MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 6, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0042

Establishment of EMab-134, a Sensitive and SpecificAnti-Epidermal Growth Factor Receptor Monoclonal
Antibody for Detecting Squamous Cell Carcinoma
Cells of the Oral Cavity

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

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, activates downstream signaling cascades inmany tumors. In this study, we established novel anti-EGFR monoclonal antibodies (mAbs) and characterized
their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We immunized mice with a
combination of the extracellular domain of EGFR and EGFR-overexpressing LN229 glioblastoma cells (LN229/
EGFR) and performed the first screening using enzyme-linked immunosorbent assay. Next, we selected mAbs
using flow cytometry. Among 156 established clones, two mAbs, EMab-51 (IgG1, kappa) and EMab-134 (IgG1,
kappa), reacted with EGFR in Western blot analysis; EMab-134 showed a much higher sensitivity compared with
EMab-51. We compared the binding affinities of EMab-51 and EMab-134 using flow cytometry; the calculated
KD values for EMab-51 and EMab-134 against SAS cells/HSC-2 cells were 9.2 · 10-9 M/9.9 · 10-9 M and
2.6 · 10-9 M/8.3 · 10-9 M, respectively, indicating that EMab-134 has a higher affinity to EGFR-expressing
cells. Immunohistochemical analysis of EMab-51 and EMab-134 showed sensitive and specific reactions
against oral cancer cells; EMab-134 demonstrated a much higher sensitivity (36/38 cases; 94.7%) to oral
squamous cell carcinomas compared with EMab-51 (6/38 cases; 15.8%). This novel anti-EGFR mAb, EMab134, could be advantageous for detecting EGFR in the pathological analysis of EGFR-expressing cancers.
Keywords: EGFR, monoclonal antibody, immunohistochemistry, oral cancer

IntroductionEpidermal growth factor receptor (EGFR) is amember of the human EGFR (HER) family of receptor
tyrosine kinases.(1–3) EGFR forms homodimers or heterodimers with other members of the HER family, such as HER2(4)and HER3,(5) controlling many biological processes. EGFR isa type-I transmembrane glycoprotein that is involved in cell
growth and differentiation.(6) Overexpression of EGFR is observed in many cancers, including head and neck, lung, colorectal, breast, pancreatic, kidney, ovary, bladder, and prostatecancers.(7)Monoclonal antibodies (mAbs) have been developed forcancer treatment, including cetuximab (a mouse–human chimeric mAb; IgG1) against head and neck and colorectal cancers,
panitumumab (a fully human mAb; IgG2) against colorectal
cancers, and necitumumab (a fully human mAb; IgG1) against
non-small cell lung cancers.(8–10) Anti-EGFR mAbs possess

various functional mechanisms: antibody-dependent cellularcytotoxicity (ADCC), complement-dependent cytotoxicity(CDC), blocking dimerization, blocking ligand binding, and
EGFR endocytosis.
Recently, we developed anti-HER2 mAb (clone: H2Mab77) using our original technology. H2Mab-77 is useful forWestern blot, flow cytometry, and immunohistochemical
analyses.(11) In this study, we established sensitive and specific mAbs against EGFR.
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1, P3X63Ag8U.1 (P3U1),
HEK-293T, Met-5A, LN229, and A431 were obtained fromthe American Type Culture Collection (ATCC; Manassas, VA).
HSC-2, HSC-3, HSC-4, HSC-3M3, Ca9-22, HO-1-u-1, andSAS were obtained from the Japanese Collection of Research

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
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2272

MABS AGAINST EGFR

Bioresources Cell Bank (Osaka, Japan). LN229/EGFR andCHO/EGFR were produced by transfecting pCAG/PAEGFR-RAP-MAP into LN229 and CHO-K1 cells using theNeon transfection system (Thermo Fisher Scientific, Inc.,
Waltham, MA) and Lipofectamine LTX (Thermo Fisher
Scientific, Inc.), respectively.(12) A few days after transfection, PA tag-positive cells were sorted using a cell sorter(SH800; Sony Corp., Tokyo, Japan). The PA tag systemcomprises a rat anti-human podoplanin mAb (clone NZ-1)
and the PA tag (GVAMPGAEDDVV) derived from theplatelet aggregation-stimulating (PLAG) domain of human
podoplanin.(13)Animals and tissues

specific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved all of the animal
experiments described. Oral cancer tissue arrays were purchased from US Biomax, Inc. (Rockville, MD).
Culture of cell lines

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, HSC-4, HSC3M3, Ca9-22, HO-1-u-1, SAS, HEK-293T, and Met-5A celllines were cultured in Dulbecco’s modified Eagle’s medium(DMEM) (Nacalai Tesque, Inc.), supplemented with 10%
heat-inactivated 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.) at 37C in
a humidified atmosphere containing 5% CO2 and 95% air.

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Four-week-old female BALB/c mice were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under

273

FIG. 1. Flow cytometry using anti-EGFR mAbs for several cells. Cells were treated with 1 mg/mL of EMab-51 (A) andEMab-134 (B), followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control.
EGFR, epidermal growth factor receptor; mAbs, monoclonal antibodies.

274
Purification of extracellular domain of EGFRThe extracellular domain of EGFR with N-terminal PA
tag and C-terminal RAP tag-MAP tag was purified from thesupernatant of LN229/sol-EGFR using anti-RAP tag, as
described previously.(14) The RAP tag system comprises amouse anti-rat podoplanin mAb (clone PMab-2) and the RAP
tag (DMVNPGLEDRIE) derived from the PLAG domain of
rat podoplanin.(14)

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Production of hybridoma cell lines

BALB/c mice were immunized using intraperitoneal injections of LN229/EGFR cells or 100 mg of sol-EGFR together with Imject Alum (Thermo Fisher Scientific, Inc.).
After several additional immunizations, a booster injectionof LN229/EGFR cells or 100 mg of sol-EGFR 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 1640 medium supplemented with hypoxanthine, aminopterin, and thymidine
selection medium supplement (Thermo Fisher Scientific,

ITAI ET AL.

Inc.). Culture supernatants were screened using enzymelinked immunosorbent assay (ELISA) with sol-EGFR, andmAbs were selected using flow cytometry, Western blot,
and immunohistochemical analyses.
Purification of mAbs

Hybridomas were cultured in Hybridoma-SFM medium(Thermo Fisher Scientific, Inc.). mAbs were purified fromsupernatants using Protein G Sepharose 4 Fast Flow (GEHealthcare 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 (PBS), the cells
were treated with 1 mg/mL of anti-EGFR for 30 minutes at
4C, followed by treatment with Alexa Fluor 488-conjugatedanti-mouse IgG (1:1000; Cell Signaling Technology, Inc.,
Danvers, MA). Fluorescence data were collected using
EC800 Cell Analyzers (Sony Corp.).

FIG. 2. Flow cytometry using anti-EGFR mAbs for squamous cell carcinoma cell lines. Cells were treated with 1 mg/mL
of EMab-51 (A) and EMab-134 (B), followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG; black line,
negative control.

MABS AGAINST EGFR
Determination of the binding
affinity using flow cytometry

SAS and HSC-2 (2 · 105 cells) were suspended in 100 mLof serially diluted mAbs (0.6 ng/mL–10 mg/mL), and Alexa
Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc.) was then added. Fluorescence data
were collected using a cell analyzer (EC800; Sony Corp.).
The dissociation constant (KD) was calculated by fitting
the binding isotherms using the built-in one-site binding
models in GraphPad PRISM 6 (GraphPad Software, Inc., La
Jolla, CA).

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Western blot analysis

Cell lysates (10 mg) were boiled in SDS sample buffer(Nacalai Tesque, Inc.). Proteins were then electrophoresed on
5%–20% polyacrylamide gels (Wako Pure Chemical Industries Ltd., Osaka, Japan) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA,
Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.), membranes were incubated with antiEGFR (1–10 mg/mL) and 1 mg/mL of anti-b-actin (clone
AC-15; Sigma-Aldrich Corp., St. Louis, MO), followed
by incubation with peroxidase-conjugated anti-mouse IgG(diluted 1:1000; Agilent Technologies, Inc., Santa Clara,

275CA), and were finally developed using ImmunoStar LD(Wako Pure Chemical Industries Ltd.) using a Sayaca-Imager(DRC Co. Ltd., Tokyo, Japan).
Immunohistochemical analyses

Histological sections (4-mm thick) were directly autoclaved in EnVision FLEX Target Retrieval Solution High pH(Agilent Technologies, Inc.) for 20 minutes or were deparaffinized in xylene and then rehydrated and autoclaved incitrate buffer, pH 6.0 (Agilent Technologies, Inc.) for 20
minutes. Sections were then incubated with 5 mg/mL of mAbs
for 1 hour at room temperature, followed by treatment withan 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+, or 3+.
Results
Establishment of anti-EGFR mAbs

EGFR is a type-I transmembrane glycoprotein, which
is similar to the platelet aggregation-inducing factor

FIG. 3. Determination of binding affinity using flow cytometry. SAS and HSC-2 were suspended in 100 mL of seriallydiluted EMab-51 (A) or EMab-134 (B) (0.6 ng/mL–10 mg/mL), and secondary anti-mouse IgG was then added. Fluorescence data were collected using a cell analyzer.

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276

ITAI ET AL.

podoplanin.(15) We previously produced anti-podoplanincancer-specific mAbs (CasMabs), clones LpMab-2(16,17) and
LpMab-23,(18,19) which particularly recognized cancer-type
podoplanin. It is critical that immunogens are produced usingcancer cell lines, such as LN229 glioblastoma cells, which
express cancer-specific glycan-attached membrane proteins.
We have used this method to develop useful mAbs against
many membrane proteins.(20)In this study, we immunized mice with LN229/EGFR or
purified recombinant EGFR (sol-EGFR). Culture supernatants were then screened for binding to sol-EGFR using
ELISA. We then used flow cytometry as a second screen
to assess reactions with LN229 and LN229/EGFR cells.
A stronger reaction against LN229/EGFR was required
because LN229 cells express endogenous EGFR.(21)
Of 156 established clones, only two mAbs (1.3%), EMab51 (IgG1, kappa) and EMab-134 (IgG1, kappa), reactedwith EGFR in Western blot analysis. Both mAbs were
produced by immunizing sol-EGFR. We purified EMab51 and EMab-134 using Protein G and characterized thesemAbs.
Characterization of anti-EGFR mAbs

In flow cytometry, EMab-51 reacted with CHO/EGFR but
not with the parental cell CHO-K1, indicating that EMab-51
had specificity for EGFR (Fig. 1A). EMab-51 also reactedwith LN229/EGFR more strongly than with endogenous
EGFR-expressing LN229 glioblastoma cells (Fig. 1A).
Moreover, EMab-51 reacted with normal cell lines, including
HEK-293T (renal epithelial cells) and Met-5A (mesothelial
cells).(22) The reactions of EMab-134 were very similar to
those of EMab-51 (Fig. 1B).
EMab-51 recognized endogenous EGFR in A431 epidermoid carcinoma cells(23) (Fig. 2A). EMab-51 also reacted with oral squamous cell carcinomas such as HSC-2,
HSC-3, HSC-4, HSC-3M3, Ca9-22, HO-1-u-1, and SAS,
indicating that EMab-51 detected each squamous cell
carcinoma cell line at a low concentration (1 mg/mL) in flow
cytometry. Another clone, EMab-134, also reacted withsquamous cell carcinoma cell lines in the same way as EMab51 (Fig. 2B).
We further determined the binding affinities of EMab51 and EMab-134 for SAS and HSC-2 cells through flow
cytometry (Fig. 3) and calculated KD values for EMab-51
and EMab-134. KD values of EMab-51 were determined
to be 9.2 · 10-9 M and 9.9 · 10-9 M against SAS and
HSC-2, respectively. In contrast, KD values of EMab-134
were determined to be 2.6 · 10-9 M and 8.3 · 10-9 M
against SAS and HSC-2, respectively, indicating that thebinding affinities of EMab-134 were 3.5 times and 1.2
times higher than those of EMab-51 against SAS and
HSC-2, respectively.
Next, we compared the reactivity between EMab-51 andEMab-134 in Western blot analysis. As shown in Figure 4,
both EMab-51 and EMab-134 reacted with LN229/EGFR.
However, the sensitivity of EMab-134 was much higher
compared with EMab-51, although their reactivity was similar in flow cytometry (Figs. 1 and 2). Both mAbs reactedwith endogenous EGFR, which was reported to be expressedin A431 cells.(23) EMab-51 detected an *180-kDa weaksignal in LN229/EGFR and a strong signal in A431 cells

FIG. 4. Western blot analysis using EMab-51 and EMab134. Cell lysates (10 mg) were electrophoresed, and proteins
were transferred onto PVDF membranes. After blocking,membranes were incubated with 10 mg/mL of EMab-51(A), 1 mg/mL of EMab-134 (B), or 1 mg/mL of anti-b-actin(clone: AC-15) (C) and then incubated with peroxidaseconjugated anti-mouse IgG. PVDF, polyvinylidene difluoride.

(Fig. 4A), whereas EMab-134 detected *180- and 130-kDa
signals in LN229/EGFR and A431 cells, respectively (Fig. 4B).
Furthermore, EMab-134 showed strong signals against fivehuman oral cancer cell lines (HSC-2, HSC-3, HSC-4, HSC3M3, and Ca9-22) and moderate signals against two humanoral cancer cell lines (HO-1-u-1 and SAS; Fig. 4B), which werecompatible with the flow cytometric data (Fig. 3). In contrast,
EMab-51 showed moderate signals against two cell lines

MABS AGAINST EGFR

(HSC-2 and HSC-3) and weak signals against the other celllines (Fig. 4A).
Immunohistochemical analysis for oral cancers

stronger signals than EMab-51 (Fig. 6C, D), although thestaining areas of EMab-134 and EMab-51 were the same. All
staining results of EMab-51 and EMab-134 for oral cancers
are shown in Table 1. EMab-51 stained 6/38 (15.8%) ofsquamous cell carcinomas, whereas EMab-134 stained 36/38(94.7%) of the carcinomas (Table 2). Several typical resultsof EMab-134 are also shown in Supplementary Figures S2(intensity: 3+) and S3 (intensity: 2+).
Discussion

To date, we have developed many mAbs against membraneproteins. We recently developed specific and sensitive antipodocalyxin mAbs using a technology developed by us anddemonstrated that the clone PcMab-47 was the most useful forflow cytometry, Western blot, and immunohistochemical
analyses among the 100 anti-podocalyxin mAb clones.
PcMab-47 has been useful for investigations of podocalyxin

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We performed immunohistochemical analysis using
EMab-134 for human oral cancers because EGFR expression
was highly observed in oral cancer cell lines (Fig. 2). First, weinvestigated the antigen retrieval conditions for immunohistochemical analysis using EMab-134. EMab-134 stained themembranes of cancer cells of oral squamous cell carcinomas(Fig. 5A–D). The sensitivity using EnVision FLEX Target
Retrieval Solution High pH (Fig. 5A, B and Supplementary
Fig. S1A, B) was much higher than that using citrate buffer(pH 6.0) as the antigen retrieval solution (Fig. 5C, D and
Supplementary Fig. S1C, D). Next, we compared the immunoreactivity of EMab-51 and EMab-134 under the same
conditions (Fig. 6). EMab-134 (Fig. 6A, B) showed much

277

FIG. 5. Immunohistochemical analysis by EMab-134 for oral cancers. After antigen retrieval, sections (Case-24)
were incubated with 5 mg/mL of primary EMab-134 for 1 hour at room temperature, followed by treatment withEnvision+ kit for 30 minutes. Color was developed using 3,3-diaminobenzidine tetrahydrochloride for 2 minutes, andsections were then counterstained with hematoxylin. (A, B) Antigen retrieval using EnVision FLEX Target Retrieval
Solution High pH; (C, D) antigen retrieval using citrate buffer, pH 6.0; (E, F) hematoxylin and eosin staining; scalebar = 100 mm.

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278

ITAI ET AL.

FIG. 6. Immunohistochemical analysis by anti-EGFR mAbs for oral cancers. After antigen retrieval using EnVision FLEX Target Retrieval Solution High pH, sections (Case-15) were incubated with 5 mg/mL of primaryEMab-134 or 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 for 2 minutes, and sections were thencounterstained with hematoxylin. (A, B) EMab-134; (C, D) EMab-51; (E, F) hematoxylin and eosin staining; scalebar = 100 mm.

expression and function in cancers and normal tissues.(20) We
also used our technology to generate mAbs that bind to various
novel epitopes of podoplanin, including LpMab-3,(24) LpMab12,(25) LpMab-19,(26) and LpMab-21.(27,28) Importantly, thesemAbs are useful for Western blot, flow cytometry, and immunohistochemical analyses for podoplanin. Recently, we
further developed anti-HER2 mAb (clone: H2Mab-77), whichis also useful for Western blot, flow cytometry, and immunohistochemical analyses.(11) Development of mAbs, which
are useful for various experiments, is often difficult because
the design of immunogens and screening methods to produce
specific mAbs differ based on the applications. Furthermore, it
is difficult to produce mAbs sensitive and specific to endogenous proteins.
In this study, we immunized mice with a combinationof LN229/EGFR and recombinant EGFR to develop specific and sensitive anti-EGFR mAbs, which were useful

for Western blotting, flow cytometry, and immunohistochemical analyses. Two established clones, EMab-51 andEMab-134, were determined to be of the IgG1 subclass,precluding confirmation of ADCC or CDC without conversion to mouse IgG2a, mouse IgG2b, or human IgG1. In
future studies, we will convert the subclass of EMab-51
and EMab-134 into ADCC/CDC-inducing subclasses formeasuring ADCC/CDC activities.(17,29,30) Furthermore,
we should determine the epitope of EMab-134 andEMab-51 and investigate the reason why EMab-134 issensitive in Western blot and immunohistochemical analyses.
In conclusion, of 156 clones of anti-EGFR mAbs, EMab134 was highly efficacious in Western blot analysis andstrongly stained oral cancers. Thus, EMab-134 could be usefulin various experiments and advantageous for the pathological
identification of EGFR in many cancers.

MABS AGAINST EGFR

279

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Table 1. Results of EMab-51 and EMab-134 Immunostaining in 48 Patients with Oral Cancers
No.

Sex

Age

Organ

Pathology diagnosis

Differentiation

Type

EMab-51

EMab-134

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
43
44
45
46
47
48

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

61
57
67
59
47
62
47
51
62
53
50
76
55
57
61
50
54
62
55
63
56
45
50
46
48
67
64
50
63
46
35
55
49
61
53
51
73
61
62
50
68
58
41
45
57
52
65
74

TongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueTongueParotid
Parotid
Parotid
Parotid
Parotid
Parotid
Parotid
Parotid

SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
SCC
ACC
MC
PA
PA
PA
MPA
MPA
MPA
AL
ACC

Well
Well
Well
Well
Well
—
Well
Well
Well
Well
Well
Well
Well
Moderately
Well
Well
Well
Well
Well
Well
Well
Well
Well
PoorlyModerately—
PoorlyModerately
Moderately
Well
Moderately
PoorlyPoorlyModerately
Moderately
PoorlyPoorlyPoorly
—
—
—
—
—
—
—
—
—
—

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
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant
Malignant

0
0
0
0
0
0
0
0
0
0
0
0
0
0
3+
0
0
0
2+
0
0
0
2+
0
0
0
1+
0
0
0
0
0
0
0
1+
0
0
1+
0
0
0
0
0
0
0
0
0
0

0
1+
2+
1+
1+
3+
2+
0
2+
3+
3+
3+
1+
1+
3+
2+
2+
1+
3+
1+
1+
3+
3+
3+
2+
2+
3+
1+
2+
2+
2+
1+
2+
1+
3+
2+
2+
3+
0
0
1+
0
0
0
1+
1+
1+
1+

ACC, adenoid cystic carcinoma; AL, adenolymphoma; F, female; M, male; MC, mucoepidermoid carcinoma; MPA, malignant
pleomorphic adenoma; PA, pleomorphic adenoma; SCC, squamous cell carcinoma.

Table 2. Summary of Immunostaining Using EMab-51 and EMab-134
EMab-51 immunostaining

Tumor
type

No. of
cases

3+

2+

1+

SCC
PA
MPA
ACC
MC
AL

38
3
3
2
1
1

1
0
0
0
0
0

2
0
0
0
0
0

3
0
0
0
0
0

EMab-134 immunostaining

0

No. of positive
cases (%)

3+

2+

1+

0

No. of positive
cases (%)

32
3
3
2
1
1

6/38 (15.8)
0/3 (0)0/3 (0)0/2 (0)0/1 (0)0/1 (0)

12
0
0
0
0
0

13
0
0
0
0
0

11
1
2
1
0
1

2
2
1
1
1
0

36/38 (94.7)
1/3 (33.3)
2/3 (66.7)
1/2 (50)
0/1 (0)1/1 (100)

280
Acknowledgments

The authors thank Takuro Nakamura, Miyuki Yanaka,
Noriko Saidoh, Saori Handa, and Yoshimi Nakamura for excellent technical assistance. This work was mainly supportedby 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 Platform Project for
Supporting Drug Discovery and Life Science Research [Basisfor Supporting Innovative Drug Discovery and Life Science
Research (BINDS)] from AMED (Y.K.) and by Project for
utilizing glycans in the development of innovative drug discovery technologies from AMED (Y.K.).
Author Disclosure Statement

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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
Received: August 8, 2017
Accepted: September 30, 2017