MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 1, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0052

Short Communications

Development of an Anti-HER2 Monoclonal Antibody
H2Mab-139 Against Colon Cancer

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Mika K. Kaneko,1 Shinji Yamada,1 Shunsuke Itai,1 and Yukinari Kato1,2

Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast,gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated withpoor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits forpatients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel antiHER2 monoclonal antibody (mAb), H2Mab-139 (IgG1, kappa) and investigated it against colon cancers using flow
cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H2Mab-139
reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO
205, HCT-8, SW1116, and DLD-1. Although H2Mab-139 strongly reacted with LN229/HER2 cells on the western
blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses
revealed sensitive and specific reactions of H2Mab-139 against colon cancers, indicating that H2Mab-139 is useful
in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.
Keywords: HER2, monoclonal antibody, immunohistochemistry, colon cancer

American Type Culture Collection (ATCC; Manassas, VA).
COLO 205, DLD-1, and HCT-15 cell lines were obtained
from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku University(Miyagi, Japan). LN229/HER2 was produced previously.(12)COLO 205, DLD-1, HCT-15, COLO 201, SW1116, and P3U1
were cultured in RPMI 1640 medium (Nacalai Tesque, Inc.,
Kyoto, Japan), and LN229, Caco-2, HCT-116, HT-29, LS 174T,and HCT-8 were cultured in DMEM (Dulbecco’s modified
Eagle’s medium) medium (Nacalai Tesque, Inc.), supplemented
with 10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc., Waltham, MA), 100 units/mL of penicillin,
100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B(Nacalai Tesque, Inc.). All cell lines were incubated at 37C in ahumidified atmosphere containing 5% CO2 and 95% air.

Introduction

Human epidermal growth factor receptor 2(HER2) has been reported to be overexpressed in >20%of patients with breast cancer and is associated with poor
clinical outcomes.(1,2) Trastuzumab and pertuzumab, humanized anti-HER2 monoclonal antibodies (mAbs), andtrastuzumab emtansine, an antibody-drug conjugate, havebeen approved for the treatment of HER2-positive breastcancer.(3–5) Trastuzumab treatment has resulted in significant
survival benefits for patients with metastatic HER2-positive
breast cancer.(6) Furthermore, the combination of pertuzumab and trastuzumab with chemotherapy has led to significant improvements in overall survival compared withtrastuzumab alone plus chemotherapy.(7) In addition, HER2
overexpression has been reported in gastric cancers,(8) lungcancers,(9) pancreatic cancers,(10) and colorectal cancers.(11)In this study, we developed a novel anti-HER2 mAb andcharacterized it against colon cancers using flow cytometry,western blot, and immunohistochemical analyses.

Hybridoma production

Female 4-week-old BALB/c mice 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. Anti-HER2 hybridomas
were produced, as described previously.(13) Briefly, BALB/cmice were immunized using intraperitoneal (i.p.) injections of 100 mg of recombinant HER2-extracellular domain

Materials and Methods
Cell lines

LN229, Caco-2, HCT-116, HT-29, LS 174T, COLO 201,HCT-8, SW1116, and P3U1 cell lines were obtained from the1
2

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

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FIG. 1. Characterization of H2Mab-139. (A) Flow cytometry with H2Mab-139; cells were treated with 10 mg/mL ofH2Mab-139, followed by Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control. (B) Western blot using
H2Mab-139; cell lysates were electrophoresed, and proteins were transferred onto PVDF membranes. After blocking, themembranes were incubated with 10 mg/mL of H2Mab-139 and 1 mg/mL of anti-b-actin (AC-15) and subsequently incubatedwith peroxidase-conjugated anti-mouse IgG. (C) Flow cytometry with H2Mab-139; colon cancer cell lines were treated with
10 mg/mL of H2Mab-139, followed by Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control. (D)Sections of colon cancer were incubated with 10 mg/mL of H2Mab-139 for 1 hour at room temperature, followed bytreatment with Envision+ kit for 30 minutes. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB)
for 2 minutes; subsequently, sections were counterstained with hematoxylin. Scale bar = 100 mm. HER2, human epidermal
growth factor receptor 2; H2Mab-139, anti-HER2 monoclonal antibody; PVDF, polyvinylidene difluoride.
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MABS AGAINST HER2

together with Imject Alum (Thermo Fisher Scientific, Inc.).
After several additional immunizations, a booster injection
was intraperitoneally administered 2 days before harvesting
spleen cells. Spleen cells were then fused with P3U1 cells
using PEG1500 (Roche Diagnostics, 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-linkedimmunosorbent assay with recombinant HER2-extracellulardomain. MAbs were purified from the supernatants of hybridomas, cultured in Hybridoma-SFM medium (ThermoFisher Scientific, Inc.) using Protein G Sepharose 4 Fast Flow
(GE Healthcare UK, Ltd.).

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Flow cytometry

Cells were harvested by brief exposure to 0.25% trypsin/1mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin/
phosphate-buffered saline, the cells were treated with 10 mg/mLof anti-HER2 (clone H2Mab-139) for 30 minutes at 4C andsubsequently with Alexa Fluor 488-conjugated anti-mouse
IgG (1:1000; Cell Signaling Technology, Inc., Danvers,MA). Fluorescence data were collected using EC800 Cell
Analyzers (Sony, Corp.).
Western blot analysis

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate(SDS) sample buffer (Nacalai Tesque, Inc.) and proteins werethen electrophoresed on 5%–20% polyacrylamide gels
(Wako Pure Chemical Industries, Ltd., Osaka, Japan) before
being transferred onto polyvinylidene difluoride (PVDF)
membranes (Merck KGaA, Darmstadt, Germany). After
blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes
were incubated with primary antibodies, such as anti-HER2(10 mg/mL; clone H2Mab-139) and anti-b-actin (1 mg/mL; clone
AC-15; Sigma-Aldrich, Corp., St. Louis, MO) and then withperoxidase-conjugated anti-mouse IgG (Agilent Technologies, Inc., Santa Clara, CA; 1:1000 diluted). Finally, membranes were developed using ImmunoStar LD (Wako Pure
Chemical Industries, Ltd.) with a Sayaca-Imager (DRC, Co.,
Ltd., Tokyo, Japan).

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check reactions with LN229 and LN229/HER2 cells. A
stronger reaction against LN229/HER2 was needed comparedwith LN229 because LN229 cells endogenously express
HER2. We obtained one clone: H2Mab-139 (IgG1, kappa).
Flow cytometry analysis revealed that H2Mab-139 reactedwith LN229/HER2 cells more strongly than with endogenous
HER2-expressing LN229 glioblastoma cells (Fig. 1A).
H2Mab-139 also reacted with Chinese hamster ovary (CHO)/
HER2 cells but did not react with CHO-K1 cells (data notshown), indicating that H2Mab-139 is specific for HER2.
Western blot analysis of H2Mab-139 against LN229 andLN229/HER2 cells revealed that the molecular weight of
H2Mab-139 was *200 kDa in LN229/HER2 cells (Fig. 1B).
H2Mab-139 recognized endogenous HER2 in colon cancer
cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS
174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1(Fig. 1C). However, H2Mab-139 did not detect HER2 incolon cancer cell lines in western blots (data not shown),indicating that H2Mab-139 is not suitable for western blot
analysis of colon cancer cell lines.
We further investigated the immunohistochemical utilityof H2Mab-139 in human colon cancers. Figure 1D shows that
H2Mab-139 stained the cell membranes of colon cancers thatwere previously diagnosed as HER2-positive. H2Mab-139
also stained the cell membranes of HER2-positive breast
cancer cells (Supplementary Fig. S1), indicating that H2Mab139 is useful for the immunohistochemical analysis for
HER2-positive cancers. In the future, we intend to determine
the positive rate of many colon cancer tissues via immunohistochemical analysis using H2Mab-139.
Acknowledgments

This work was supported in part by the Basic Science andPlatform Technology Program for Innovative Biological
Medicine from Japan Agency for Medical Research andDevelopment, AMED (Y.K.), by Project for utilizing glycans
in the development of 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 (Y.K.).
Author Disclosure Statement

Immunohistochemical analyses

Colon cancer tissue arrays were purchased from US Biomax, Inc. (Rockville, MD). Histologic 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 were then incubated with 10 mg/mL of
H2Mab-139 for 1 hour at room temperature and treated using
an Envision+ kit (Agilent Technologies, Inc.) for 30 minutes.
Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2
minutes; subsequently, sections were counterstained withhematoxylin (Wako Pure Chemical Industries, Ltd.).
Results and Discussion

We immunized mice with the purified recombinant extracellular domain of HER2. We performed flow cytometry toY.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-kuSendaiMiyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: September 19, 2017
Accepted: November 30, 2017