MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 4, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0014

Short Communications

Epitope Mapping of an Antihuman EGFR Monoclonal
Antibody (EMab-134) Using the REMAP Method

Downloaded by Yukinari Kato from www.liebertpub.com at 08/23/21. For personal use only.

Masato Sano,1 Mika K. Kaneko,1 Teizo Aasano,1 and Yukinari Kato1,2,i

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor that plays an important role innormal epidermal cell physiology. EGFR is overexpressed in cancer cells and has a number of mutations that
implicate tumor malignancy, development, and poor patient prognosis; thus, EGFR is an attractive target forcancer therapy. At present, anti-EGFR monoclonal antibodies (mAbs) have been approved and are used fortreating patients with a variety of EGFR-expressing cancers. Epitope mapping is important in identifying thetherapeutic mechanism of anti-EGFR mAbs; however, the development of epitope mapping techniques lags
behind the development of antimolecular target mAbs, including anti-EGFR mAbs. Hence, in this study, anovel epitope mapping method, RIEDL insertion for epitope mapping (REMAP) method, was developed. The
results of this study demonstrated that the critical epitope of anti-EGFR mAb EMab-134 is Gly378, Asp379,
Ser380, Phe381, Thr382, His383, Thr384, Pro385, and Pro386 of EGFR. The REMAP method could be usefulfor determining the critical epitope of functional mAbs against many target molecules.
Keywords: EGFR, EMab-134, epitope mapping, monoclonal antibody, RIEDL tag

IntroductionT

he epidermal growth factor receptor (EGFR) is atyrosine kinase receptor, closely related to human epidermal receptor 2 (HER2), HER3, and HER4.(1,2) EGFR is
activated by ligand binding and it promotes cell progression
by activating the ERK, MAPK, AKT-PI3K, and PLC-g1PKC pathways.(3–6) EGFR is overexpressed in various cancers and is usually associated with the survival, proliferation,
invasion, and drug resistance of cancer cells.(7,8) Therefore,
the EGFR expression is a poor prognostic factor for cancer
patients and EGFR is an attractive target for cancer therapy.
Although EGFR-targeting therapies, such as EGFR tyrosine
kinase inhibitors and anti-EGFR monoclonal antibodies(mAbs), show some antitumor efficacy in clinical trials, tumors can evolve with EGFR-resistant mutations, thereby
causing failure of these therapies.(9–14) Thus, EGFR-targeting
drugs are needed to overcome these mutations.
Four anti-EGFR antibodies, cetuximab, panitumumab,
nimotuzumab, and necitumumab, have been developed forclinical use in the treatment of colorectal cancer, head andneck squamous cell carcinoma, glioblastoma, gastric cancer,
and squamous nonsmall cell lung cancer.(15–22) Epitope
mapping of these mAbs using structural analysis reveals thatthey bind to different amino acid (aa) residues of domain III

of EGFR and inhibit binding of the EGFR ligands, such as
EGF.(23–26) The structural analysis revealed that panitumumab binds to the cetuximab-resistant EGFR K467T mutant,whereas necitumumab binds to cetuximab- andpanitumumab-resistant EGFR mutants.(23,24,26) Therefore,
epitope mapping is important in determining the therapeuticand molecular mechanisms of mAbs.
Epitopes of mAbs are generally divided into two types:
linear and conformational epitopes. Linear epitopes areformed by continuous aa residues (£16 aa), whereas conformational epitopes are not continuous and are formed by threedimensional peptides combined by protein folding.(27) There
are several conformational epitope mapping methods, such as
X-ray cocrystallography, cryogenic electron microscopy,
site-directed mutagenesis mapping, high-throughput shotgun
mutagenesis epitope mapping, and hydrogen–deuterium exchange mass spectrometry.(28–32) X-ray cocrystallography
and cryogenic electron microscopy can precisely determine
the antibody binding sites, but these methods are highly
technical, time consuming, and expensive. In site-directedmutagenesis mapping and high-throughput shotgun mutagenesis epitope mapping, it is challenging to map a part of aconformational epitope because a single aa residue mutation
is not enough to disrupt the antibody–antigen interaction.
Currently, large number of mAbs have been obtained as

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
ORCID ID (https://orcid.org/0000-0001-5385-8201).

2
i

191

Downloaded by Yukinari Kato from www.liebertpub.com at 08/23/21. For personal use only.

FIG. 1. Epitope mapping using RIEDL tag-inserted mutants of EGFR/dN313. The RIEDL tag-inserted mutants wereincubated with (A) NZ-1 (an anti-PAtag mAb), (B) LpMab-7 (an anti-RIEDL tag mAb), or (C) EMab-134. Black lines:
control (without 1st mAb). EGFR, epidermal growth factor receptor; mAb, monoclonal antibody.
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EPITOPE MAPPING OF EMAB-134

193

therapeutic drugs against many diseases; however, the speedof epitope mapping techniques has not kept pace with theincreasing number of isolated mAbs.(31,33)
In this study, a novel epitope mapping technique using the
RIEDL tag system(34) was developed. Then, the epitope ofanti-EGFR mAb (clone EMab-134)(35) was characterizedusing the RIEDL insertion for epitope mapping (REMAP)
method.

que, Inc.). After washing the cells with 0.1% bovine serum
albumin in phosphate-buffered saline, they were treated withprimary mAbs (1 or 10 lg/mL) for 30 minutes at 4C and thenwith Alexa Fluor 488-conjugated antimouse immunoglobulin G (IgG) or antirat IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA). Fluorescence data were collectedusing the EC800 Cell Analyzer (Sony Corp.).

Materials and Methods

Results and Discussion

Cell lines

In our previous study, the critical epitope of an antiEGFR mAb (EMab-134)(35) was determined to be
377-RGDSFTHTPP-386 from domain III of the extracellularregion of EGFR using site-directed mutagenesis epitope
mapping.(39) In this study, the epitope of EMab-134 wascharacterized using a novel epitope mapping technique: REMAP method.
We produced 14 EGFR/dN313 transfectants (an N-terminal
PA tag and EGFR from aa 313 to 1210), in which a RIEDL tag
was inserted into the expected epitope region at each possible
position of 375-AFRGDSFTHTPPLDP-389 (Fig. 1). The
LpMab-7 (an anti-RIEDL tag mAb) recognized the five aa-long
RIEDL tag.(34) 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 positive control
NZ-1 (an anti-PA tag mAb) detected wild type (WT) and 14
mutants of EGFR/dN313 (Fig. 1A). Because RIEDL sequence was not inserted into WT, LpMab-7 did not react with
WT, but reacted with 14 mutants of EGFR/dN313 (Fig. 1B).
In contrast, EMab-134 did not react with eight mutants, such
as 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 six mutants, suchas Ala375_RIEDL_Phe376 (A375_R*_F376), Phe376_
RIEDL_Arg377 (F376_R*_R377), Arg377_RIEDL_Gly378

Chinese hamster ovary (CHO)-K1 cells were obtained
from the America Type Culture Collection (ATCC, Manassas, VA). The EGFR mutation plasmids were transfected
into the CHO-K1 cells using the Neon Transfection System(Thermo Fisher Scientific, Inc., Waltham, MA) and stabletransfectants were sorted using a cell sorter (SH800; Sony
Corp., Tokyo, Japan). The CHO-K1 cells and transfectants
were cultured in RPMI 1640 medium (Nacalai Tesque, Inc.,
Kyoto, Japan) supplemented with 10% heat-inactivated fetal
bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL
penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere containing 5% CO2. The transfectants were
cultivated in a medium containing 0.5 mg/mL Zeocin (InvivoGen, San Diego, CA).
Plasmid preparation

The EGFR open reading frames were amplified as described
previously.(36) The EGFR deletion mutants (EGFR/dN313)
were produced using the HotStar HiFidelity Polymerase Kit(Qiagen, Inc., Hilden, Germany) with oligonucleotides containing the desired mutations. The PA tag (GVAMPGAEDDVV) was added at the N-terminus,(37) which isrecognized by an anti-PA tag mAb (NZ-1).(38) The RIEDL
tag(34) was inserted into the EGFR sequence using the HotStar
HiFidelity Polymerase Kit with oligonucleotides containing
the RIEDL tag insertions at the desired position in 375-AFRGDSFTHTPPLDP-389 of EGFR/dN313. For example,
Ala375_RIEDL_Phe376 (A375_R*_F376) was produced byinserting the RIEDL sequence between Ala375 and Phe376 of
EGFR/dN313. The polymerase chain reaction fragments withthe desired mutations were inserted into the pCAG-Ble vector
using the In-Fusion HD Cloning Kit (Takara Bio, Inc.,
Shiga, Japan). The following RIEDL tag insertion mutants
were produced: 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_Leu 387 (P386_
R*_L387), Leu387_RIEDL_Asp388 (L387_R*_D388), andAsp388_RIEDL_Pro389 (D388_R*_P389).
Flow cytometry

The cells were harvested by a brief exposure to 0.25%
trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tes-

FIG. 2. Schematic illustration of EMab-134 epitopes.
‘‘GDSFTHTPP’’ is a critical epitope of EMab-134. PA, PA
tag.

194

(R377_R*_G378), Pro386_RIEDL_Leu387 (P386_R*_L387),
Leu387_RIEDL_Asp388 (L387_R*_D388), and Asp388_
RIEDL_Pro389 (D388_R*_P389) (Fig. 1C), indicatingthat EMab-134 might bind to EGFR through nine aas(378-GDSFTHTPP-386). These results are summarized in
Figure 2.
As described in this study, the REMAP method may also
be suitable for the mapping of linear and conformational
epitopes of a number of anti-EGFR mAbs, including thoseused in preclinical or clinical trials. This could reveal novel
therapeutic mechanisms of anti-EGFR mAbs. Furthermore,
there are a number of mAbs for which the antigen binding site
has not been identified. The REMAP method might be able tocharacterize the epitope of these mAbs in a future study.
Author Disclosure Statement

Downloaded by Yukinari Kato from www.liebertpub.com at 08/23/21. For personal use only.

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under Grant
Nos. JP21am0401013 (Y.K.) and JP21am0101078 (Y.K.)
and by Japan Society for the Promotion of Science ( JSPS)
KAKENHI under Grant Nos. 21K16322 (M.S.), 21K07168(M.K.K.), 21K15523 (T.A.), and 21K07705 (Y.K.).
References

1. Olayioye MA, Neve RM, Lane HA, and Hynes NE: The
ErbB signaling network: Receptor heterodimerization indevelopment and cancer. EMBO J 2000;19:3159–3167.
2. Pines G, Köstler WJ, and Yarden Y: Oncogenic mutant
forms of EGFR: Lessons in signal transduction and targets
for cancer therapy. FEBS Lett 2010;584:2699–2706.
3. Lewis TS, Shapiro PS, and Ahn NG: Signal transduction
through MAP kinase cascades. Adv Cancer Res 1998;74:
49–139.
4. Morrison DK: MAP kinase pathways. Cold Spring Harb
Perspect Biol 2012;4:a011254.
5. Gschwind A, Fischer OM, and Ullrich A: The discovery of
receptor tyrosine kinases: Targets for cancer therapy. Nat
Rev Cancer 2004;4:361–370.
6. Fan QW, Cheng C, Knight ZA, Haas-Kogan D, Stokoe D,
James CD, McCormick F, Shokat KM, and Weiss WA:
EGFR signals to mTOR through PKC and independently of
Akt in glioma. Sci Signal 2009;2:ra4.
7. Sigismund S, Avanzato D, and Lanzetti L: Emerging
functions of the EGFR in cancer. Mol Oncol 2018;12:3–20.
8. Aratani K, Komatsu S, Ichikawa D, Ohashi T, Miyamae M,
Okajima W, Imamura T, Kiuchi J, Nishibeppu K, Kosuga
T, Konishi H, Shiozaki A, Fujiwara H, Okamoto K, Tsuda
H, and Otsuji E: Overexpression of EGFR as an independent prognostic factor in adenocarcinoma of the esophagogastric junction. Anticancer Res 2017;37:3129–3135.
9. Nan X, Xie C, Yu X, and Liu J: EGFR TKI as first-line
treatment for patients with advanced EGFR mutationpositive non-small-cell lung cancer. Oncotarget 2017;8:75712–75726.
10. Isomoto K, Haratani K, Hayashi H, Shimizu S, Tomida S,
Niwa T, Yokoyama T, Fukuda Y, Chiba Y, Kato R, Tanizaki J, Tanaka K, Takeda M, Ogura T, Ishida T, Ito A,
and Nakagawa K: Impact of EGFR-TKI treatment on the

SANO ET AL.

tumor immune microenvironment in EGFR mutation–
positive non–small cell lung cancer. Clin Cancer Res 2020;
26:2037–2046.
11. Shi P, Oh YT, Deng L, Zhang G, Qian G, Zhang S, Ren H,
Wu G, Legendre B, Jr., Anderson E, Ramalingam SS,
Owonikoko TK, Chen M, and Sun SY: Overcoming acquired resistance to AZD9291, a third-generation EGFR
inhibitor, through modulation of MEK/ERK-dependent
Bim and Mcl-1 degradation. Clin Cancer Res 2017;23:
6567–6579.
12. Shi P, Oh YT, Zhang G, Yao W, Yue P, Li Y, Kanteti R,
Riehm J, Salgia R, Owonikoko TK, Ramalingam SS, Chen
M, and Sun SY: Met gene amplification and protein hyperactivation is a mechanism of resistance to both first andthird generation EGFR inhibitors in lung cancer treatment.
Cancer Lett 2016;380:494–504.
13. Zang H, Qian G, Zong D, Fan S, Owonikoko TK, Ramalingam SS, and Sun SY: Overcoming acquired resistance of
epidermal growth factor receptor-mutant non-small celllung cancer cells to osimertinib by combining osimertinibwith the histone deacetylase inhibitor panobinostat(LBH589). Cancer 2020;126:2024–2033.
14. Zhang T, Qu R, Chan S, Lai M, Tong L, Feng F, Chen H,
Song T, Song P, Bai G, Liu Y, Wang Y, Li Y, Su Y, ShenY, Sun Y, Chen Y, Geng M, Ding K, Ding J, and Xie H:
Discovery of a novel third-generation EGFR inhibitor andidentification of a potential combination strategy to overcome resistance. Mol Cancer 2020;19:90.
15. Jonker DJ, O’Callaghan CJ, Karapetis CS, Zalcberg JR, Tu
D, Au H-J, Berry SR, Krahn M, Price T, Simes RJ, Tebbutt
NC, Van Hazel G, Wierzbicki R, Langer C, and Moore MJ:
Cetuximab for the treatment of colorectal cancer. N Engl J
Med 2007;357:2040–2048.
16. Weber J, and McCormack PL: Panitumumab: In metastatic
colorectal cancer with wild-type KRAS. BioDrugs 2008;22:
403–411.
17. Li S, Schmitz KR, Jeffrey PD, Wiltzius JJ, Kussie P, andFerguson KM: Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell
2005;7:301–311.
18. Chen Z-P, Yang Q, and Guo C-C: Profile of nimotuzumabin the treatment of high-grade glioma. Onco Targets Ther
2015:8:819–825.
19. Hirsch F, and Genova C: Clinical potential of necitumumabin non-small cell lung carcinoma. Onco Targets Ther 2016;
9:5427–5437.
20. Crombet Ramos T, Mestre Fernández B, Mazorra Herrera
Z, and Iznaga Escobar NE: Nimotuzumab for patients withinoperable cancer of the head and neck. Front Oncol 2020;
10:817.
21. Maron SB, Alpert L, Kwak HA, Lomnicki S, Chase L, Xu
D, O’Day E, Nagy RJ, Lanman RB, Cecchi F, HembroughT, Schrock A, Hart J, Xiao S-Y, Setia N, and Catenacci
DVT: Targeted therapies for targeted populations: AntiEGFR treatment for EGFR-amplified gastroesophageal
adenocarcinoma. Cancer Discov 2018;8:696–713.
22. Cai WQ, Zeng LS, Wang LF, Wang YY, Cheng JT, Zhang
Y, Han ZW, Zhou Y, Huang SL, Wang XW, Peng XC,
Xiang Y, Ma Z, Cui SZ, and Xin HW: The latest battles
between EGFR monoclonal antibodies and resistant tumor
cells. Front Oncol 2020;10:1249.
23. Bagchi A, Haidar JN, Eastman SW, Vieth M, Topper M,
Iacolina MD, Walker JM, Forest A, Shen Y, Novosiadly
RD, and Ferguson KM: Molecular basis for necitumumabDownloaded by Yukinari Kato from www.liebertpub.com at 08/23/21. For personal use only.

EPITOPE MAPPING OF EMAB-134

inhibition of EGFR variants associated with acquired cetuximab resistance. Mol Cancer Ther 2018;17:521–531.
24. Sickmier EA, Kurzeja RJ, Michelsen K, Vazir M, Yang E,
and Tasker AS: The panitumumab EGFR complex reveals
a binding mechanism that overcomes cetuximab induced
resistance. PLoS One 2016;11:e0163366.
25. Talavera A, Friemann R, Gomez-Puerta S, Martinez-Fleites
C, Garrido G, Rabasa A, Lopez-Requena A, Pupo A, Johansen RF, Sanchez O, Krengel U, and Moreno E: Nimotuzumab, an antitumor antibody that targets the epidermal
growth factor receptor, blocks ligand binding while permitting the active receptor conformation. Cancer Res 2009;69:5851–5859.
26. Voigt M, Braig F, Göthel M, Schulte A, Lamszus K,
Bokemeyer C, and Binder M: Functional dissection of theepidermal growth factor receptor epitopes targeted bypanitumumab and cetuximab. Neoplasia 2012;14:1023–
1031.
27. Forsström B, Bisławska Axnäs B, Rockberg J, Danielsson
H, Bohlin A, and Uhlen M: Dissecting antibodies withregards to linear and conformational epitopes. PLoS One
2015;10:e0121673.
28. Toride King M, and Brooks CL. Epitope Mapping of
Antibody-Antigen Interactions with X-Ray Crystallography. Springer, New York, 2018, pp. 13–27.
29. Long F, Fong RH, Austin SK, Chen Z, Klose T, Fokine A,
Liu Y, Porta J, Sapparapu G, Akahata W, Doranz BJ,
Crowe JE, Diamond MS, and Rossmann MG: Cryo-EM
structures elucidate neutralizing mechanisms of antichikungunya human monoclonal antibodies with therapeutic activity. Proc Natl Acad Sci U S A 2015;112:13898–
13903.
30. Benjamin DC, and Perdue SS: Site-directed mutagenesis inepitope mapping. Methods 1996;9:508–515.
31. Davidson E, and Doranz BJ: A high-throughput shotgun
mutagenesis approach to mapping B-cell antibody epitopes.
Immunology 2014;143:13–20.
32. Wei H, Mo J, Tao L, Russell RJ, Tymiak AA, Chen G,
Iacob RE, and Engen JR: Hydrogen/deuterium exchange
mass spectrometry for probing higher order structure ofprotein therapeutics: Methodology and applications. Drug
Discov Today 2014;19:95–102.
33. Bourquard T, Musnier A, Puard V, Tahir S, Ayoub MA,
Jullian Y, Boulo T, Gallay N, Watier H, Bruneau G, Reiter E,

195

Crépieux P, and Poupon A: MAbTope: A method for improved epitope mapping. J Immunol 2018;201:3096–3105.
34. Asano T, Kaneko MK, and Kato Y: RIEDL tag: A novel
pentapeptide tagging system for transmembrane protein
purification. Biochem Biophys Rep 2020;23:100780.
35. Itai S, Yamada S, Kaneko MK, Chang YW, Harada H, andKato Y: Establishment of EMab-134, a sensitive and specific anti-epidermal growth factor receptor monoclonal
antibody for detecting squamous cell carcinoma cells of theoral cavity. Monoclon Antib Immunodiagn Immunother2017;36:272–281.
36. Fujii Y, Kaneko MK, and Kato Y: MAP tag: A novel
tagging system for protein purification and detection.
Monoclon Antib Immunodiagn Immunother 2016;35:293–
299.
37. Fujii Y, Kaneko M, Neyazaki M, Nogi T, Kato Y, andTakagi J: PA tag: A versatile protein tagging system using a
super high affinity antibody against a dodecapeptide derived from human podoplanin. Protein Expr Purif 2014;95:
240–247.
38. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K,
Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H, Mishima K, and Osawa M: Inhibition of tumor cell-induced
platelet aggregation using a novel anti-podoplanin antibody
reacting with its platelet-aggregation-stimulating domain.
Biochem Biophys Res Commun 2006;349:1301–1307.
39. Kaneko MK, Yamada S, Itai S, Chang YW, Nakamura T,
Yanaka M, and Kato Y: Elucidation of the critical epitopeof an anti-EGFR monoclonal antibody EMab-134. Biochem Biophys Rep 2018;14:54–57.

Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: March 25, 2021
Accepted: May 17, 2021