MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 6, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0050

Short Communications

Establishment of H2Mab-119, an Anti-Human Epidermal
Growth Factor Receptor 2 Monoclonal Antibody,
Against Pancreatic Cancer

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Shinji Yamada,1 Shunsuke Itai,1,2 Takuro Nakamura,1 Yao-Wen Chang,1 Hiroyuki Harada,2Hiroyoshi Suzuki,3 Mika K. Kaneko,1 and Yukinari Kato1,4

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor
clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal,lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival
benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established anovel anti-HER2 monoclonal antibody (mAb), H2Mab-119 (IgG1, kappa), and characterized its efficacy against
pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H2Mab-119 reactedwith pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 inflow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for
MIA PaCa-2, although H2Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical
analyses with H2Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did notreact with pancreatic cancers, indicating that H2Mab-119 is useful for detecting HER2 overexpression inpancreatic cancers using flow cytometry and Western blot analyses.
Keywords: HER2, monoclonal antibody, immunohistochemistry, pancreatic cancer

IntroductionHuman epidermal growth factor receptor 2 (HER2)
is involved in the progression of breast cancers.(1,2)
HER2 overexpression is reported in >20% of patients withbreast cancer and is associated with poor clinical outcomes.
Humanized anti-HER2 monoclonal antibodies (mAbs), suchas trastuzumab and pertuzumab; antibody-drug conjugate,including trastuzumab emtansine; and tyrosine kinase inhibitors, such as lapatinib, have been approved for the treatment
of HER2-positive breast cancers.(3–6) Trastuzumab treatments have resulted in significant survival benefits in patientswith metastatic HER2-positive breast cancers.(7) Furthermore,
the combination of pertuzumab and trastuzumab with chemotherapy has led to significant improvements in overall
survival compared with trastuzumab alone plus chemotherapy.(8) In addition, overexpression of HER2 has been reported
in gastric cancers,(9) colorectal cancers,(10) lung cancers,(11)and pancreatic cancers.(12) In this study, we developed a novelanti-HER2 mAb and investigated its utility using flow cytometry, Western blot, and immunohistochemical analyses forpancreatic cancers.
Materials and Methods
Cell lines, tissues, and animals

LN229, Capan-2, and P3U1 cell lines were obtained fromthe American Type Culture Collection (Manassas, VA). KLM1, PANC-1, and MIA PaCa-2 cell lines were obtained fromthe Cell Resource Center for Biomedical Research Institute ofDevelopment, Aging and Cancer Tohoku University (Miyagi,Japan). LN229/HER2 was produced by transfecting pCAG/
PA-HER2-RAP-MAP(13) into LN229 cells using the neontransfection system (Thermo Fisher Scientific, Inc., Waltham, MA). A few days after transfection, PA tag-positivecells(14) were sorted using a cell sorter (SH800; Sony Corp.,Tokyo, Japan). P3U1, KLM-1, and PANC-1 cell lines werecultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto,1

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 Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
4
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
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Japan); Capan-2 was cultured in McCoy’s-5A medium(GE Healthcare UK Ltd., Buckinghamshire, England);
whereas LN229, LN229/HER2, and MIA PaCa-2 cell lines
were cultured in Dulbecco’s Modified Eagle Medium(DMEM) (Nacalai Tesque, Inc.) supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific, Inc.),
100 U/mL of penicillin, 100 mg/mL of streptomycin, and25 mg/mL of amphotericin B (Nacalai Tesque, Inc.). All celllines were incubated at 37C in a humidified atmosphere
containing 5% CO2 and 95% air. One breast cancer patient who
underwent surgery at Sendai Medical Center was used for thisstudy.(15) Colon cancer tissue arrays (40 cases) and pancreaticcancer tissue arrays (26 cases) were purchased from U.S.
Biomax, Inc. (Rockville, MD). Female 4-week-old BALB/cmice were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under specific pathogen-free conditions.
The Animal Care and Use Committee of Tohoku University
approved all the animal experiments described in this study.
Hybridoma production

Anti-HER2 hybridomas were produced, as described
previously.(15) In brief, BALB/c mice were immunized using intraperitoneal (i.p.) injections of 100 mg of recombinant
HER2-extracellular domain together with Imject Alum
(Thermo Fisher Scientific, Inc.). After several additional
immunizations, a booster injection was intraperitoneallyadministered 2 days before harvesting spleen cells. Spleencells were then fused with P3U1 cells using PEG1500 (RocheDiagnostics, Indianapolis, IN). 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 recombinant HER2-extracellular domain.
MAbs were purified from the supernatants of hybridomas,
cultured in Hybridoma-SFM (Thermo Fisher Scientific, Inc.)
using Protein G Sepharose 4 Fast Flow (GE Healthcare UKLtd.).
Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin/
1 mM ethylenediaminetetraacetic acid (EDTA; NacalaiTesque, Inc.). After washing with 0.1% bovine serum albumin/phosphate-buffered saline, the cells were treated with
10 mg/mL of anti-HER2 (clone H2Mab-119) for 30 minutes at4C and subsequently with Alexa Fluor 488-conjugated antimouse IgG (1:1000; Cell Signaling Technology, Inc.,
Danvers, MA). Fluorescence data were collected using
EC800 Cell Analyzers (Sony Corp.).
Western blot analysisCell lysates (10 mg) were boiled in Sodium Dodecyl Sulfate (SDS) sample buffer (Nacalai Tesque, Inc.) and proteins
were then electrophoresed on 5%–20% polyacrylamide gels
(Wako Pure Chemical Industries Ltd., Osaka, Japan) before
being transferred onto PVDF membranes (Merck KGaA,Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.), membranes were incubated withprimary antibodies, such as anti-HER2 (10 mg/mL; clone
H2Mab-119), anti-MAP tag (1 mg/mL; clone PMab-1),(13) andYAMADA ET AL.

anti-b-actin (1 mg/mL, clone AC-15; Sigma-Aldrich Corp., St.
Louis, MO) and then with peroxidase-conjugated antimouse
IgG or antirat IgG (1:1000 diluted; Agilent Technologies, Inc.,
Santa Clara, CA). Finally, membranes were developed using
ImmunoStar LD (Wako Pure Chemical Industries Ltd.) with a
Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Immunohistochemical analyses

Histological sections of 4 mm thickness were deparaffinized in xylene and subsequently rehydrated and autoclaved in EnVision FLEX Target Retrieval Solution, high pH(Agilent Technologies, Inc.), for 20 minutes. Sections werethen incubated with 10 mg/mL of H2Mab-119 for 1 hour atroom temperature and treated using an Envision+ kit (AgilentTechnologies, Inc.) for 30 minutes. Color was developed
using 3,3-diaminobenzidine tetrahydrochloride (AgilentTechnologies, Inc.) for 2 minutes; subsequently, sections
were counterstained with hematoxylin (Wako Pure ChemicalIndustries Ltd.).
Results and Discussion

In this study, we immunized mice with purified recombinant extracellular domain of HER2. Culture supernatants
were then screened using ELISA. We used flow cytometry
analyses to assess reactions with LN229 and LN229/HER2cells. A stronger reaction against LN229/HER2 was necessary because LN229 cells express endogenous HER2. We
obtained one clone H2Mab-119 (IgG1, kappa).
In flow cytometric analyses, H2Mab-119 reacted with
LN229/HER2 more strongly than with endogenous HER2expressing LN229 glioblastoma cells (Fig. 1A). H2Mab-119
also reacted with CHO/HER2 but did not react with the parental cell strain CHO-K1 (data not shown), indicating that
H2Mab-119 is specific for HER2. H2Mab-119 recognized endogenous HER2 in pancreatic cancer cell lines, such as KLM1, Capan-2, and MIA PaCa-2, but did not react with PANC-1(Fig. 1B). In Western blots against LN229 and LN229/HER2
as well as pancreatic cancer cell lines, H2Mab-119 detected a
180–200 kDa protein in LN229/HER2 (Fig. 1C). In contrast,
H2Mab-119 detected a moderate signal in KLM-1, a weaksignal in MIA PaCa-2, and a faint signal in both PANC-1 andCapan-2, indicating that H2Mab-119 is useful for Western blot
analysis of pancreatic cancer cell lines.
Finally, we investigated the immunohistochemical utility of
H2Mab-119 in human breast cancers and pancreatic cancers.
As shown in Figure 1D, H2Mab-119 stained the cancer cell
membranes of breast cancers and colon cancers that were diagnosed previously as HER2-positive breast cancer. In contrast, H2Mab-119 did not stain HER2-positive pancreaticcancer tissues (data now shown), indicating that H2Mab-119 isapplicable only for flow cytometry and Western blot analyses
against pancreatic cancers in this study, although it is useful for
immunohistochemical analysis against breast cancers and colon cancers. In the future, we should stain many pancreaticcancer tissues to determine the positive rate in immunohistochemistry using H2Mab-119.
Acknowledgments

We thank Miyuki Yanaka, Noriko Saidoh, Saori Handa,
Yoshimi Nakamura, Yoshikazu Satoh, and Maki Takahashi

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MABS AGAINST HER2FIG. 1. Characterization of H2Mab-119. (A, B) Flow cytometry with H2Mab-119; cells were treated with 10 mg/mL ofH2Mab-119, followed by Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control. (C) Western blotting
using H2Mab-119; cell lysates were electrophoresed and proteins were transferred onto PVDF membranes. After blocking,membranes were incubated with 10 mg/mL of H2Mab-119, 1 mg/mL of anti-MAP tag (PMab-1), or 1 mg/mL of anti-b-actin(AC-15) and subsequently incubated with peroxidase-conjugated antimouse IgG against H2Mab-119 and AC-15 or antirat
IgG against PMab-1. (D) Sections were incubated with 10 mg/mL of H2Mab-119 for 1 hour at room temperature, followed
by treatment with Envision+ kit for 30 minutes. Color was developed using 3,3-diaminobenzidine tetrahydrochloride for 2minutes; subsequently, sections were counterstained with hematoxylin. Scale bar = 100 mm.
for excellent technical assistance. This work was supported,in part, by the Basic Science and Platform Technology
Program for Innovative Biological Medicine from Japan
Agency for Medical Research and Development, AMED
(Y.K.), by Project for utilizing glycans in the developmentof innovative drug discovery technologies from AMED(Y.K.), and by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting
Innovative Drug Discovery and Life Science Research,BINDS) from AMED.

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Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd. All other authors have nothing to disclose.

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Address correspondence to:
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
SendaiMiyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: September 15, 2017
Accepted: October 30, 2017