MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 4, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0010

Original Articles

Epitope Mapping of an Anti-Human Epidermal Growth
Factor Receptor Monoclonal Antibody (EMab-51)
Using the RIEDL Insertion for Epitope Mapping Method

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Ren Nanamiya,1 Masato Sano,1 Teizo Asano,1 Miyuki Yanaka,1 Takuro Nakamura,1 Masaki Saito,2Tomohiro Tanaka,1 Hideki Hosono,1 Nami Tateyama,1 Mika K. Kaneko,1 and Yukinari Kato1–3,iThe classic method for identifying the epitope that monoclonal antibodies (mAbs) bind uses deletion mutants
and point mutants of the target protein. However, determining the epitope of mAbs-reactive membrane proteinsis often challenging. We recently developed the RIEDL insertion for epitope mapping (REMAP) method to identify mAb-binding epitopes. Herein, we first checked the reactivity of an anti-epidermal growth factor receptor(EGFR) mAb (EMab-51) to several EGFR deletion mutants such as EGFR/dN152, EGFR/dN313, EGFR/
dN370, EGFR/dN375, EGFR/dN380, and EGFR/dN482. We found the N-terminus of the EMab-51-binding epitope between residues 375 and 380 of EGFR. We next produced EGFR/dN313 mutants with the RIEDL peptide
tag inserted at each possible position of 375-AFRGDSFTHTPPLDP-389. EMab-51 lost its reactivity with the mutants having a RIEDL tag inserted at each position of 377-RGDSFTHTPP-386, whereas LpMab-7 (an anti-RIEDL
mAb) detected every mutant. Thus, using the REMAP method, we identified the EMab-51-binding epitope of
EGFR as 377-RGDSFTHTPP-386.
Keywords: EGFR, EMab-51, epitope mapping, monoclonal antibody, RIEDL tag
IntroductionT

he epidermal growth factor receptor (EGFR)
belongs to the human epidermal growth factor receptor(HER) family.(1) The HER family is composed of four different receptors with a common structure, including EGFR(HER1/ErbB1), HER2 (ErbB2/neu), HER3 (ErbB3), andHER4 (ErbB4).(2) When the ligand (EGF) binds the EGFR, it
stabilizes the receptor dimer. The two subunits phosphorylate
each other in the C-terminal region of the cytoplasmic side,which transmits signals downstream.(3,4) Besides, EGFR canform homodimers or heterodimers with the other HER family
members (HER2, HER3, and HER4).(5,6) The downstream
effectors of EGFR include PI3K/AKT/mTOR, RAS/RAF/
MAPK, and JAK/STAT, which act on division, migration,
anti-apoptosis, and cell cycle progression.(7) Many cancers
overexpress EGFR, including squamous cell carcinoma of
the esophagus,(8) brain tumor,(9,10) lung cancer,(11) head andneck cancer,(12) colorectal cancer,(13,14) breast cancer,(15)bladder cancer,(16) clear cell renal cell carcinoma,(17) ovariancancer,(18) prostate cancer,(19) pancreatic cancer,(20) and melanoma.(21) Therefore, monoclonal antibodies (mAbs) that can
detect EGFR with high sensitivity are essential.

Because mAbs usually recognize epitopes consisting ofseveral amino acids, they often might cross-react with unexpected proteins. Therefore, identifying the epitopes thatmAbs recognize is important to avoid unexpected crossreactivity, and is helpful for the development of antibody
drugs. Epitope identification methods include site-directedmutagenesis mapping, array-based oligopeptide scanning, and
X-ray co-crystallography.(22) Because X-ray co-crystallography
allows to directly visualize the mAb-antigen interaction, it
gives the clearest epitope identification. However, crystallizing the antigen-mAb complex is usually costly and timeconsuming. Although the array-based oligopeptide scanningand site-directed mutagenesis mapping can easily identify
linear epitopes, they are not appropriate for conformational
epitopes.
We previously developed a novel anti-EGFR mAb, EMab51 (IgG1, kappa),(23) by immunizing mice with the purified
recombinant ectodomain of EGFR (EGFRec) from culture
supernatants of LN229/EGFRec cells. Importantly, this mAb
is useful for flow cytometry, Western blotting, and immunohistochemical analyses against EGFR. Accordingly, apractical use of EMab-51 will likely be EGFR detection invarious tumors. However, we failed to identify the EMab-51-

Departments of 1Antibody Drug Development and 2Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai,
Japan.
3
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
i
ORCID ID (https://orcid.org/0000-0001-5385-8201).

149

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binding epitope using conventional epitope-mapping methods such as alanine scanning. In this study, we aimed to
identify the EGFR epitope recognized by EMab-51 using the
RIEDL insertion for epitope mapping (REMAP) method.(24)Materials and Methods
Cell lines(SH800; Sony Corp., Tokyo, Japan). We cultured the CHOK1 cells and transfectants in RPMI 1640 medium (Nacalai
Tesque, Inc., Kyoto, Japan) supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific,Inc.), 100 U/mL of penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.) at 37C in
a humidified atmosphere containing 5% CO2. We cultivated
the transfectants in a medium containing 0.5 mg/mL Zeocin(InvivoGen, San Diego, CA).
Plasmid preparation

We amplified the EGFR open reading frame as described
previously.(25) We produced the EGFR deletion mutants

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We obtained Chinese hamster ovary (CHO)-K1 cells fromthe America Type Culture Collection (ATCC, Manassas,
VA). We transfected the EGFR mutation plasmids into CHOK1 cells using Neon Transfection System (Thermo Fisher
Scientific, Inc., Waltham, MA) and sorted the stable transfectants using an anti-PA tag mAb (NZ-1) with a cell sorter

NANAMIYA ET AL.

FIG. 1. Epitope mapping using EGFR deletion mutants. (A, B) We analyzed the EGFR deletion mutants using flow
cytometry. We expressed the EGFR deletion mutants in CHO-K1 cells and then incubated them with EMab-51 (A), anti-PA
tag mAb (NZ-1) (B), or buffer control (A, B) for 30 min at 4C. Finally, we treated them with the corresponding secondary
antibodies. (C) Schematic illustration of EGFR and three deletion mutants with an N-terminal PA tag. Deletion mutants of
EGFR: dN152, dN313, and dN482. EMab-51 recognized dN152 and dN313 (in black) but not dN482 (in white). CHO,
Chinese hamster ovary; EGFR, epidermal growth factor receptor; IC, intracellular domain; mAbs, monoclonal antibodies;SS, signal sequence; TM, transmembrane domain.

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EPITOPE MAPPING OF EMAB-51

151

FIG. 2. Epitope mapping using EGFR deletion mutants. (A, B) We analyzed the EGFR deletion mutants using flow
cytometry. We expressed the EGFR deletion mutants in CHO-K1 cells and then incubated them with EMab-51 (A), anti-PA
tag mAb (NZ-1) (B), or buffer control (A, B) for 30 minutes at 4C. Finally, we treated them with the corresponding
secondary antibodies. (C) Schematic illustration of three deletion mutants with an N-terminal PA tag. Deletion mutants of
EGFR: dN370, dN375, and dN380. EMab-51 recognized dN370 and dN375 (in black) but not dN380 (in white).
(EGFR/dN152, EGFR/dN313, EGFR/dN370, EGFR/dN375,
EGFR/dN380, and EGFR/dN482) using HotStar HiFidelity
Polymerase Kit (Qiagen, Inc., Hilden, Germany) with oligonucleotides containing the desired mutations. At the Nterminus, we added the PA tag (GVAMPGAEDDVV),(26)which is recognized by NZ-1.(27) We inserted the RIEDL
tag(24) in the EGFR sequence using a HotStar HiFidelity
Polymerase Kit with oligonucleotides containing the RIEDL
tag insertions at the desired position in 375-AFRGDSFTHTPPLDP-389 of EGFR/dN313. For example, we produced

Ala375_RIEDL_Phe376 (A375_R*_F376) by inserting the
RIEDL sequence between Ala375 and Phe376 of EGFR/
dN313. We inserted the polymerase chain reaction fragments bearing the desired mutations into the pCAG-Ble
vector using an In-Fusion HD Cloning Kit (Takara Bio, Inc.,
Shiga, Japan). The RIEDL tag insertion mutants produced are
Ala375_RIEDL_Phe376 (A375_R*_F376), Phe376_RIEDL_Arg377 (F376_R*_R377), Arg377_RIEDL_Gly378(R377_R*_G378), Gly378_RIEDL_Asp379 (G378_R*_
D379), Asp379_RIEDL_Ser380 (D379_R*_S380), Ser380_‰
FIG. 3. Epitope mapping using RIEDL tag insertion mutants of EGFR/dN313. RIEDL tag was inserted into EGFR/
dN313. The RIEDL tag insertion mutants are as follows: Ala375_RIEDL_Phe376 (A375_R*_F376), Phe376_RIEDL_
Arg377 (F376_R*_R377), Arg377_RIEDL_Gly378 (R377_R*_G378), Gly378_RIEDL_Asp379 (G378_R*_D379), Asp379_
RIEDL_Ser380 (D379_R*_S380), Ser380_RIEDL_Phe381 (S380_R*_F381), Phe381_RIEDL_Thr382 (F381_R*_T382),
Thr382_RIEDL_His383 (T382_R*_H383), His383_RIEDL_Thr384 (H383_R*_T384), Thr384_RIEDL_Pro385 (T384_
R*_P385), Pro385_RIEDL_Pro386 (P385_R*_P386), Pro386_RIEDL_Leu387 (P386_R*_L387), Leu387_RIEDL_
Asp388 (L387_R*_D388), and Asp388_RIEDL_Pro389 (D388_R*_P389). We analyzed the RIEDL tag insertion mutants using flow cytometry. We expressed the RIEDL tag insertion mutants in CHO-K1 cells and incubated them with(A) EMab-51, (B) anti-PA tag mAb (NZ-1), (C) anti-RIEDL tag mAb (LpMab-7), or buffer control (A–C) for 30
minutes at 4C. Finally, we treated them with the corresponding secondary antibodies.

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EPITOPE MAPPING OF EMAB-51

RIEDL_Phe381 (S380_R*_F381), Phe381_RIEDL_Thr382(F381_R*_T382), Thr382_RIEDL_His383 (T382_R*_
H383), His383_RIEDL_Thr384 (H383_R*_T384), Thr384_
RIEDL_Pro385 (T384_R*_P385), Pro385_RIEDL_Pro386(P385_R*_P386), Pro386_RIEDL_Leu387 (P386_R*_
L387), Leu387_RIEDL_Asp388 (L387_R*_D388), andAsp388_RIEDL_Pro389 (D388_R*_P389).

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Flow cytometry

We harvested the cells by brief exposure to 0.25%
trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai
Tesque, Inc.). After washing in 0.1% bovine serum albuminin phosphate-buffered saline (blocking buffer), the cells weretreated with primary mAbs, such as EMab-51 (mouse IgG1,
kappa), NZ-1 (rat IgG2a, lambda), or LpMab-7 (mouse IgG1,
kappa) at a concentration of 1 lg/mL for 30 minutes at 4C;
subsequently, with Alexa Fluor 488-conjugated anti-mouse
IgG or Alexa Fluor 488-conjugated anti-rat IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA). We collected
fluorescence data using an EC800 Cell Analyzer (SonyCorp.).
Results
Determination of the EMab-51-recognized epitope
using EGFR deletion mutants

EMab-51 might recognize an epitope located at the extracellular region of EGFR because we obtained it by immunizing mice with EGFRec from culture supernatants of
LN229/EGFRec cells.(23) First, we produced N-terminal deletion mutants (EGFR/dN152, EGFR/dN313, and EGFR/
dN482) with a PA tag at their N-terminus and investigated thereactivity between EMab-51 and each deletion mutant by
flow cytometry analysis. EMab-51 recognized EGFR/wild
type, EGFR/dN152, and EGFR/dN313, but not EGFR/dN482(Fig. 1A). In contrast, NZ-1 detected all the deletion mutants(EGFR/dN152, EGFR/dN313, and EGFR/dN482) (Fig. 1B).
These results show that the N-terminus of the EMab-51 epitope exists between residues 313 and 482 (Fig. 1C).
Next, we produced additional N-terminal deletion mutants(EGFR/dN370, EGFR/dN375, and EGFR/dN380) with a PA
tag at their N-terminus and investigated the reactivity between EMab-51 and each deletion mutant by flow cytometry
analysis. EMab-51 recognized EGFR/dN370 and EGFR/

153

dN375, but not EGFR/dN380 (Fig. 2A). In contrast, NZ-1
detected all the deletion mutants (EGFR/dN370, EGFR/
dN375, and EGFR/dN380) (Fig. 2B). These results show that
the N-terminus of the EMab-51 epitope exists between residues 375 and 380 (Fig. 2C).
Determination of the EMab-51-recognized epitope
using the REMAP method

To identify the amino acids composing the conformational
epitope binding EMab-51, we used the REMAP method.(24)
We constructed 14 EGFR/dN313 mutants, in which we inserted a RIEDL tag into the expected epitope region at eachpossible position of 375-AFRGDSFTHTPPLDP-389. The antiRIEDL tag mAb (clone LpMab-7) recognized the five aminoacid-long RIEDL tag.(24) For example, we produced Ala375_
RIEDL_Phe376 (A375_R*_F376) by inserting the RIEDL
sequence between Ala375 and Phe376 of EGFR/dN313.
Flow cytometry analysis showed that EMab-51 did notreact with nine mutants, such as Arg377_RIEDL_Gly378(R377_R*_G378), Gly378_RIEDL_Asp379 (G378_R*_
D379), Asp379_RIEDL_Ser380 (D379_R*_S380), Ser380_
RIEDL_Phe381 (S380_R*_F381), Phe381_RIEDL_Thr382(F381_R*_T382), Thr382_RIEDL_His383 (T382_R*_H383),
His383_RIEDL_Thr384(H383_R*_T384),
Thr384_RIEDL_Pro385 (T384_R*_P385), and Pro385_RIEDL_
Pro386 (P385_R*_P386) although it strongly detected fourmutants, such as Ala375_RIEDL_Phe376 (A375_R*_F376),
Phe376_RIEDL_Arg377 (F376_R*_R377), Leu387_RIEDL_
Asp388 (L387_R*_D388), and Asp388_RIEDL_Pro389 (D388_R*_P389), and weakly detected Pro386_RIEDL_Leu387(P386_R*_L387) (Fig. 3A), indicating that EMab-51 might bindto EGFR through 10 amino acids (377-RGDSFTHTPP-386). The
positive controls NZ-1 and LpMab-7 detected all 14 mutants(Fig. 3B, C). Thus, using the REMAP method, we determinedthat EMab-51 binds to the EGFR epitope 377-RGDSFTHTPP-386(Fig. 4).
Discussion

mAb-binding epitope investigations often use alaninescanning mutagenesis and peptide screening.(22,28–41) Although these methods are effective for identifying linear
epitopes, they are inapplicable to conformational epitopes.
By immunizing mice with EGFRec, we previously developed

FIG. 4. Schematic illustration of EMab-51 epitope. The EMab-51 epitope is 377-RGDSFTHTPP-386, which is located in
the extracellular domain III of EGFR.

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154

a novel anti-EGFR mAb (clone EMab-51) (23). EMab-51 hasapplications not only in flow cytometry and Western blotting
but also in immunohistochemical analysis to detect EGFR
in various cancers. Unfortunately, we could not determine
the EMab-51-binding epitope using conventional epitopemapping methods such as alanine scanning. This is because
EMab-51 recognizes a conformational epitope and a singleamino acid substitution may insufficiently disrupt the epitope
conformation region to inhibit EMab-51 binding.
In this study, N-terminal deletion mutant analyses showedthat the N-terminus of the EMab-51-binding epitope is located between EGFR residues 375 and 380 (Fig. 2). To identify
the critical epitope amino acids, we employed the REMAP
method.(24) The flow cytometry analysis showed that EMab51 lost its reactivity to some RIEDL tag insertion mutants(Fig. 3). Thus, the RIEDL tag insertion may have partiallydisrupted the EGFR conformation and the binding of EMab51 to EGFR. Using the REMAP method, we successfully
determined that the EMab-51 epitope is 377-RGDSFTH
TPP-386, which is located in the extracellular domain III of
EGFR (Fig. 4). These amino acids are continuous, but may
not form a linear structure. The epitope identification of
EMab-51 will be helpful for the development of EGFRtargeting therapeutic antibodies in the future.
Authors’ Contributions

R.N., M.Sano., T.A., M.Y., T.N., M.Saito, T.T., H.H., and
N.T. performed experiments; M.K.K. designed the experiments; R.N. and Y.K. wrote the article.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
Numbers JP21am0401013 (to Y.K.) and JP21am0101078(to Y.K.), and by the Japan Society for the Promotionof Science ( JSPS) Grants-in-Aid for Scientific Research(KAKENHI) Grant Numbers 21K15523 (to T.A.), 21K07168(to M.K.K.), 20K16322 (to M.Sano), and 19K07705 (to Y.K.).
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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 9, 2021
Accepted: June 5, 2021