MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 5, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0028

Downloaded by Yukinari Kato from online.liebertpub.com at 10/20/17. For personal use only.

Development of EMab-51, a Sensitive
and Specific Anti-Epidermal Growth Factor Receptor
Monoclonal Antibody in Flow Cytometry,
Western Blot, and Immunohistochemistry
Shunsuke Itai,1,2,* Mika K. Kaneko,1,* Yuki Fujii,3,* Shinji Yamada,1 Takuro Nakamura,1Miyuki Yanaka,1 Noriko Saidoh,1 Saori Handa,1 Yao-Wen Chang,1 Hiroyoshi Suzuki,4Hiroyuki Harada,2 and Yukinari Kato1,3,5

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor(HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers
or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades inmany cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized
their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the fulllength or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or
ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third
screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51(IgG1, kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect
EGFR in pathological analyses of EGFR-expressing cancers.
Keywords: EGFR, monoclonal antibody, immunohistochemistry, oral cancer
IntroductionT

he epidermal growth factor receptor (EGFR) isa transmembrane receptor that is involved in cell
growth and differentiation.(1–3) EGFR is a member of thehuman epidermal growth factor receptor (HER) family ofreceptor tyrosine kinases. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3,
activate downstream signaling cascades and control many
biological processes. These pathways are frequently dysregulated via overexpression of EGFR in many cancers,
including brain tumors, head and neck cancers, lung cancers,
breast cancers, pancreatic cancers, kidney cancers, prostate cancers, ovary cancers, bladder cancers, and colorectal
cancers.(4)EGFR is the first receptor target against which monoclonal
antibodies (mAbs), including cetuximab (a mouse-humanchimeric mAb; IgG1) against colorectal cancers and headand neck cancers, panitumumab (a fully human mAb; IgG2)
against colorectal cancers, and necitumumab (a fully humanmAb; IgG1) against nonsmall cell lung cancers, have beendeveloped for cancer treatment.(5–7) Anti-EGFR mAbs possess
several functional mechanisms, including antibody-dependent
cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), blocking dimerization or ligand binding, and
EGFR endocytosis.
We have produced antipodoplanin (PDPN) cancer-specificmAbs (CasMabs), clone LpMab-2(8,9) and LpMab-23,(10,11)
which specifically recognize cancer-type PDPN in tumor
tissues. For this technology, it is critical that immunogens areproduced using cancer cell lines, such as LN229 glioblastoma
cells, which express cancer-specific glycan-attached membrane proteins. This method could be used to develop usefulmAbs against multiple membrane proteins. In this study, we

1

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

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MABS AGAINST EGFR

produced sensitive and specific mAbs against EGFR using
this technology.
Materials and Methods

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Cell lines

LN229, A431, Chinese hamster ovary (CHO)-K1, HEK293T, MCF-10A, Met-5A, and P3U1 cell lines were obtained
from the American Type Culture Collection (ATCC, Manassas, VA). HSC-2 and HSC-3 were obtained from the Japanese Collection of Research Bioresources ( JCRB) Cell Bank
(Osaka, Japan). LN229/EGFR and CHO/EGFR were produced by transfecting pCAG/PA-EGFR-RAP-MAP(12) 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-positive cells(13) were
sorted using a cell sorter (SH800; Sony Corp., Tokyo, Japan).
CHO-K1, CHO/EGFR, and P3U1 cell lines were cultured
in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan),and LN229, LN229/EGFR, A431, HSC-2, HSC-3, HEK293T, MCF-10A, 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 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

The ectodomain of EGFR with N-terminal PA tag(13) andC-terminal RAP tag(14)-MAP tag(12) (EGFRec) was purified
from supernatant of LN229/EGFRec using anti-RAP tag
as described previously.(14) BALB/c mice were immunizedusing intraperitoneal (i.p.) injections of LN229/EGFR cells or
100 mg of EGFRec together with Imject Alum (Thermo Fisher
Scientific, Inc.). After several additional immunizations, abooster injection of LN229/EGFR cells or 100 mg of EGFRec
was intraperitoneally administered 2 days before harvesting
spleen cells. 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 enzymelinked immunosorbent assay (ELISA) with EGFRec, andmAbs were selected using flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). MAbs were purified
from supernatants of hybridomas cultured in HybridomaSFM medium (Thermo Fisher Scientific, Inc.) using Protein
G Sepharose 4 Fast Flow (GE Healthcare UK Ltd., Buckinghamshire, England).

215
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 (PBS), the cells weretreated with 1 mg/mL of anti-EGFR (clone EMab-51) for 30
minutes at 4C and then with Alexa Fluor 488-conjugatedanti-mouse IgG (1:1000; Cell Signaling Technology, Inc.,
Danvers, MA). Fluorescence data were collected using
EC800 or SA3800 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) andwere transferred onto polyvinylidene difluoride (PVDF)
membranes (Merck KGaA, Darmstadt, Germany). After
blocking with 4% skim milk (Nacalai Tesque, Inc.), membranes were incubated with 10 mg/mL of anti-EGFR (clone
EMab-51) and 1 mg/mL of anti-b-actin (clone AC-15;
Sigma-Aldrich Corp., St. Louis, MO), then with peroxidaseconjugated 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

LN229 and A431 (2 · 105 cells) were suspended in 100 mLof serially diluted mAbs (6 ng/mL–50 mg/mL), and Alexa
Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.) 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

Oral cancer tissues were purchased from US Biomax, Inc.
(Rockville, MD). Histologic sections of 4-mm thickness weredeparaffinized in xylene and were then rehydrated and autoclaved in EnVision FLEX Target Retrieval Solution HighpH (Agilent Technologies, Inc.) for 20 minutes. Sections
were then incubated with 5 mg/mL of EMab-51 for 1 hour atroom temperature and were 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 of staining was evaluated as —, 1+, 2+, 3+.
Results
Production of anti-EGFR mAbs

In this study, we immunized mice with LN229/EGFR or
purified recombinant EGFRec from culture supernatants of
LN229/EGFRec cells. A booster i.p. injection of LN229/
EGFR or EGFRec was also administered, and culture supernatants were then screened for binding to purified EGFRec

216

tions with purified EGFRec. Of 100 mAbs, 94 clones reactedwith A431 cells, which express endogenous EGFR.(16) Subsequently, mAbs were further selected according to theirefficacy on Western blot analysis. These analyses identified
only one clone EMab-51 (IgG1, kappa) from EGFRec immunizations that were useful for Western blot analysis.

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using ELISA. As a second screening, we used flow cytometry
to assess reactions with LN229 and LN229/EGFR cells.
LN229 cells express endogenous EGFR(15); therefore, astronger reaction against LN229/EGFR was required.
A total of 38 clones were generated from immunizations
with LN229/EGFR, and 62 were generated from immuniza-

ITAI ET AL.

FIG. 1. Characterization of EMab-51. (A) Western blot using EMab-51; cell lysates (10 mg) were electrophoresed andproteins were transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking, membranes were incubatedwith 10 mg/mL of EMab-51 or 1 mg/mL of anti-b-actin (AC-15) and then incubated with peroxidase-conjugated anti-mouse
IgG. (B, C) Flow cytometry with EMab-51; cells were treated with 1 mg/mL of EMab-51 followed by Alexa Fluor 488conjugated anti-mouse IgG; black line, negative control.

MABS AGAINST EGFR

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Characterization of EMab-51

On Western blot analyses against LN229,(15) LN229/
EGFR,(12) and A431 cells,(16) EMab-51 detected *180-kDa
strong signal in LN229/EGFR and A431 cells, and the weaksignal in LN229, indicating that EMab-51 is very useful forWestern blot analyses (Fig. 1A). Subsequent flow cytometry
demonstrated that EMab-51 reacted with LN229/EGFR morestrongly than with endogenous EGFR-expressing LN229
brain tumor cells (Fig. 1B). EMab-51 also reacted with CHO/
EGFR and did not react with the parental cell CHO-K1, indicating that EMab-51 is specific for EGFR (Fig. 1B). EMab51 also recognized endogenous EGFR in A431 epidermoid
carcinoma cells,(16) HSC-2 and HSC-3 oral squamous cell
carcinomas,(11) HEK-293T renal epithelial cells,(17) Met-5A
mesothelial cells,(18) and MCF-10A breast epithelial cells(19)(Fig. 1C).
We further determined binding affinity of EMab-51 for
LN229 and A431 cells using flow cytometry (Fig. 2) andcalculated KD values for EMab-51 of 1.5 · 10-8 M against
LN229 or 1.3 · 10-8 M against A431, indicating that EMab51 possesses moderate affinity for EGFR-expressing celllines.

FIG. 3. Immunohistochemical analysis by EMab-51
against oral cancers. Sections were incubated with 5 mg/mLof primary EMab-51 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–D) Case No. 15; (E–H) case No. 23; (A, B, E, F) immunostaining by EMab-51;(C, D, G, H) hematoxylin and eosin staining; scale bar =100 mm.
Immunohistochemical analysis against oral cancers

We investigated the immunohistochemical utility of
EMab-51 in human oral cancers because high EGFR expression was observed in oral cancer cell lines, such as HSC2 and HSC-3 (Fig. 1C). As depicted in Figure 3, EMab-51
stained the membranes of cancer cells of oral squamous cell
carcinomas. EMab-51 stained 6 of 38 (15.8%) squamous cell
carcinomas in oral cancer tissue array (Supplementary
Table S1) and did not stain any other tumor types of oral
cancers (Supplementary Table S2).
Discussion

FIG. 2. Binding affinity of EMab-51 was determined using flow cytometry. LN229 and A431 cells were suspended
in 100 mL of serially diluted EMab-51 (6 ng/mL–50 mg/mL),and secondary anti-mouse IgG was then added. Fluorescence data were collected using a cell analyzer.

Establishment of mAbs, which are useful for Western blot,
flow cytometry, and immunohistochemical analyses, is often
difficult because the design of immunogens and screening
methods to develop specific mAbs is different between Western blot and immunohistochemical applications. Furthermore,Downloaded by Yukinari Kato from online.liebertpub.com at 10/20/17. For personal use only.

218

it is very difficult to produce sensitive and specific mAbs
against endogenous proteins. Recently, we developed sensitiveand specific antipodocalyxin (PODXL) mAbs using our original technology, and we demonstrated that clone PcMab-47 isuseful for flow cytometry, Western blot, and immunohistochemical analyses among anti-PODXL mAbs, warranting
consideration of PcMab-47 for investigations of PODXL
expression and function in cancers and normal tissues.(20)
We further used this technology to generate mAbs that bindto various novel epitopes of PDPN, including LpMab-3,(21)LpMab-7,(22–24) LpMab-10,(25) LpMab-12,(26) LpMab-13,(27)LpMab-17,(28) LpMab-19,(29) and LpMab-21.(30,31) Importantly, these mAbs are useful for Western blot, flow
cytometry, and immunohistochemical analyses against
PDPN. In this study, we immunized mice with LN229/EGFR
or purified recombinant EGFRec from culture supernatants of
LN229/EGFRec cells to develop anti-EGFR mAbs, which areuseful for Western blot, flow cytometry, and immunohistochemical analyses. Finally, we developed clone EMab-51.
Among 100 anti-EGFR mAbs, 90 clones (90%) were identified as belonging to the IgG1 subclass, 5 to the IgG2a subclass,
1 to the IgG2b subclass, and 4 to the IgM class. EMab-51 wasalso determined to be of the IgG1 subclass, precluding confirmation of ADCC or CDC using EMab-51. Thus, in futurestudies, we will convert the subclass of EMab-51 into mouse
IgG2a or IgG2b subclasses or human IgG1 and assess their applications for measuring ADCC/CDC activities.(9,24,32)
In this study, we used EnVision FLEX Target Retrieval
Solution (high pH) for antigen retrieval.(33–35) For immunohistochemical analysis, we typically used citrate buffer (pH6.0) for antigen retrieval; however, citrate buffer did not
improve the signal from EMab-51 use (data not shown).
In conclusion, of 100 clones of anti-EGFR mAbs, EMab-51
was highly efficacious in Western blot analyses and produced
strong staining in oral cancers. Hence, EMab-51 could be usefulin all present experiments and will likely be an advantageous tool
for the pathological identification of EGFR in many cancers.
Acknowledgments

We thank Yoshimi Nakamura for excellent 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 [Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)] 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.).
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd.
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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
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp