/ml streptomycin,

Abbreviations: mAb, monoclonal antibody; SCC, squamous cell carcinoma; DMEM, Dulbecco's Modiﬁed Eagle's Medium; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum
albumin; PBS, phosphate-buﬀered saline; FBS, fetal bovine serum; DAB, 3,3-diaminobenzidine tetrahydrochloride⁎
Corresponding author.
E-mail addresses: yukinarikato@med.tohoku.ac.jp, yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2018.03.010
Received 22 March 2018; Received in revised form 28 March 2018; Accepted 30 March 2018
2405-5808/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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M.K. Kaneko et al.

and 25 μg/ml amphotericin B (Nacalai Tesque, Inc.), and incubated at
37 °C in a humidiﬁed atmosphere of 5% CO2 and 95% air.
2.2. Enzyme-linked immunosorbent assay (ELISA)Synthesized EGFR (Accession No.: NP_005219) peptides using
PEPScreen (Sigma-Aldrich Corp., St. Louis, MO) and extracellular domain of EGFR (EGFRec) were immobilized on Nunc Maxisorp 96-wellimmunoplates (Thermo Fisher Scientiﬁc Inc.) at 10 μg/ml for 30 min at
37 °C or over night at 4 °C. After blocking with SuperBlock T20 (PBS)
Blocking Buﬀer (Thermo Fisher Scientiﬁc Inc.), the plates were incubated with puriﬁed EMab-134 (10 μg/ml), followed by a 1:2000 dilution of peroxidase-conjugated anti-mouse IgG (Agilent TechnologiesInc., Santa Clara, CA). The enzymatic reaction was conducted using 1Step Ultra TMB-ELISA (Thermo Fisher Scientiﬁc Inc.). Optical density
was measured at 655 nm using an iMark microplate reader (Bio-RadLaboratories, Inc., Berkeley, CA). These reactions were performed at37 °C with a total sample volume of 50–100 μl.
2.3. Flow cytometry
Cells were harvested after brief exposure to 0.25% trypsin/1 mM
ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). Afterwashing with 0.1% bovine serum albumin in PBS, the cells were treatedwith EMab-134 (10 μg/ml) or EMab-134 (10 μg/ml) plus peptides(10 μg/ml) for 30 min at 4 °C, followed by treatment with Alexa Fluor
488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology,Inc., Danvers, MA). Fluorescence data were acquired using the CellAnalyzer SA3800 (Sony Corp., Tokyo, Japan).
2.4. Immunohistochemical analyses
Fig. 1. (A) ELISA with EGFR peptides. (B) Schematic illustration of the EMab134-epitope. EGFRec, EGFR extracellular domain; SS, signal peptide; TM,transmembrane; IC, intracellular.

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 andapproved the use of human cancer tissues. Written informed consent
was obtained for the use of human cancer tissue samples. HistologicalSections (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) orEMab-134 (5 μg/ml) plus peptides (5 μg/ml) for 1 h at room temperature, treated using an Envision+ kit (Agilent Technologies Inc.) for30 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).

targeted therapy against EGFR.
In this study, three deletion mutants of EGFR were expressed transiently in CHO-K1 cells, including dN152 [corresponding to 152–1210aa], dN313 (corresponding to 313–1210 aa), and dN482 (corresponding to 482–1210 aa) (data not shown). All deletion mutants ofEGFR 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 theN-terminus of EMab-134 epitope existed between aa 313 and 482.
We next synthesized a series of EGFR peptides (SupplementaryTable 1). EMab-134 reacted with the 375–394 aa (375-AFRGDSFTHTPPLDPQELDI−394) sequence among the peptides and with the extracellular domain of EGFR (EGFRec) on ELISA (Fig. 1 and Fig. 1B).
We performed a blocking assay using ﬂow cytometry. EMab-134
reacted with the HSC-3 and LN229/EGFR cell lines (Fig. 2). This reaction was partially neutralized by a 375–394-aa sequence. In contrast,
the 365–384-aa (365-ISGDLHILPVAFRGDSFTHT−384) and 385–404-aa(385-PPLDPQELDILKTVKEITGF−404) sequences did not block the reaction of EMab-134 with both HSC-3 and LN229/EGFR, thereby conﬁrming that the 375–394-aa sequence is a critical epitope of EMab-134.
We compared the binding aﬃnity of EMab-134 against a 375–394-aa
peptide with that against EGFRec in ELISA. As a result, the binding
aﬃnity against EGFRec (KD: 1.1 × 10−9) is much higher than thatagainst a 375–394-aa peptide (KD: 1.6 × 10−7). Therefore, a 375–394aa peptide might not neutralize EMab-134 reaction completely against
HSC-3 and LN229/EGFR cell lines in ﬂow cytometry.
We next performed a blocking assay using immunohistochemistry
and found that EMab-134 reacted with oral cancer tissues (Fig. 3) andthat the reaction was neutralized by the 375–394-aa sequence. The

2.5. Determination of binding-aﬃnity by ELISAEGFRec 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 TechnologiesInc.). The dissociation constants (KD) were obtained by ﬁtting thebinding isotherms using the built-in one-site binding models in Prism
software.
3. 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 andWestern 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 molecular55

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M.K. Kaneko et al.

Fig. 2. Flow cytometry of HSC-3 and LN229/EGFR cells. The cells were reacted with EMab-134 (10 μg/ml) plus peptides (10 μg/ml) for 30 min at 4 °C, followed bytreatment with Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence data were acquired using the cell analyzer SA3800. Gray peak, negative control; blue peak,EMab-134; red peak, EMab-134 plus each peptide.

In conclusion, our results indicated that the critical epitope ofEMab-134 is the 377-RGDSFTHTPP−386 sequence. Our ﬁndings can beused for the production of more functional anti-EGFR mAbs, which
would be advantageous for eliciting antitumor eﬀects against EGFRexpressing cancers.

365–384-aa and 385–404-aa sequences did not block the reaction ofEMab-134, thereby conﬁrming the results of ﬂow cytometric blocking
assays.
Finally, we synthesized a series of point mutations of EGFR peptidesof length 375–394 aa (Supplementary Table 1). EMab-134 reacted withA375G, F376A, L387A, D388A, P389A, Q390A, E391A, L392A, D393A,
and I394A, but not with R377A, G378A, D379A, S380A, F381A, T382A,H383A, T384A, P385A, and P386A, indicating that the EGFR peptides
from aa 377 to 386 (377-RGDSFTHTPP−386) constituted the criticalminimum epitope of EMab-134 (Fig. 4 and Supplementary Fig. 1). Although we checked whether the RGDSFTHTPP sequence is conserved inHER family, the identical sequence was not observed in HER2, HER3,
and HER4 (Supplementary Fig. 2).

Acknowledgments
We thank Saori Handa and Yoshimi Nakamura for excellent technical assistance.

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M.K. Kaneko et al.

Fig. 4. (A) ELISA with EGFR peptides. (B) Schematic illustration of EGFR andthe EMab-134-epitope. The critical epitope of EMab-134 is the 377-RGDSFTHTPP−386 sequence. SS, signal peptide; TM, transmembrane; IC, intracellular.

Appendix A. Transparency document
Supplementary data associated with this article can be found in theonline version at http://dx.doi.org/10.1016/j.bbrep.2018.03.010.
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Fig. 3. Immunohistochemistry for oral cancers. The sections were incubatedwith EMab-134 (5 μg/ml) or EMab-134 (5 μg/ml) plus peptides (5 μg/ml) andtreated using the Envision+ kit. Color development was obtained using 3,3diaminobenzidine tetrahydrochloride. The sections were then counterstainedwith hematoxylin. Scale bar = 100 µm.

Conﬂict of interest
The authors declare no conﬂicts of interest involving this article.

Funding
This research was supported in part by AMED under Grant numbers:
JP18am0301010 (Y.K.), JP18am0101078 (Y.K.), and JP18ae0101028(Y.K.), and by JSPS KAKENHI Grant number 17K07299 (M.K.K.) andGrant number 16K10748 (Y.K.).
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