Short Communications

MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 6, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0029

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Epitope Mapping of an Anti-HER2 Monoclonal Antibody(H2Mab-181) Using Enzyme-Linked Immunosorbent Assay

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Teizo Asano,1 Junko Takei,1 Hiroyuki Suzuki,2 Mika K. Kaneko,1 and Yukinari Kato1,2,i

Human epidermal growth factor receptor 2 (HER2) is a type I transmembrane 185 kDa protein expressed invarious types of normal or cancer cells. Overexpression of HER2 is found in many cancers and is related tocell proliferation, differentiation, and migration. We recently developed a novel anti-HER2 monoclonal
antibody, H2Mab-181, by immunizing mice with the purified recombinant extracellular domain of HER2.
H2Mab-181 can specifically and sensitively detect HER2 not only in flow cytometry and Western blotting
for gastric cancer cell lines, but also in immunohistochemical analyses for gastric cancer tissues. In thisstudy, we analyzed the binding epitope of H2Mab-181 to HER2 using enzyme-linked immunosorbent assay(ELISA). Results showed that the H2Mab-181 epitope was determined to be Gly383, Asp384, Ala386,
Asn388, and Pro391 by ELISA.
Keywords: HER2, monoclonal antibody, epitope mapping, ELISA

IntroductionEpidermal growth factor receptor (EGFR) family
protein is a type I transmembrane glycoprotein composed of three regions: extracellular, single transmembrane,
and intracellular regions.(1,2) The extracellular region contains four domains, namely, I, II, III, and IV domains, and theintracellular region contains a kinase domain.(3) The EGFR
family comprises four closely related members, namely,
EGFR (HER1/ErbB1), human epidermal growth factor receptor 2 (HER2; ErbB2/Neu), HER3 (ErbB3), and HER4
(ErbB4).(4) The EGFR family members transduce extracellular signals into intracellular signaling pathways throughactivation of the tyrosine kinase domain. These receptor tyrosine kinases play a critical role in the regulation of cellproliferation, survival, migration, and differentiation.(1) The
ligand binding to the extracellular domain promotes the formation of homo- or heterodimers between the EGFR family
receptors.(5) However, the ligand of HER2 has not been
identified among the HER family and is represented as anactivated structure without ligand binding.(6–8) Homodimers

or heterodimers are formed between HER2 and other EGFR
family members.(6) Dimers including HER2 have a higher
tyrosine kinase activity. Among them, HER2/HER3 heterodimer has the greatest transforming and mitogenic signalingability.(9,10)
HER2 is expressed in various cell types, excluding thecells of hematopoietic origin.(11) Overexpression and mutation of HER2 have been reported in many cancers, including
breast,(12,13) ovarian,(14) pancreatic,(15) lung,(16) colorectal,(17)and gastric cancers.(18) In HER2-positive breast cancer patients, this oncogene overexpression is associated with poor
prognosis and aggressive tumor phenotypes.(12,13)
Recently, trastuzumab and pertuzumab, which are antiHER2 monoclonal antibodies (mAbs), have been widelyused. Trastuzumab, a humanized murine mAb, was the first
approved anti-HER2 mAb drug for breast cancer in 1998
by the U.S. Food and Drug Administration (FDA).(19–21) The
use of trastuzumab in gastric cancers began in 2010.(22)Pertuzumab is a fully humanized mAb, and it was approvedin 2012 by FDA for breast cancer combination therapy
with trastuzumab and docetaxel.(23,24) Trastuzumab binds to

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

255

256

mice with the purified recombinant protein for the HER2
extracellular domain including domains I–IV.(29-37) H2Mab181 can specifically and sensitively detect HER2 in flow
cytometry and Western blotting applications in gastric cancer
cell lines. It can also be used in immunohistochemical analysis of gastric cancer tissues. In this study, we investigatedthe epitope of H2Mab-181 for HER2 using enzyme-linked
immunosorbent assay (ELISA).
Materials and Methods
PeptidesHER2 (Accession No.: M11730) peptides, including 62
deletion mutants (Fig. 1) and 20 alanine-substituted peptides(Table 1), were synthesized by utilizing PEPScreen (SigmaAldrich Corp., St. Louis, MO, USA).

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domain IV of HER2 that contains the binding pocket for theextended domain II loop of the inactive states of the EGFR
and HER3. The interaction of trastuzumab is mediated bythree loop regions on HER2, formed by residues 557–561,
570–573, and 593–603.(25) Pertuzumab binds the center ofdomain II of HER2, and pertuzumab binding should block theregion necessary for dimerization with the other EGFR
family.(26) The difference in the epitope region caused
different inhibition mechanisms and antitumor effects.Trastuzumab inhibits the growth of tumors highly overexpressing HER2. In contrast, pertuzumab inhibits tumor
growth of both low- and high-expressing HER2 levels.(27)
Therefore, identification of the epitope is important to develop
the mAb drugs.
Previously, we developed a novel anti-HER2 mAb,
H2Mab-181 (IgG1, kappa),(28) through the immunization of

ASANO ET AL.

FIG. 1. Epitope mapping of H2Mab-181 using deletion mutants of HER2 by ELISA. The synthesized peptides were
immobilized on immunoplates. The plates were incubated with H2Mab-181 (1 mg/mL), followed by incubation withperoxidase-conjugated anti-mouse immunoglobulins. HER2 and synthesized peptides were illustrated. A black bar indicate
the peptide detected by H2Mab-181. ELISA, enzyme-linked immunosorbent assay; HER2, human epidermal growth factor
receptor 2; IC, intracellular domain; SS, signal sequence; TM, transmembrane domain.

EPITOPE MAPPING OF ANTI-HER2 MAB

257
Epitope mapping by ELISA using
alanine-substituted peptides

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Table 1. Identification of H2Mab-181 Epitope
Using Enzyme-Linked Immunosorbent Assay
PeptideSequence

H2Mab-181

373–392
S373A
L374A
A375G
F376A
L377A
P378A
E379A
S380A
F381A
D382A
G383A
D384A
P385A
A386G
S387A
N388A
T389A
A390G
P391A
L392A

SLAFLPESFDGDPASNTAPLALAFLPESFDGDPASNTAPLSAAFLPESFDGDPASNTAPLSLGFLPESFDGDPASNTAPLSLAALPESFDGDPASNTAPLSLAFAPESFDGDPASNTAPLSLAFLAESFDGDPASNTAPLSLAFLPASFDGDPASNTAPLSLAFLPEAFDGDPASNTAPLSLAFLPESADGDPASNTAPLSLAFLPESFAGDPASNTAPLSLAFLPESFDADPASNTAPLSLAFLPESFDGAPASNTAPLSLAFLPESFDGDAASNTAPLSLAFLPESFDGDPGSNTAPLSLAFLPESFDGDPAANTAPLSLAFLPESFDGDPASATAPLSLAFLPESFDGDPASNAAPLSLAFLPESFDGDPASNTGPLSLAFLPESFDGDPASNTAALSLAFLPESFDGDPASNTAPA

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

Next, we synthesized 20 alanine-substituted HER2
peptides of 373–392 aa (Table 1). H2Mab-181 reactedwith S373A, L374A, A375G, F376A, L377A, P378A,
E379A, S380A, F381A, D382A, P385A, S387A, T389A,
A390G, L392A, and wild-type 373–392 aa, but not with
G383A, D384A, A386G, N388A, and P391A (Table 1 andFig. 2A), indicating that Gly383, Asp384, Ala386,
Asn388, and Pro391 are the critical epitope of H 2Mab-181(Fig. 2B).
Although we also tried to determine the binding epitope of
H2Mab-181 using surface plasmon resonance (SPR), thosedata were immature probably because the binding affinity is
low for the SPR analysis (data not shown). We should further
optimize the condition of SPR for the biological characterization in a future study.
Discussion+, OD655 ‡ 0.1; -, OD655 < 0.1.

Enzyme-linked immunosorbent assay (ELISA)

Synthesized HER2 peptides using PEPScreen (Table 1)
were immobilized on Nunc Maxisorp 96-well immunoplates(Thermo Fisher Scientific, Inc., Waltham, MA, USA) at aconcentration of 10 lg/mL, respectively, for 30 minutes at
37C. After washing with phosphate-buffered saline
containing 0.05% Tween20 (PBST), wells were blockedwith 1% bovine serum albumin-containing PBST for 30
minutes at 37C. The plates were then incubated with
H2Mab-181 (1 lg/mL), followed by a 1:2000 dilution ofperoxidase-conjugated anti-mouse immunoglobulins(Agilent Technologies, Inc., Santa Clara, CA, USA). Enzymatic reactions were performed using the ELISA POD
Substrate TMB Kit (Nacalai Tesque, Inc., Kyoto, Japan).
Optical density was measured at 655 nm using an iMark
microplate reader (Bio-Rad Laboratories, Inc., Berkeley,
CA, USA). These reactions were performed with a total
sample volume of 50–100 lL.

Domains I and III of HER2 are named as ligand-binding
domains although there is no ligand for HER2; domain II
mediates the dimer formation, and domain IV contains thebinding pocket for the extended domain II loop of
the inactive states EGFR and HER3.(38) Trastuzumab
binds to domain IV and induces antibody-dependent cellmediated cytotoxicity.(25) Meanwhile, pertuzumab binds
to domain II and inhibits the ligand-induced dimerization.(26) Previously, some mAbs-targeted domain III
of HER2 were reported. (39,40) A novel anti-HER2 mAb(hHERmAb-F0178C1) effectively blocked HER2/HER3
heterodimerization and signaling. Its combined use with
pertuzumab had a synergistic effect. (39) Another novelanti-HER2 mAb (3E10) inhibited HER2 heterodimerization with EGFR and HER3 through a different mechanism
from trastuzumab and pertuzumab.(40) Furthermore, synergistic inhibition of HER2 heterodimerization and signaling was observed with 3E10 in combination with either
trastuzumab or pertuzumab.(40) These studies show thattargeting domain III of HER2 may be a new target forcancer treatment. Moreover, we showed that H2Mab-181
is suitable for various applications, including flow cytometry, Western blotting, and immunohistochemical
analyses for gastric cancer.(28) Therefore, we will study
the antitumor effect of H2Mab-181 against gastric cancer
in the future.
Authors’ Contributions

Results
Epitope mapping by ELISA using deletion mutants

Determination of binding epitope with deletion mutants
using PEPScreen is a basic method for determining the linear
epitope of mAbs. Furtheremore, alanine scanning using
PEPScreen is also very easy to exactly determine the binding
epitope.
We synthesized 62 HER2 peptides of 20 amino acids (aa)length (Fig. 1) and performed ELISA. Results showed that
H2Mab-181 reacted with the 373–392 aa (373-SLAFLPESFDGDPASNTAPL-392) sequence (Fig. 1), indicating that the
H2Mab-181 epitope is located in domain III of HER2.

T.A., J.T., T.N., and H.S. performed experiments;
M.K.K. designed the experiments; and T.A. 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) under257

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258

ASANO ET AL.

FIG. 2. (A) Determination of H2Mab-181 epitope on HER2 by ELISA using point mutants. The 373–392 peptide (positive
control) and synthesized alanine-substituted peptides were immobilized on immunoplates. The plates were incubatedwith H2Mab-181 (1 mg/mL), followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins. The opticaldensity was measured at 655 nm. (B) Schematic illustration of the epitope recognized by H2Mab-181 using ELISA.
Underlined amino acids indicate the critical epitope of H2Mab-181 determined using ELISA.
Grant Nos.: 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 Nos. 21K15523 (to T.A.), 21K07168(to M.K.K.), and 19K07705 (to Y.K.).
References

1. Gschwind A, Fischer OM, and Ullrich A: The discoveryof receptor tyrosine kinases: Targets for cancer therapy. Nat
Rev Cancer 2004;4:361–370.
2. Yarden Y, and Sliwkowski MX: Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001;2:127–137.
3. Lax I, Johnson A, Howk R, Sap J, Bellot F, Winkler M,
Ullrich A, Vennstrom B, Schlessinger J, and Givol D:
Chicken epidermal growth factor (EGF) receptor: cDNA
cloning, expression in mouse cells, and differential bindingof EGF and transforming growth factor alpha. Mol Cell
Biol 1988;8:1970–1978.

4. Olayioye MA, Neve RM, Lane HA, and Hynes NE: The
ErbB signaling network: Receptor heterodimerization indevelopment and cancer. Embo J 2000;19:3159–3167.
5. Ogiso H, Ishitani R, Nureki O, Fukai S, Yamanaka M,
Kim JH, Saito K, Sakamoto A, Inoue M, Shirouzu M, andYokoyama S: Crystal structure of the complex of humanepidermal growth factor and receptor extracellular domains. Cell 2002;110:775–787.
6. Garrett TP, McKern NM, Lou M, Elleman TC, Adams TE,
Lovrecz GO, Kofler M, Jorissen RN, Nice EC, Burgess AW,
and Ward CW: The crystal structure of a truncated ErbB2
ectodomain reveals an active conformation, poised to interact
with other ErbB receptors. Mol Cell 2003;11:495–505.
7. Linggi B, and Carpenter G: ErbB receptors: New insights onmechanisms and biology. Trends Cell Biol 2006;16:649–656.
8. Harris RC, Chung E, and Coffey RJ: EGF receptor ligands.
Exp Cell Res 2003;284:2–13.
9. Tzahar E, Waterman H, Chen X, Levkowitz G, KarunagaranD, Lavi S, Ratzkin BJ, and Yarden Y: A hierarchical net-

Downloaded by Yukinari Kato from www.liebertpub.com at 12/27/21. For personal use only.

EPITOPE MAPPING OF ANTI-HER2 MAB

work of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol 1996;16:5276–5287.
10. Citri A, Skaria KB, and Yarden Y: The deaf and the dumb:
The biology of ErbB-2 and ErbB-3. Exp Cell Res 2003;
284:54–65.
11. Hynes NE, and Stern DF: The biology of erbB-2/neu/
HER-2 and its role in cancer. Biochim Biophys Acta 1994;
1198:165–184.
12. Howlader N, Altekruse SF, Li CI, Chen VW, Clarke CA,
Ries LA, and Cronin KA: US incidence of breast cancer
subtypes defined by joint hormone receptor and HER2
status. J Natl Cancer Inst 2014;106.
13. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A,
and McGuire WL: Human breast cancer: Correlation of
relapse and survival with amplification of the HER-2/neu
oncogene. Science 1987;235:177–182.
14. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG,
Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, and
Press MF: Studies of the HER-2/neu proto-oncogene inhuman breast and ovarian cancer. Science 1989;244:707–
712.
15. Harder J, Ihorst G, Heinemann V, Hofheinz R, Moehler M,
Buechler P, Kloeppel G, Röcken C, Bitzer M, Boeck S,
Endlicher E, Reinacher-Schick A, Schmoor C, and Geissler
M: Multicentre phase II trial of trastuzumab and capecitabine in patients with HER2 overexpressing metastatic
pancreatic cancer. Br J Cancer 2012;106:1033–1038.
16. Hirsch FR, Varella-Garcia M, Franklin WA, Veve R, Chen
L, Helfrich B, Zeng C, Baron A, and Bunn PA, Jr.: Evaluation of HER-2/neu gene amplification and protein expression in non-small cell lung carcinomas. Br J Cancer
2002;86:1449–1456.
17. Richman SD, Southward K, Chambers P, Cross D, Barrett
J, Hemmings G, Taylor M, Wood H, Hutchins G, Foster
JM, Oumie A, Spink KG, Brown SR, Jones M, Kerr D,
Handley K, Gray R, Seymour M, and Quirke P: HER2
overexpression and amplification as a potential therapeutictarget in colorectal cancer: Analysis of 3256 patients enrolled in the QUASAR, FOCUS and PICCOLO colorectal
cancer trials. J Pathol 2016;238:562–570.
18. Gravalos C, and Jimeno A: HER2 in gastric cancer: A new
prognostic factor and a novel therapeutic target. Ann Oncol
2008;19:1523–1529.
19. Li SG, and Li L: Targeted therapy in HER2-positive breast
cancer. Biomed Rep 2013;1:499–505.
20. Lambert JM, and Chari RV: Ado-trastuzumab Emtansine (T-DM1): An antibody-drug conjugate (ADC) for
HER2-positive breast cancer. J Med Chem 2014;57:
6949–6964.
21. Valabrega G, Montemurro F, and Aglietta M: Trastuzumab:
Mechanism of action, resistance and future perspectives in
HER2-overexpressing breast cancer. Ann Oncol 2007;18:
977–984.
22. Sitarz R, Skierucha M, Mielko J, Offerhaus GJA, Maciejewski R, and Polkowski WP: Gastric cancer: Epidemiology, prevention, classification, and treatment. Cancer ManagRes 2018;10:239–248.
23. Amiri-Kordestani L, Wedam S, Zhang L, Tang S, Tilley
A, Ibrahim A, Justice R, Pazdur R, and Cortazar P: First
FDA approval of neoadjuvant therapy for breast cancer:
Pertuzumab for the treatment of patients with HER2positive breast cancer. Clin Cancer Res 2014;20:5359–
5364.

259

24. Blumenthal GM, Scher NS, Cortazar P, Chattopadhyay S,
Tang S, Song P, Liu Q, Ringgold K, Pilaro AM, Tilley A,
King KE, Graham L, Rellahan BL, Weinberg WC, Chi B,
Thomas C, Hughes P, Ibrahim A, Justice R, and Pazdur R:
First FDA approval of dual anti-HER2 regimen: Pertuzumab in combination with trastuzumab and docetaxel for
HER2-positive metastatic breast cancer. Clin Cancer Res
2013;19:4911–4916.
25. Cho HS, Mason K, Ramyar KX, Stanley AM, Gabelli SB,
Denney DW, Jr., and Leahy DJ: Structure of the extracellular region of HER2 alone and in complex with theHerceptin Fab. Nature 2003;421:756–760.
26. Franklin MC, Carey KD, Vajdos FF, Leahy DJ, de Vos
AM, and Sliwkowski MX: Insights into ErbB signalingfrom the structure of the ErbB2-pertuzumab complex.
Cancer Cell 2004;5:317–328.
27. Agus DB, Akita RW, Fox WD, Lewis GD, Higgins B,
Pisacane PI, Lofgren JA, Tindell C, Evans DP, Maiese K,
Scher HI, and Sliwkowski MX: Targeting ligand-activated
ErbB2 signaling inhibits breast and prostate tumor growth.
Cancer Cell 2002;2:127–137.
28. Takei J, Asano T, Tanaka T, Sano M, Hosono H, Nanamiya R, Tateyama N, Saito M, Suzuki H, Harada H,
Kaneko MK, and Kato Y: Development of a novel antiHER2 monoclonal antibody H2Mab-19-181 for gastriccancer. Monoclon Antib Immunodiagn Immunother2021;40:168–176.
29. Kato Y, Ohishi T, Sano M, Asano T, Sayama Y, Takei J,
Kawada M, and Kaneko MK: H2Mab-19 anti-human epidermal growth factor receptor 2 monoclonal antibody
therapy exerts antitumor activity in pancreatic cancer xenograft models. Monoclon Antib Immunodiagn Immunother 2020;39:61–65.
30. Kato Y, Ohishi T, Takei J, Nakamura T, Kawada M, andKaneko MK: An antihuman epidermal growth factor receptor 2 monoclonal antibody (H2Mab-19) exerts antitumor activity in glioblastoma xenograft models.
Monoclon Antib Immunodiagn Immunother 2020;39:
135–139.
31. Kato Y, Ohishi T, Takei J, Nakamura T, Sano M, Asano T,
Sayama Y, Hosono H, Kawada M, and Kaneko MK: An
anti-human epidermal growth factor receptor 2 monoclonal
antibody H2Mab-19 exerts antitumor activity in mousecolon cancer xenografts. Monoclon Antib Immunodiagn
Immunother 2020;39:123–128.
32. Takei J, Kaneko MK, Ohishi T, Kawada M, Harada H, andKato Y: H2Mab-19, an anti-human epidermal growth factor
receptor 2 monoclonal antibody exerts antitumor activity inmouse oral cancer xenografts. Exp Ther Med 2020;20:846–
853.
33. Kato Y, Ohishi T, Yamada S, Itai S, Takei J, Sano M,
Nakamura T, Harada H, Kawada M, and Kaneko MK:
Anti-human epidermal growth factor receptor 2
monoclonal antibody H 2 Mab-41 exerts antitumor activity in a mouse xenograft model of colon cancer.
Monoclon Antib Immunodiagn Immunother 2019;38:
157–161.
34. Tateyama N, Asano T, Ohishi T, Takei J, Hosono H,
Nanamiya R, Tanaka T, Sano M, Saito M, Kawada M,
Kaneko MK, and Kato Y: An anti-HER2 monoclonal
antibody H2Mab-41 exerts antitumor activities in mouse
xenograft model using dog HER2-overexpressed cells.
Monoclon Antib Immunodiagn Immunother 2021;40:184–
190.

Downloaded by Yukinari Kato from www.liebertpub.com at 12/27/21. For personal use only.

260

35. Itai S, Fujii Y, Kaneko MK, Yamada S, Nakamura T,
Yanaka M, Saidoh N, Chang YW, Handa S, Takahashi M,
Suzuki H, Harada H, and Kato Y: H2Mab-77 is a sensitiveand specific anti-HER2 monoclonal antibody against breast
cancer. Monoclon Antib Immunodiagn Immunother 2017;36:143–148.
36. Yamada S, Itai S, Nakamura T, Chang YW, Harada H,
Suzuki H, Kaneko MK, and Kato Y: Establishment of
H2Mab-119, an anti-human epidermal growth factor receptor 2 monoclonal antibody, against pancreatic cancer.
Monoclon Antib Immunodiagn Immunother 2017;36:
287–290.
37. Kaneko MK, Yamada S, Itai S, and Kato Y: Developmentof an anti-HER2 monoclonal antibody H2Mab-139 againstcolon cancer. Monoclon Antib Immunodiagn Immunother2018;37:59–62.
38. Ferguson KM: Structure-based view of epidermal growth
factor receptor regulation. Annu Rev Biophys 2008;37:
353–373.
39. Fu W, Wang Y, Zhang Y, Xiong L, Takeda H, Ding L, Xu
Q, He L, Tan W, Bethune AN, and Zhou L: Insights into

ASANO ET AL.

HER2 signaling from step-by-step optimization of antiHER2 antibodies. MAbs 2014;6:978–990.
40. Meng Y, Zheng L, Yang Y, Wang H, Dong J, Wang C,
Zhang Y, Yu X, Wang L, Xia T, Zhang D, Guo Y, and Li
B: A monoclonal antibody targeting ErbB2 domain III inhibits ErbB2 signaling and suppresses the growth of ErbB2overexpressing breast tumors. Oncogenesis 2016;5:e211.

Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: June 5, 2021
Accepted: September 25, 2021