MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 4, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0026

Original Articles

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H2Mab-77 is a Sensitive and Specific Anti-HER2
Monoclonal Antibody Against Breast Cancer
Shunsuke Itai,1,2,* Yuki Fujii,3,* Mika K. Kaneko,1 Shinji Yamada,1 Takuro Nakamura,1Miyuki Yanaka,1 Noriko Saidoh,1 Yao-Wen Chang,1 Saori Handa,1 Maki Takahashi,4
Hiroyoshi Suzuki,4 Hiroyuki Harada,2 and Yukinari Kato1,3,5

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, andHER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanizedantibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In
this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy
in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full lengthor ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or
LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of
HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening),
Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones,
only three mAbs reacted with HER2 in Western blot, and clone H2Mab-77 (IgG1, kappa) was selected. Finally,
immunohistochemical analyses with H2Mab-77 showed sensitive and specific reactions against breast cancer
cells, warranting the use of H2Mab-77 to detect HER2 in pathological analyses of breast cancers.
Keywords: HER2, monoclonal antibody, immunohistochemistry, breast cancer

IntroductionHuman epidermal growth factor receptor 2(HER2) plays an important role in the progression ofbreast cancers, and its overexpression was reported in >20%
of patients with breast cancer.(1,2) HER2 overexpression isassociated with poor clinical outcomes, and the monoclonal
antibodies (mAbs) trastuzumab(3) and pertuzumab,(4) the tyrosine kinase inhibitor lapatinib,(5) and the antibody–drugconjugate (ADC) of trastuzumab and the maytansinoid microtubule assembly inhibitor trastuzumab emtansine(6) havebeen approved for the treatment of HER2-positive breast
cancers. Treatment with trastuzumab demonstrated significant survival benefits for patients with HER2-positive metastatic breast cancers.(7) Furthermore, dual blockade of HER2
with the combination of pertuzumab and trastuzumab, as well
as chemotherapy, led to significant improvements in overall
survival compared with trastuzumab plus chemotherapy.(8)Previously, we developed an original technology for the
production of cancer-specific monoclonal antibodies (Cas-

Mabs) and anti-glycopeptide mAbs (GpMabs) against
membrane proteins.(9) We also developed sensitive andspecific mAbs against several other membrane proteins using
the same technology.(10–13) In this study, we developed antiHER2 mAbs for use in flow cytometry, Western blot, andimmunohistochemical analyses.
Materials and Methods
Cell lines

LN229, A431, SK-BR-3, Chinese hamster ovary (CHO)K1, HEK-293T, Met-5A, and P3U1 cell lines were obtained
from the American Type Culture Collection (ATCC, Manassas, VA). LN229/HER2 and CHO/HER2 were produced by transfecting pCAG/PA-HER2-RAP-MAP(14) into
LN229 and CHO-K1 cells using Neon transfection system(Thermo Fisher Scientific, Inc., Waltham, MA) and Lipofectamine LTX (Thermo Fisher Scientific, Inc.), respectively. A few days after transfection, PA tag-positive1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Yushima, Bunkyo-ku, Tokyo, Japan.
3
Department of Regional Innovation, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi, Japan.
4
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Miyagino, Miyagino-ku, Sendai, Miyagi, Japan.
5
New Industry Creation Hatchery Center, Tohoku University, Aoba-ku, Sendai, Miyagi, Japan.
*Both authors contributed equally to this work.
2

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cells(15) were sorted using a cell sorter (SH800; Sony Corp.,
Tokyo, Japan).
CHO-K1, CHO/HER2, and P3U1 cell lines were cultured
in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan),and LN229, LN229/HER2ec, LN229/HER2, A431, SK-BR3, HEK-293T, and Met-5A cell lines were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Inc.),
supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc.), 100 units/mL of penicillin,
100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B(Nacalai Tesque, Inc.) at 37C in a humidified atmosphere
containing 5% CO2 and 95% air.
Animals

Female 4-week-old BALB/c mice were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed underpathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved all of the animal experiments described herein.
Tissues

One breast cancer patient who underwent surgery at Sendai
Medical Center was recruited for examinations, and theethics committee of Sendai Medical Center approved thisstudy. Normal and cancer tissues were purchased from BioChain Institute, Inc. (Newark, CA) and US Biomax, Inc.
(Rockville, MD), respectively.
Hybridoma production

The ectodomain of HER2 with N-terminal PA tag(15) andC-terminal RAP tag(16) -MAP tag(14) (HER2ec) was purified
from supernatant of LN229/HER2ec using anti-MAP tag as
described previously.(14) BALB/c mice were immunized using
intraperitoneal (i.p.) injections of LN229/HER2 cells or 100 mg
of HER2ec together with Imject Alum (Thermo Fisher Scientific, Inc.). After several additional immunizations, a booster
injection of LN229/HER2 cells or 100 mg of HER2ec wasintraperitoneally administered 2 days before harvesting spleencells. Spleen cells were then fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN) or
GenomONE-CF (Ishihara Sangyo Kaisha, Ltd., Osaka, Japan).
The resulting hybridomas were grown in RPMI medium
supplemented with hypoxanthine, aminopterin, and thymidine
selection medium supplement (Thermo Fisher Scientific, Inc.).
Culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA) with HER2ec, and mAbs werescreened using flow cytometry (second screening), Western
blot (third screening), and immunohistochemical analyses(fourth screening). MAbs were purified from supernatants ofhybridomas cultured in Hybridoma-SFM medium (Thermo
Fisher Scientific, Inc.) using Protein G Sepharose 4 Fast Flow
(GE Healthcare UK Ltd, Buckinghamshire, England).
Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin/
1-mM ethylenediaminetetraacetic acid (EDTA) (Nacalai
Tesque, Inc.). After washing with 0.1% bovine serum albumin (BSA)/phosphate-buffered saline, the cells were treatedwith 1 mg/mL of anti-HER2 (clone H2Mab-77) for 30 minutes
at 4C and then with Oregon green-conjugated anti-mouse

ITAI ET AL.

IgG (1:1000 diluted; Thermo Fisher Scientific, Inc.). Fluorescence data were collected using EC800 or SA3800 Cell
Analyzers (Sony Corp.).
Western blot analysis

Cell lysates (10 mg) were boiled in the sodium dodecyl
sulfate (SDS) sample buffer (Nacalai Tesque, Inc.) and proteins were then electrophoresed on 5%–20% polyacrylamidegels (Wako Pure Chemical Industries Ltd., Osaka, Japan),and were transferred onto polyvinylidene difluoride (PVDF)
membranes (Merck KGaA, Darmstadt, Germany). After
blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes
were incubated with 1 mg/mL of primary antibodies, such as
anti-HER2 (clone H2Mab-77) and anti-b-actin (clone AC-15;
Sigma-Aldrich Corp., St. Louis, MO), and then withperoxidase-conjugated anti-mouse IgG (1:1000 diluted; Agilent
Technologies, Inc., Santa Clara, CA), and were finally developed using ImmunoStar LD (Wako Pure Chemical Industries
Ltd.) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Determination of the binding affinity
using flow cytometry

A431 and SK-BR-3 (2 · 105 cells) were suspended in
100 mL of serially diluted mAbs (0.6 ng/mL–10 mg/mL), andAlexa Fluor 488-conjugated anti-mouse IgG (1:1000; Cell
Signaling Technology, Inc., Danvers, MA) was then added.
Fluorescence data were collected using a cell analyzer(EC800; Sony Corp.). The dissociation constants (KD) were
calculated by fitting the binding isotherms using the built-in
one-site binding models in GraphPad PRISM 6 (GraphPad
software, Inc., La Jolla, CA).
Immunohistochemical analyses

Histologic sections of 4-mm thickness were deparaffinizedin xylene and then rehydrated and autoclaved in citrate buffer(pH 6.0; Agilent Technologies, Inc.) for 20 minutes. Sections
were then incubated with 1 mg/mL of primary mAbs for
1 hour at room temperature and then treated using an
Envision+ kit (Agilent Technologies, Inc.) for 30 minutes.
Color was developed using 3,3-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes,
and sections were then counterstained with hematoxylin(Wako Pure Chemical Industries Ltd.). The intensity ofstaining was evaluated as 0, 1+, 2+, and 3+.
Results
Production of anti-HER2 mAbs

Herein, we immunized mice with LN229/HER2 or purified
recombinant HER2ec from culture supernatants of LN229/
HER2ec cells. A booster i.p. injection of LN229/HER2 or
HER2ec was also administered and culture supernatants werethen screened for binding to purified HER2ec using ELISA.
As a second screen, we used flow cytometry analyses to assess reactions with LN229 and LN229/HER2 cells. Because
LN229 cells express endogenous HER2, a stronger reaction
against LN229/HER2 was necessary.
A total of 29 clones were generated from immunizations with LN229/HER2, and 71 were generated from
immunizations with purified HER2ec. Among 100 clones, 96

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FIG. 1. Characterization of H2Mab-77. (A, B) Flow cytometry with H2Mab-77; cells were treated with 1 mg/mL of
H2Mab-77 followed by Oregon green-conjugated anti-mouse IgG; black line, negative control. (C) Western blot using
H2Mab-77; cell lysates (10 mg) were electrophoresed and proteins were transferred onto PVDF membranes. After blocking,membranes were incubated with 1 mg/mL of H2Mab-77 or anti-b-actin (AC-15), and then incubated with peroxidaseconjugated anti-mouse IgG. (D) Binding affinity of H2Mab-77 was determined using flow cytometry. A431 and SK-BR-3
cells were suspended in 100 mL of serially diluted H2Mab-77 (0.6 ng/mL–10 mg/mL) and secondary anti-mouse IgG wasthen added. Fluorescence data were collected using a cell analyzer. PVDF, polyvinylidene difluoride.
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ITAI ET AL.

clones reacted with SK-BR-3 cells, which express endogenous HER2 (data not shown). Then, mAbs were further selected according to their efficacy on Western blot analysis.
These analyses identified only one clone from LN229/HER2
immunizations and two clones from HER2ec immunizations
that were useful for Western blot analyses, and the mostsensitive clone H2Mab-77 (IgG1, kappa; from HER2ec immunization) was selected for subsequent studies.

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Characterization of H2Mab-77

In flow cytometric analyses, H2Mab-77 reacted with
LN229/HER2 more strongly than with endogenous HER2expressing LN229 glioblastoma cells (Fig. 1A). H2Mab-77
also reacted with CHO/HER2 and did not react with theparental cell strain CHO-K1, indicating that H2Mab-77 is
specific for HER2 (Fig. 1A). H2Mab-77 also recognizedendogenous HER2 in A431 epidermoid carcinoma cells,
SK-BR-3 breast cancer cells, HEK-293T kidney epithelial
cells, and Met-5A mesothelial cells (Fig. 1B). In subsequentWestern blot analyses against LN229, LN229/HER2, and SKBR-3 cells (Fig. 1C), H2Mab-77 detected a 180–200 kDa
protein in LN229/HER2 and SK-BR-3 cells, indicating that
H2Mab-77 is very useful for Western blot analyses. We further determined the binding affinity of H2Mab-77 for A431
and SK-BR-3 cells using flow cytometry (Fig. 1D) and calculated KD values for H2Mab-77 of 2.1 · 10-9 M against A431
and 7.3 · 10-9 M against SK-BR-3, indicating that H2Mab-77
possesses high affinity, particularly for A431 cells.
Immunohistochemical analysis against breast cancers

Finally, we investigated the immunohistochemical utilityof H2Mab-77 in human breast cancers. As shown in
Figure 2A, H2Mab-77 stained membranes of cancer cells inthis patient, who was previously diagnosed with HER2positive breast cancer using HercepTest. In contrast, H2Mab77 did not stain any adjacent breast tissues (Fig. 2B).
In immunohistochemical analyses using H2Mab-77
against two breast cancer tissue microarrays (Supplementary
Table S1), H2Mab-77 stained 13 of 58 (22.4%) invasive
ductal carcinomas and one medullary carcinoma (Supplementary Table S2). Typical staining patterns are presented in
Supplementary Figure S1.
Discussion

Establishment of mAbs that are useful for flow cytometry,
Western blot, and immunohistochemical analyses is generally difficult. Moreover, designs of immunogens andscreening methods to produce specific mAbs vary between
Western blot and immunohistochemical applications, and it
is very difficult to produce specific and sensitive mAbs that
detect endogenous proteins. In this study, we used our original method for producing mAbs and immunized mice with
LN229/HER2 or purified HER2ec from LN229/HER2ec
cells. Among the 100 anti-HER2 mAbs developed herein,
only three were applicable to Western blot analyses.
Among 100 clones of anti-HER2 mAbs, 97 (97%) wereidentified as belonging to the IgG1 subclass, and the remaining three to the IgG2b subclass. H2Mab-77 was also
determined to be of the IgG1 subclass, precluding confirmation of antibody-dependent cellular cytotoxicity (ADCC) or

FIG. 2. Immunohistochemical analysis by H2Mab-77
against breast cancers. Sections were incubated with 1 mg/
mL of primary H2Mab-77 for 1 hour at room temperature
followed by treatment with Envision+ kit for 30 minutes.
Color was developed using 3, 3-diaminobenzidine tetrahydrochloride (DAB) for 2 minutes, and sections were thencounterstained with hematoxylin. (A) Breast cancer; (B)normal breast tissues; scale bar = 100 mm.
complement-dependent cytotoxicity (CDC) activities using
H2Mab-77. Thus, in future studies, we will convert the subclass H2Mab-77 into mouse IgG2a or IgG2b subclasses orhuman IgG1, and assess their applications to measure ADCC/
CDC activities.(12,17,18)
We previously produced an anti-podoplanin cancer-specificmAb (CasMab), clone LpMab-2(9,18) or LpMab-23,(19) which
specifically recognizes cancer-type podoplanin in tumor tissues.
For this technology, it is critical that immunogens are produced
using cancer cell lines, such as LN229 glioblastoma cells, which
express cancer-specific glycan-attached membrane proteins.
This technology was also useful for generating anti-glycopeptide
mAbs (GpMabs), including LpMab-3,(20) LpMab-9,(21) LpMab12,(22) LpMab-19,(23) and LpMab-21.(24,25) Furthermore, weused this technology to generate mAbs that bind to various novel
epitopes of podoplanin, including LpMab-7,(10–12) LpMab10,(26) LpMab-13,(27) and LpMab-17.(28) We have also developed sensitive and specific anti-podocalyxin mAbs using
the same methods.(13) Among anti-podocalyxin mAbs, clone
PcMab-47 was reportedly useful in flow cytometry, Western
blot, and immunohistochemical analyses, warranting consideration of PcMab-47 in investigations of podocalyxin
expression and function in cancers and normal tissues.
Moreover, this method could be used to develop useful mAbs
against multiple membrane proteins.
Many other studies about antibody production and antibody engineering targeting HER2 have been performed. LuoDownloaded by 202.162.86.108 from online.liebertpub.com at 08/15/17. For personal use only.

MABS AGAINST HER2

et al. recently reported the glycoengineering of pertuzumab
and its impact on the pharmacokinetic/pharmacodynamic
properties.(29) They showed that fucose was critical for
ADCC activity by influencing the interaction between pertuzumab and FcgRIIIa, and removal of sialic acid using sialidase hydrolysis increased the ADCC and CDC activity of
pertuzumab. Lopez-Albaitero et al. demonstrated that a novel
HER2/CD3 bispecific antibody (HER2-BsAb) recruits andactivates nonspecific circulating T cells, promoting T celltumor infiltration and ablating HER2-positive tumors, evenwhen these are resistant to standard HER2-targeted therapies.(30) Menderes et al. reported that SYD985, a novel
HER2-targeting ADC, is more potent than trastuzumab emtansine (T-DM1) against epithelial ovarian cancer.(31) Sokolova et al. reported that immunotoxin 4D5scFv-PE40,
which comprises 4D5scFv anti-HER2 antibody as a targeting
module and fragment of Pseudomonas exotoxin A as an effector (toxic), showed significant antitumor effect on HER2positive cancer cells in vitro and on HER2-positive xenograftmodels in vivo.(32) In the same way with those studies, ourestablished H2Mab-77 should be converted to ADC, immunotoxin, bispecific antibody with CD3, or glycoengineeringmAbs to target effectively HER2-positive breast cancers.
In conclusion, from 100 mAb clones, H2Mab-77 washighly efficacious in Western blot analyses and produced
strong staining in breast cancers. Hence, the mAb H2Mab-77
was useful in all the present experiments and will likely be auseful tool for the pathological identification of HER2 breast
cancers.
Acknowledgments

We thank Yoshimi Nakamura and Yoshikazu Satoh forexcellent technical assistance. This work was mainly supported by the Basic Science and Platform Technology Program for Innovative Biological Medicine from Japan Agency
for Medical Research and development, AMED (Y.K.). This
work was also supported, in part, by the Platform Project for
Supporting Drug Discovery and Life Science Research from
AMED (Y.K.), by Project for utilizing glycans in the development of innovative drug discovery technologies from
AMED (Y.K.), by the Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) from AMED
(Y.K.), and by JSPS KAKENHI Grant Number 17K07299(M.K.K.) and 16K10748 (Y.K.). This work was performed,
in part, under the Cooperative Research Program of Institutefor Protein Research, Osaka University, CR-17-05, and by
the Grant for Joint Research Project of the Institute ofMedical Science, the University of Tokyo. The authors would
like to thank Enago (www.enago.jp) for the English language
review.
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd.
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Address correspondence to:
Dr. Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
yukinarikato@med.tohoku.ac.jp
Received: May 11, 2017
Accepted: June 14, 2017