MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 4, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0021

Development of a Novel Anti-HER2 Monoclonal
Antibody H2Mab-181 for Gastric Cancer

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Junko Takei,1,2 Teizo Asano,1 Tomohiro Tanaka,1 Masato Sano,1 Hideki Hosono,1
Ren Nanamiya,1 Nami Tateyama,1 Masaki Saito,3 Hiroyoshi Suzuki,4 Hiroyuki Harada,2Mika K. Kaneko,1 and Yukinari Kato1,3,5,i

Human epidermal growth factor receptor 2 (HER2) is a type I transmembrane 185 kDa protein. HER2 isexpressed in a variety of normal tissue types and cancer cells. HER2 is associated with cell proliferation, differentiation, and migration. The overexpression of HER2 has been observed in a number of cancers, including
breast and gastric cancers. Gastric cancer is one of the most common cancers worldwide, with an annual caserate of *1 million people diagnosed with the disease. Trastuzumab is a humanized anti-HER2 monoclonal
antibody (mAb) that has been utilized in gastric cancer therapy. In this study, we have developed a novel antiHER2 mAb, H2Mab-181 (IgG1, kappa), through the immunization of mice with a purified recombinant extracellular domain of HER2. H2Mab-181 can specifically and sensitively detect HER2 in both flow cytometry
and Western blot applications in gastric cancer cell lines and can also be utilized in immunohistochemical
analyses of gastric cancer tissues. Together, H2Mab-181 could be useful for the diagnosis and therapy in gastriccancers.
Keywords: HER2, monoclonal antibody, gastric cancer

IntroductionHuman epidermal growth factor receptor 2 (HER2)/
ErbB2/Neu is a 185 kDa type I transmembrane glycoprotein that is composed of an extracellular region, a single
transmembrane region, and an intracellular region.(1) The
extracellular region consists of four domains named I, II, III,and IV.(2) Among the HER family, which consists of EGFR,
HER2, HER3, and HER4, the HER2 receptor ligand has notbeen identified and is represented as an activated structure
without ligand binding.(3) Homodimers or heterodimers areformed between HER2 and other HER family members,(4)and these complexes promote signaling cascades that affectcell proliferation, differentiation, and migration.(5) Dimersthat include HER2 have a higher tyrosine kinase activity, andamong them, HER2/HER3 heterodimer has the greatest
transforming and mitogenic signaling ability.(6,7)
HER2 is expressed in a variety of cell types, excluding
the hematopoietic origin.(8) Overexpression and mutation of

HER2 have been reported in many cancers, including breast,
ovarian,(9) lung,(10) colorectal,(11) and gastric cancers.(12) Recently, drugs that specifically target HER2, such as trastuzumab and pertuzumab, have been widely used. Trastuzumab
is a humanized murine monoclonal antibody (mAb) and was
the first approved anti-HER2 agent for breast cancer in
1998.(13) Pertuzumab, which is a fully-humanized mAb thattargets a different site (domain II) to trastuzumab (domain IV),
was approved for breast cancer combination therapy withtrastuzumab and docetaxel in 2012.(14) The use of trastuzumab in gastric cancers began in 2010.(15)
Gastric cancer is the fifth most common newly diagnosed
cancer and is the fourth leading cause of death according to
Global Cancer Statistics 2020.(16) Incidence rates are particularly high in East Asia and Eastern European regions. As
the initial symptoms are clinically silent or asymptomatic,
gastric cancer is often diagnosed at an advanced stage.(17)
Although the 5-year survival rate is 80% for those patientswith stage I, the survival rate is less than 20% in those1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
3
Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Sendai, Japan.
4
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
5
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
i
ORCID ID (https://orcid.org/0000-0001-5385-8201).
2

168

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ANTI-HER2 MAB FOR GASTRIC CANCER

diagnosed with stage IV.(18) Improvement to lifestyle habits
and early diagnosis have resulted in a decrease in the numberof cases and mortality(19); however, incidence rates havestarted to increase recently within the young population.(20)Treatment for gastric cancer involves resection alone forearly-stage disease, and chemotherapy with or without radiotherapy is also included for those diagnosed at an advanced
stage.(21) Chemotherapy involves combinations of platinumbased drugs, where fluoropyrimidine and anthracycline arecommonly used.(15) For those tumors that are unresectableor recurrent cases with HER2-positive gastric cancers,
trastuzumab-containing regimens are recommended.(21)HER2 expression occurs in 10%–20% of gastric cancer cases,
and some studies have reported that overexpression of HER2
is associated with poor outcomes.(12) The use of trastuzumab
improved the prognosis of these patients; however, resistanceoften occurs in a relatively short time (median PFS 6.7
months).(22) The combination use of pertuzumab is currently
undergoing clinical trials(23,24); however, Tabernero et al.
reported that combining pertuzumab and trastuzumab withchemotherapy did not significantly improve overall survival in a phase 3 study.(24) Therefore, it is pertinent to develop
new therapeutic strategies for the treatment of gastric cancer.
In this study, we have established a novel anti-HER2 mAb
through the immunization of mice with the purified recombinant protein for the HER2 extracellular domain. We subsequently investigated its use in a variety of applications,
including flow cytometry, Western blot, and immunohistochemical analyses.
Materials and Methods
Cell lines

The following cell lines were all purchased from American Type Culture Collection (Manassas, VA): LN229, Chinese hamster ovary (CHO)-K1, and P3X63Ag8U.1 (P3U1).
The cell lines MKN-7 and Kato III were obtained from the
Cell Resource Center for Biomedical Research Institute ofDevelopment, Aging, and Cancer, Tohoku University(Miyagi, Japan). The amino acid sequences of tag system in this study were as follows: PA tag,(25) 12 aminoacids (GVAMPGAEDDVV); RAP tag,(26) 12 amino acids(DMVNPGLEDRIE); and MAP tag,(27) 12 amino acids(GDGMVPPGIEDK). The cell lines LN229/HER2 andCHO/HER2 had been previously established by transfecting
pCAG/PA-HER2-RAP-MAP into LN229 and CHO-K1 cells
using the Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA) and Lipofectamine LTX (Thermo
Fisher Scientific, Inc.), respectively.(28) LN229/HER2ec had
been previously established by transfecting pCAG/PAHER2ec-RAP-MAP into LN229 cells using the Neon transfection system (Thermo Fisher Scientific, Inc.).(28) CHO-K1,
CHO/HER2, P3U1, MKN-7, and Kato III were cultured in
RPMI-1640 media (Nacalai Tesque, Inc., Kyoto, Japan),while LN229, LN229/HER2, and LN229/HER2ec were
cultured in Dulbecco’s modified Eagle’s medium (Nacalai
Tesque, Inc.). Media were supplemented with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin (Nacalai Tesque, Inc.),
100 lg/mL streptomycin (Nacalai Tesque, Inc.), and0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.) and incubated at 37C in a humidified atmosphere containing 5% CO2.

169
Hybridoma production

Female 4-week-old BALB/c mice were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under
specific pathogen-free conditions, and the Animal Care and
Use Committee of Tohoku University approved all animal
experimentations described.
The ectodomain of HER2 with N-terminal PA tag andC-terminal RAP tag-MAP tag (HER2ec) was purified fromthe culture supernatant of LN229/HER2ec using an anti-MAP
tag mAb (clone PMab-1) as previously described.(28) Intraperitoneal injections were utilized to immunize BALB/cmice with 100 lg of HER2ec and Imject Alum (Thermo
Fisher Scientific, Inc.). Additional immunizations were provided with a booster injection intraperitoneally administered
2 days before harvesting the spleen cells. Spleen cells were
subsequently fused with P3U1 cells using PEG1500 (Roche
Diagnostics, Indianapolis, IN). RPMI-1640 medium, supplemented with 10% heat-inactivated FBS (Thermo Fisher
Scientific, Inc.), 100 U/mL of penicillin (Nacalai Tesque, Inc.),
100 lg/mL streptomycin (Nacalai Tesque, Inc.), 0.25 lg/mL
amphotericin B (Nacalai Tesque, Inc.), and hypoxanthine/aminopterin/thymidine selection medium supplement (Thermo
Fisher Scientific, Inc.), was used to culture the resulting hybridomas. Enzyme-linked immunosorbent assay (ELISA)
was utilized to screen the culture supernatants with HER2ec.
ELISA

A concentration of 1 lg/mL of HER2ec was immobilizedon Nunc MaxiSorp 96-well immuno plates (Thermo Fisher
Scientific, Inc.) for 30 minutes at 37C. Plates were washedwith phosphate-buffered saline containing 0.05% Tween 20(PBST), and the wells were subsequently blocked with 1%
bovine serum albumin (BSA; Nacalai Tesque, Inc.) containing PBST for 30 minutes at 37C. The culture supernatant
was then added to the plate along with a 1:2000 dilution ofperoxidase-conjugated anti-mouse immunoglobulins (Agilent Technologies, Inc., Santa Clara, CA). We used 1-Step
Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.) for the
enzymatic reactions. The optical density was measured at
655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).
Flow cytometric analyses

Cells were trypsinized with 0.25% trypsin in 1-mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.) and subsequently harvested. After washing with 0.1% BSA in PBS,
cells were treated with 1 lg/mL of anti-HER2 mAb (H2Mab181) for 30 minutes at 4C, with subsequent addition of Alexa
Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA). Fluorescence data werecollected using EC800 Cell Analyzers (Sony Corp., Tokyo,
Japan).
Western blot analysis

Protein samples were obtained from cell lysates (10 lg) by
boiling in sodium dodecyl sulfate sample buffer (Nacalai
Tesque, Inc.) and electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan) and, subsequently, transferred onto polyvinylidene difluoride membranes (Merck KGaA, Darmstadt,

170

Germany). The membranes were blocked with 4% skim
milk (Nacalai Tesque, Inc.) and then incubated with either
10 lg/mL of H2Mab-181 or 1 lg/mL of anti-b-actin (clone
AC-15; Sigma-Aldrich Corp., St. Louis, MO) and then withperoxidase-conjugated anti-mouse immunoglobulins (1:2000
dilution for H2Mab-181, 1:1000 dilution for anti-b-actin;
Agilent Technologies, Inc.). An ImmunoStar LD (FUJIFILM
Wako Pure Chemical Corporation) was used to develop theblots, and they were imaged with a Sayaca-Imager (DRC Co.
Ltd., Tokyo, Japan).

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Determination of the binding affinity

Cells were suspended in 100 lL of serially diluted H2Mab181 (0.006–100 lg/mL). To this Alexa Fluor 488-conjugatedanti-mouse IgG (1:200; Cell Signaling Technology, Inc.) was
added. Fluorescence data were collected using the EC800
Cell Analyzer (Sony Corp.). For the calculation of the dissociation constant (KD), the binding isotherms were fitted tobuilt-in one-site binding models in GraphPad Prism 8(GraphPad Software, Inc., La Jolla, CA).

FIG. 1. Characterization of H2Mab-181. (A) Flow cytometry with H2Mab-181; CHO-K1, CHO/HER2, MKN-7, andKato III were treated with 1 mg/mL of H2Mab-181 followed
by Alexa Fluor 488-conjugated anti-mouse IgG; the black line
indicates the negative control. (B) Western blot using H2Mab181; cell lysates (10 mg) were electrophoresed, and proteins were transferred onto PVDF membranes. After blocking,membranes were incubated with 10 mg/mL of H2Mab-181
or 1 mg/mL of anti-b-actin (AC-15) and then incubatedwith peroxidase-conjugated anti-mouse immunoglobulines.
CHO, Chinese hamster ovary; HER2, human epidermal
growth factor receptor 2; PVDF, polyvinylidene difluoride.

TAKEI ET AL.
Immunohistochemical analyses

A microarray of the histologic sections (4 lm thickness) of
stomach adenocarcinoma tissue (Catalog No.: BS01012e; US
Biomax, Inc., Rockville, MD) was directly autoclaved in
EnVision FLEX Target Retrieval Solution High pH (Agilent
Technologies, Inc.) for 20 minutes. Sections were thenblocked with SuperBlock T20 (PBS) Blocking Buffer(Thermo Fisher Scientific, Inc.) and then incubated with
5 lg/mL H2Mab-181 for 1 hour at room temperature and thentreated with an EnVision+ kit (Agilent Technologies, Inc.)for 30 minutes. The color was developed for 2 minutes using
3,3¢-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.), and hematoxylin (FUJIFILM Wako Pure Chemical Corporation) was used to counterstain the sections.
Hematoxylin and eosin (HE; FUJIFILM Wako Pure Chemical Corporation) staining was also performed. The intensity of staining was evaluated as -, 1+, 2+, 3+.

FIG. 2. Determination of the binding affinity by flow cytometry. CHO/HER2, MKN-7, and Kato III cells were suspended in 100 mL of serially diluted H2Mab-181 (6 ng/mL
to 100 mg/mL), and secondary anti-mouse IgG was subsequently added. Fluorescence data were collected using a cell
analyzer.

171

Sex

F
M
M
M
F
F
M
F
F
M
M
M
M
M
M
M
M
M
F
F
F
M
M
M
M
M
M
M
F
M
M
F
M
M
M
F
M
F
M
M
M
F

No.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42

49
68
55
75
60
48
66
55
47
61
70
60
54
72
71
63
66
53
68
64
50
72
50
69
59
35
43
60
70
64
45
35
57
72
68
72
62
68
43
55
61
49

Age

StomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachOrgan
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Mucinous adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Mucinous adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma

Pathology diagnosis
T3N1M0
T3N0M0
T4N2M0
T3N1M0T2N1M0T3N2M0
T3N0M0T2N0M0
T3N1M0
T3N1M0
T3N1M0T2N1M0T2N1M0T2N1M0T2N1M0T2N1M0T1N2M0
T3N1M0T2N1M0T2N1M0
T3N0M0T2N0M0
T3N0M0T2N0M0T2N0M0
T3N0M0
T3N1M0
T3N1M0T2N0M0T2N1M0
T3N1M0T3N2M0
T3N1M0
T3N1M0
T3N1M0
T3N0M0T2N2M0T2N1M0T2N0M0T3N2M0
T3N0M0
T3N1M0TNM
Moderately
Moderately
Moderately
PoorlyModerately
PoorlyModerately
Well*moderately
PoorlyModerately
PoorlyModerately
Moderately
Moderately
Moderately
Moderately
Moderately
PoorlyModerately
Moderately
PoorlyModerately*poorly
PoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyModerately

Grade
IIB
IIA
IIIB
IIB
IIA
IIIA
IIA
IB
IIB
IIB
IIB
IIA
IIA
IIA
IIA
IIA
IIA
IIB
IIA
IIA
IIA
IB
IIA
IB
IB
IIA
IIB
IIB
IB
IIA
IIB
IIIA
IIB
IIB
IIB
IIA
IIB
IIA
IB
IIIA
IIA
IIB

Stage

Table 1. Immunohistochemical Analysis of Gastric Cancers Using H2Mab-181

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Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
MalignantType(continued)

2+
3+
—
—
—
—
—
—
2+
2+
—
—
—
—
2+
—
2+
—
—
—
—
—
—
—
—
—
—
—
—
2+
—
—
—
—
—
—
—
—
—
—
—
1+

H2Mab-181 intensity

172

Sex

M
M
M
M
F
M
M
F
M
M
M
M
M
M
M
M
M
M
F
M
F
M
M
M
M
M
F
M

No.

43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70

49
57
63
60
46
50
63
37
45
34
54
64
40
33
70
46
55
58
24
35
47
64
56
75
48
48
74
45

Age

StomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachStomachOrgan
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Mucinous adenocarcinoma
Adenocarcinoma
Mucinous adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma
Adenocarcinoma

Pathology diagnosis
T2N1M0
T3N1M0T3N2M0T2N1M0
T3N1M0T2N1M0T2N1M0T2N2M0T2N1M0T2N1M0
T3N1M0T2N1M0T3N2M0
T3N1M0T2N0M0
T3N0M0
T3N1M0T2N1M0
T3N1M0
T3N1M0
T3N1M0T2N1M0T3N2M0T2N0M0T2N0M0T2N0M0T2N0M0T4N0M1TNM

Grade
PoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyPoorlyModerately
PoorlyPoorlyPoorlyPoorlyPoorlyTable 1. (Continued)

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IIA
IIB
IIIA
IIA
IIB
IIA
IIA
IIB
IIA
IIA
IIB
IIA
IIIA
IIB
IB
IIA
IIB
IIA
IIB
IIB
IIB
IIA
IIIA
IB
IB
IB
IB
IV

Stage
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant

Type

3+
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3+
—
—
—
—
—

H2Mab-181 intensity

ANTI-HER2 MAB FOR GASTRIC CANCER
Results
Establishment of an anti-HER2 mAb

Mice were immunized with the recombinant extracellulardomain of HER2 (HER2ec) and subsequently analyzed foranti-HER2 mAbs for binding to HER2ec using an ELISA. We
next performed a flow cytometric analysis as a second evaluation to access the reaction in LN229 and LN229/HER2
cells. A stronger reaction against LN229/HER2 was necessary due to LN229 cells expressing endogenous HER2.(28)We finally established one clone H2Mab-181 (IgG1, kappa),
which strongly reacted with LN229/HER2 and weakly with
LN229 cells (data not shown).
Flow cytometric analyses

which indicate that H2Mab-181 is specific to HER2. H2Mab181 also recognized endogenous HER2 in MKN-7 and Kato
III cells (Fig. 1A). The reaction of H2Mab-181 in MKN-7
cells was higher than that for Kato III cells. These results
show that H2Mab-181 is suitable for the detection of gastriccancer cells.
Western blot analyses

H2Mab-181 detected HER2 as a 180–200 kDa band in
CHO/HER2 cells and did not detect any other bands within
the CHO-K1 cells by Western blot analyses (Fig. 1B), which
again suggests that H2Mab-181 can specifically detect HER2
in Western blot analyses. H2Mab-181 also detected endogenous HER2 in MKN-7 cells (Fig. 1B). In contrast, H2Mab181 did not detect HER2 in Kato III cells, which may be due
to low HER2 expression level in these cells for the detectionby H2Mab-181 using Western blot analyses (Fig. 1B).

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The flow cytometry data showed that H2Mab-181 recognized CHO/HER2 cells but not CHO-K1 cells (Fig. 1A),

173

FIG. 3. Immunohistochemical analysis using H2Mab-181. Histological sections of stomach adenocarcinoma tissue microarrays were directly autoclaved in EnVision FLEX Target Retrieval Solution High pH. After blocking, sections wereincubated with 5 mg/mL of H2Mab-181 (A, B) or blocking buffer control (C, D) and then treated with an EnVision+ kit. The
color was developed using DAB. Sections were counterstained with hematoxylin. (E, F) HE staining was also performed.
Scale bar = 100 mm. DAB, 3,3¢-diaminobenzidine tetrahydrochloride; HE, hematoxylin and eosin.

174
Determination of the binding affinity

Kinetic analysis of the interactions between H2Mab-181
and CHO/HER2, MKN-7, and Kato III cells was performed
using flow cytometry. As shown in Figure 2, the dissociation
constants (KD) for the interaction of H2Mab-181 with CHO/
HER2, MKN-7, and Kato III cells were 5.5 · 10-9, 8.5 · 10-9,
and 3.8 · 10-9 M, respectively, suggesting that H2Mab-181
has a high affinity to HER2.

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Immunohistochemical analyses

Finally, immunohistochemical analysis was performed
using formalin-fixed paraffin-embedded tissue sections ofstomach adenocarcinomas. Typical staining patterns (PatientNo. 1 in Table 1) are shown in Figure 3A and B. The plasma
membrane was strongly stained by H2Mab-181 in the cancer
cells. In 10 out of 70 cases (14.3%) of the stomach adenocarcinoma tissue array (Table 1), H2Mab-181 staining wasobserved. No staining was observed in those sections stainedwith the buffer negative control (Fig. 3C, D). HE staining isshown in Figure 3E and F.
Discussion

The domain-I and domain-III of the extracellular region
are ligand-binding sites for EGFR, HER3, and HER4. In
contrast, no ligand can bind to HER2, and domain-I and -IIIof HER2 connect each other.(3,29) Heterodimers are formed
between HER2 and other ligand-bound HER family members using largely domain-II-mediated dimerization interface, whereas heterodimerization with ligand-free HER
family members mainly occurs through the domain IV.(30,31)
Pertuzumab binds near the center of domain II of HER2 andblocks ligand-induced receptor dimerization and the subsequent signaling cascade.(32) Trastuzumab binds to the juxtamembrane region of domain IV, resulting in the inhibitionof ligand-free receptor dimerization, stimulation of HER2
endocytosis, and removal of HER2 from the cell surface.(29)
Pertuzumab and trastuzumab also possess antibodydependent cellular cytotoxicity either alone or in combination with each other.(33)
Some studies have also previously reported the establishment of mAbs, which target domain III of HER2. Fu et al.
developed a novel anti-HER2 mAb (hHERmAb-F0178C1)
using the phage display method. hHERmAb-F0178C1 had
efficacy in blocking HER2/HER3 heterodimerization andsignaling, and its combinatory use with pertuzumab had asynergistic effect.(34) Meng et al. also established anothernovel anti-HER2 mAb (3E10) using hybridoma technology.
In that study, 3E10 inhibited HER2 heterodimerization with
EGFR and HER3 through a different mechanism to that of
trastuzumab and pertuzumab. Synergistic inhibition of HER2
dimerization and signaling was observed with 3E10 in combination with either trastuzumab or pertuzumab. Moreover,
triple-drug treatment of trastuzumab, pertuzumab, and 3E10
had even greater antitumor activity in HER2-overexpressing
breast cancer models.(35) Together, these studies provide
evidence that targeting the domain III of the HER2 extracellular region may be a new target for cancer treatment.
In this study, we have successfully developed a novel antiHER2 mAb, H2Mab-181, suitable for a range of applications,
including flow cytometry, Western blot, and immunohisto-

TAKEI ET AL.

chemical analyses. Previously, we also established otheranti-HER2 mAbs, such as H2Mab-19,(36) H2Mab-41,(37)H2Mab-77,(28) H2Mab-119,(38) and H2Mab-139,(39) which
are suitable for the same applications. However, the epitopeof those anti-HER2 mAbs has not been clarified. For futurestudies, the epitope of H2Mab-181 will be determined, andH2Mab-181 will be investigated for antitumor activities
within gastric cancers.
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 Yukinari Kato) andJP21am0101078 (to Yukinari Kato) and by the Japan Society
for the Promotion of Science ( JSPS) Grants-in-Aid for Scientific Research (KAKENHI) grant numbers 21K15523(to Teizo Asano), 21K07168 (to Mika K. Kaneko), 20K16322(to Masato Sano), and 19K07705 (to Yukinari Kato).
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TAKEI ET AL.

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: May 10, 2021
Accepted: June 5, 2021