Cancer Medicine

Open Access

ORIGINAL RESEARCH

Development and characterization of anti-­glycopeptide
monoclonal antibodies against human podoplanin, using
glycan-­deficient cell lines generated by CRISPR/Cas9 and
TALEN
Mika K. Kaneko1, Takuro Nakamura1, Ryusuke Honma1,2, Satoshi Ogasawara1, Yuki Fujii1, Shinji
Abe3,4, Michiaki Takagi2, Hiroyuki Harada5, Hiroyoshi Suzuki6, Yasuhiko Nishioka4 & Yukinari Kato1
1Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
2Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan
3Department of Clinical Pharmacy Practice Pedagogy, Graduate School of Biomedical Sciences, Tokushima University, 1-78-1 Sho-machi, Tokushima

770-8505, Japan
4Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto-cho,
Tokushima 770-8503, Japan
5Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima,
Bunkyo-ku, Tokyo 113-8510, Japan
6Department of Pathology and Laboratory Medicine, Sendai Medical Center, 2-8-8, Miyagino, Miyagino-ku, Sendai, Miyagi 983-0045, Japan

Keywords
Epitope, glycopeptide, monoclonal antibody,
podoplanin, sialic acid
Correspondence
Yukinari Kato, Tohoku University Graduate
School of Medicine, 2-1 Seiryo-machi,
Aoba-ku, Sendai, Miyagi 980-8575, Japan.
Tel: +81 22 717 8207; Fax: 81 22 717 8207;
E-mails: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Funding Information
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 the Platform for Drug
Discovery, Informatics, and Structural Life
Science (PDIS) from AMED (Y.K.), by the
Practical Research for Innovative Cancer Control
from AMED (Y.K.); by JSPS KAKENHI Grant
Number 26440019 (M.K.K.), 25462242 (Y.K.),
and 16K10748 (Y.K.), and by the Regional
Innovation Strategy Support Program from the
Ministry of Education, Culture, Sports, Scienceand Technology (MEXT) of Japan (Y.K.).

Abstract
Human podoplanin (hPDPN), which binds to C-­
type lectin-­like receptor-­
2(CLEC-­2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer-­associated fibroblasts indicates poor
prognosis. Human lymphatic endothelial cells, lung-­type I alveolar cells, andrenal glomerular epithelial cells express hPDPN. Although numerous monoclonal
antibodies (mAbs) against hPDPN are available, they recognize peptide epitopesof hPDPN. Here, we generated a novel anti-­hPDPN mAb, LpMab-­21. To characterize the hPDPN epitope recognized by the LpMab-­21, we established glycan-­deficient CHO-­S and HEK-­293T cell lines, using the CRISPR/Cas9 or TALEN.
Flow cytometric analysis revealed that the minimum hPDPN epitope, in which
sialic acid is linked to Thr76, recognized by LpMab-­21 is Thr76–Arg79. LpMab­21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, andseminoma cells as well as in normal lymphatic endothelial cells. However,
LpMab-­21 did not react with renal glomerular epithelial cells or lung type I
alveolar cells, indicating that sialylation of hPDPN Thr76 is cell-­type-­specific.
LpMab-­21 combined with other anti-­hPDPN antibodies that recognize different
epitopes may therefore be useful for determining the physiological function of
sialylated hPDPN.

Received: 7 September 2016; Revised: 5
October 2016; Accepted: 9 October 2016
Cancer Medicine 2017; 6(2):382–396
doi: 10.1002/cam4.954

382© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited.

M. K. Kaneko et al.

Introduction
Human podoplanin (hPDPN) is expressed in many cancers, including malignant gliomas, lung cancers, esophageal
cancers, malignant mesotheliomas, testicular cancers, bladder cancers, and osteosarcomas [1–13], and the expressionof hPDPN in cancer-­associated fibroblasts contributes topoor prognosis [14–19]. Human PDPN (known as theplatelet aggregation-­
inducing factor Aggrus) is involvedin cancer metastasis [11, 20, 21]. We identified C-­
type
lectin-­like receptor-­2 (CLEC-­2) as an endogenous receptorof hPDPN [22, 23]. Moreover, our comparative crystallographic studies of the hPDPN–CLEC-­
2 complex [24]revealed that CLEC-­2 binds to hPDPN through residues
Glu47 and Asp48 within its platelet aggregation-stimulating(PLAG) domain as well as to the α2-­6-­linked sialic acidlinked to Thr52.
Highly sensitive and specific anti-hPDPN mAb are
required to clarify the physiological function of hPDPN
in normal tissues and cancers. Although many anti-­hPDPN
mAbs are available, most react with the PLAG1-PLAG3
of hPDPN [6, 25–29]. We previously established the original technology to produce anti-­
glycopeptide mAbs
(GpMabs) against hPDPN [30–39]. Here, we generated anovel anti-­
hPDPN mAb designated LpMab-­
21 that recognizes a sialylated glycopeptide epitope.
Furthermore, to characterize the hPDPN epitope recognized by the LpMab-­21, we need glycan-­deficient CHO-­S
or HEK-­293T cell lines. We report the establishment ofglycan-­deficient cell lines using the CRISPR/Cas9 or
TALEN.

Materials and Methods
Cell lines, mice, and human tissues
As described in detail previously [36, 39], the cell lines
LN229, HEK-­293T, NCI-­H226, U-­2 OS, Met-­5A, Chinese
hamster ovary (CHO)-­K1, and P3U1 were obtained fromthe American Type Culture Collection (ATCC, Manassas,
VA). The HSC-­2 and HSC-­4 cell lines were obtained fromthe Japanese Collection of Research Bioresources (JCRB)
Cell Bank (Osaka, Japan). The MG-­63 cell line was obtained
from the Cell Resource Center for Biomedical ResearchInstitute of Development, Aging and Cancer Tohoku
University (Miyagi, Japan). The LN319 cell line was provided by Prof. Kazuhiko Mishima (Saitama Medical
University, Saitama, Japan) [40]. Human lymphatic
endothelial cells (LECs), CHO-­
S cells, and PC-­
10 cells
were purchased from Cambrex Corp., East Rutherford,
NJ, Thermo Fisher Scientific Inc., (Waltham, MA), andImmuno-­Biological Laboratories Co., Ltd. (Gunma, Japan),respectively. LN229 and CHO-­
K1 cells were transfected© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Anti-­glycopeptide mAb LpMab-­21 against Podoplaninwith the hPDPN plasmids using Lipofectamine 2000
(Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions [30].
CHO-­K1, CHO-­K1/hPDPN, CHO-­S, NCI-­H226, PC-­10,
and P3U1 cells were cultured in RPMI 1640 medium
containing l-­glutamine (Nacalai Tesque, Inc., Kyoto,
Japan). LN229, LN229/hPDPN, LN319, HSC-­
2, HSC-­
4,and HEK-­293T cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco’s Modified
Eagle’s Medium (DMEM) medium containing L-­glutamine(Nacalai Tesque, Inc.) and 10% heat-­inactivated fetal bovine
serum (FBS) (Thermo Fisher Scientific Inc.). LECs were
cultured in endothelial cell medium EGM-­2MV supplemented with 5% FBS (Cambrex Corp. East Rutherford,
NJ). All media contained 100 units/mL of penicillin, 100 μg/
mL of streptomycin, and 25 μg/mL of amphotericin B(Nacalai Tesque, Inc.).
Three female BALB/c mice (4-­week-­old) were purchasedfrom CLEA Japan (Tokyo, Japan) and were housed underpathogen-­free conditions. The Animal Care and Use
Committee of Tohoku University approved the animal
experiments described herein.
The Tokyo Medical and Dental University Institutional
Review Board and the Sendai Medical Center Review Board
reviewed and approved the use of human cancer tissues.
Written informed consent was obtained for using thehuman cancer tissue samples. Microarrays of normal humantissues were purchased from Cybrdi, Inc. (Frederick, MD).

Production of glycan-­deficient or PDPN-­
knock out cell lines
The HEK-­293T/hPDPN-­knockout (KO) cell line (PDIS-­2)and the LN319/hPDPN-­KO cell line (PDIS-­6) were generated by transfection using CRISPR/Cas9 plasmids (Target
ID: HS0000333287) that target PDPN (Sigma-­Aldrich, St.
Louis, MO). Plasmids were transfected, using a Gene Pulser
Xcell electroporation system (Bio-­
Rad Laboratories Inc.,
Berkeley, CA) [31, 34]. PDIS-­
2 and PDIS-­
6 cells werescreened using the NZ-­1 mAb [5]. The cell lines CHO-­S/
GnT-­1-­KO (PDIS-­9) and CHO-­S/SLC35A1-­KO (PDIS-­14)
were generated by transfecting TALEN or CRISPR/Cas9
plasmids, which target hsMgat1 (Wako Pure Chemical
Industries Ltd., Osaka, Japan) and SLC35A1 (Target ID:
HS0000168432; Sigma-­Aldrich), respectively, using a Gene
Pulser Xcell electroporation system. The cell lines HEK-­
293T/GnT-­1-­KO (PDIS-­1 or PDIS-­12) and HEK-­293T/
SLC35A1-­
KO (PDIS-­22) were generated by transfecting
TALEN or CRISPR/Cas9 plasmids, which target hsMgat1(Wako Pure Chemical Industries Ltd., Osaka, Japan) and
SLC35A1 (Target ID: HS0000168432; Sigma-­
Aldrich),respectively, using a Gene Pulser Xcell electroporation
system. PDIS-­1, PDIS-­9, and PDIS-­12 cells were screened383

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

M. K. Kaneko et al.

using leukoagglutinin from Phaseolus vulgaris (L-­PHA).
PDIS-­14 and PDIS-­22 cells were screened using Maackia
amrensis Lectin II (MAL-­
II). PDIS-­
9 and PDIS-­
14 cells
were transfected with the human PDPN plasmids using
Lipofectamine LTX (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions. Glycan-­deficientcell lines were cultured in RPMI 1640 medium.

performed, using a QuikChange Lightning site-­
directedmutagenesis kit (Agilent Technologies Inc., Santa Clara,CA) using oligonucleotides containing the desired mutations. CHO-­S or CHO-­K1 cells were transfected with theplasmids using a Gene Pulser Xcell electroporation system(Bio-­Rad Laboratories Inc.). Point mutants were cultured
in RPMI 1640 medium containing l-­glutamine.

Generation of deletion mutantsHybridoma production

Amplified hPDPN cDNA was subcloned into a pCAG-­
Ble(Zeo) vector (Wako Pure Chemical Industries Ltd.)
with a MAP-­tag, detected by PMab-­1 [41, 42], which wasadded to the N-­terminus using the In-­Fusion HD Cloning
Kit (Clontech, Palo Alto, CA). Deletion mutants of hPDPN
were generated using the primers as follows:
Sense primers and designation of the corresponding
mutant5′-­AGAAGACAAAAAGCTTGCCAGCACAGGCCAGC
C, dN23
5′-­AGAAGACAAAAAGCTTGAAGGCGGCGTTGCCA
T, dN37
5′-­AGAAGACAAAAAGCTTGCCGAAGATGATGTGGT
G, dN46
5′-­AGAAGACAAAAAGCTTACCAGCGAAGACCGCT
A, dN55
5′-­AGAAGACAAAAAGCTTACAACTCTGGTGGCAAC
A, dN64
5′-­AGAAGACAAAAAGCTTGTAACAGGCATTCGCATC, dN75
5′-­AGAAGACAAAAAGCTTACTTCAGAAAGCACAGT
CC, N85
5′-­AGAAGACAAAAAGCTTCAAAGTCCAAGCGCCA
C, dN95
5′-­AGAAGACAAAAAGCTTGCCACCAGTCACTCCA
C, dN105
Antisense primer
5′-­TCTAGAGTCGCGGCCGCTTACTTGTCGTCATCGT
CHO-­K1 cells were transfected with these plasmids using
Lipofectamine LTX (Thermo Fisher Scientific Inc.). Deletionmutants were cultured in RPMI 1640 medium containingl-­glutamine (Nacalai Tesque, Inc.) and 10% heat-­inactivated
FBS at 37°C in a humidified atmosphere containing 5%
CO2. Stable transfectants of CHO-­
K1/ssMAP-­
hPDPNdNmutants were selected by culturing them in medium containing 0.5 mg/mL Zeocin (InvivoGen, San Diego, CA).

Three 4-­week-­old female BALB/c mice were immunized byintraperitoneal (i.p.) injection of 1 × 108 LN229/hPDPN
cells together with Imject Alum (Thermo Fisher ScientificInc.) [30]. A booster injection was administered i.p. 2 days
before the mice were euthanized by cervical dislocation.
Spleen cells were harvested and fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were cultured in RPMI 1640 medium containing
hypoxanthine, aminopterin, and thymidine selection medium
supplement (Thermo Fisher Scientific Inc.). The culture
supernatants were screened, using an enzyme-­linked immunosorbent assay (ELISA) and recombinant human PDPN
purified from LN229/hPDPN cells [30]. Proteins (1 μg/mL)
were immobilized on Nunc Maxisorp 96-­well immunoplates(Thermo Fisher Scientific Inc.) for 30 min. After blocking
with 1% bovine serum albumin (BSA) in 0.05% Tween20/
phosphate-­buffered saline (PBS) (Nacalai Tesque, Inc.), theplates were incubated with culture supernatants, followedby the addition of peroxidase-­conjugated anti-­
mouse IgG
diluted 1:2000 (Dako; Agilent Technologies, Inc.). The enzymatic reaction was conducted using a 1-­
Step Ultra TMB-­ELISA (Thermo Fisher Scientific Inc.). Optical density wasmeasured at 655 nm using an iMark microplate reader(Bio-­Rad Laboratories Inc.).

Flow cytometry
Cell lines were harvested after brief exposure to 0.25%
Trypsin/1 mmol/L EDTA (Nacalai Tesque, Inc.). Afterwashing with 0.1% BSA in PBS, the cells were treatedwith primary mAbs for 30 min at 4°C, followed by treatment with Oregon Green 488-­
conjugated to goat anti-­
mouse IgG or anti-­rat IgG (Thermo Fisher Scientific Inc.).
Fluorescence data were acquired using a Cell Analyzer
EC800 (Sony Corp., Tokyo, Japan).

Immunohistochemical analyses
Production of point mutants
The amplified hPDPN cDNA was subcloned into a pcDNA3
vector (Thermo Fisher Scientific Inc.), and a FLAG epitope
tag was added to the C-­terminus. Substitutions of aminoacid residues to Ala or Gly in the hPDPN sequence were384

Four-­μm-­thick tissue sections were deparaffinized using xylene
and rehydrated. After antigen retrieval, (autoclaving using
citrate buffer, pH 6.0), sections were incubated with 1 μg/
mL of LpMab-­21 for 1 h at room temperature, and immunocomplexes were treated with an Envision+ Kit (Dako)© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

M. K. Kaneko et al.

for 30 min, color was developed using 3, 3-­diaminobenzidine
tetrahydrochloride (DAB, Dako) for 5 min. Sections werethen counterstained with hematoxylin (Wako Pure Chemical
Industries Ltd.).

Results
Generation of a novel anti-­hPDPN mAb(LpMab-­21)
We first immunized one mouse with the LN229/hPDPN,
and harvested spleen cells were fused with P3U1. The ELISA
screening was performed with supernatants from 960 hybridomas. Among 135 ELISA-­positive wells, 19 wells reactedwith LN229/hPDPN, but not with LN229 in flow cytometry.
We performed single-­cell cloning for 19 wells by limiting
dilution, and could obtain 14 hybridomas. Among them,
we previously reported five clones including LpMab-­
10,
LpMab-­12, LpMab-­13, LpMab-­17, and LpMab-­19 [30–36,
38, 39, 43]. In this study, we newly report LpMab-­21 (IgG2a,kappa), which was the first IgG2a mouse anti-­hPDPN mAb
in our study. Flow cytometry revealed that LpMab-­21 reactedwith LN229/hPDPN cells but not with LN229 cells thatwere hPDPN-­negative (Fig. 1A). LpMab-­21 detected endogenous hPDPN, which is expressed in the glioblastoma cellline LN319, but not in LN319/hPDPN-­KO cells (PDIS - 6),
indicating that LpMab-21 is specific against hPDPN(Fig. 1B).

Figure 1. Flow cytometric analysis using LpMab-­21 to detect hPDPN
expression. (A) LN229 and LN229/hPDPN cells were treated with
LpMab-­21 (1 μg/mL, red) or PBS (black) for 30 min at 4°C followed bytreatment with anti-­
mouse IgG-­
Oregon green. (B) LN319 and
LN319/hPDPN-­KO cells (PDIS-­6) were treated with LpMab-­21 (1 μg/mL,red) or PBS (black) for 30 min at 4°C followed by addition of anti-­mouse
IgG-­Oregon green. Fluorescence data were collected using a Cell Analyzer
EC800. Geometric Mean was described. hPDPN, Human podoplanin.

© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

LpMab-­21 detected the expression of hPDPN in normal
cells such as a lymphatic endothelial cell (LEC) and amesothelial cell line Met-5A (Fig. 2A). The positive-­control
LpMab-­
17 reacted with LEC and Met-5A (Fig. 2B).
LpMab-­21 detected endogenous hPDPN, which is expressedin the kidney epithelial cell line HEK-­293T, but not in
HEK-­
293T/hPDPN-­
KO cells (PDIS-2), indicating that
LpMab-21 is specific against hPDPN (Fig. 2C).
We next investigated whether LpMab-­21 was suitablefor immunohistochemical analyses (Fig. 3). Consistent
with the expression of hPDPN by lymphatic endothelial
cells [44], LpMab-­21 reacted with lymphatic endothelial
cells of the esophagus (Fig. 3A), colon (Fig. 3C), lung

Figure 2. Flow cytometric analysis using LpMab-­21 to detect hPDPN
expression in normal cells. (A) Human lymphatic endothelial cell (LEC)
and human mesothelial cells (Met-­5A) were reacted with LpMab-­21(1 μg/mL, red) or PBS (black) for 30 min at 4°C, followed by treatment
with anti-­mouse IgG-­Oregon green. (B) LEC and Met-­5A cells weretreated with LpMab-­17 (1 μg/mL, red) or PBS (black) for 30 min at 4°C,
followed by treatment with anti-­
mouse IgG-­
Oregon green. (C) The
human embryonic renal epithelial cell line (HEK-­
293T) and HEK-­
293T/hPDPN-­KO cells (PDIS-­2) were reacted with LpMab-­21 (1 μg/mL,red) or PBS (black) for 30 min at 4°C, followed by addition of anti-­
mouse IgG-­Oregon green. Fluorescence data were acquired using a Cell
Analyzer EC800. Geometric Mean was described. LEC, lymphatic
endothelial cell.

385

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

M. K. Kaneko et al.

A

D

B

E

C

F

Figure 3. Immunohistochemical analysis using LpMab-­21 to detect PDPN expression in normal human tissues. Tissues harvested from the esophagus(A), breast (B), colon (C), lung (D), kidney (E), and rectum (F). After antigen retrieval procedure, sections were incubated with 1 μg/mL of LpMab-­21,
reacted with the Envision+ kit, color was developed, using DAB, and samples were then counterstained with hematoxylin. Arrowheads, lymphatic
endothelial cells. Scale bar , 100 μm. 3, DAB, 3-­diaminobenzidine tetrahydrochloride.

(Fig. 3D), kidney (Fig. 3E), and rectum (Fig. 3F). LpMab-­21
detected hPDPN expressed by basal keratinocytes of theesophagus (Fig. 3A) and myoepithelial cells of breast
glands (Fig. 3B). In contrast, LpMab-­21 did not detect
hPDPN expression in type I alveolar cells of lung (Fig. 3D)
and podocytes of the renal glomerulus (Fig. 3E). These
results indicate that the epitope recognized by LpMab-­21
is tissue-specific [45].

Flow cytometric and immunohistochemical
analyses using LpMab-­21 to detect hPDPN
expression in cancers
PDPN is expressed by cancers such as brain tumors,
mesotheliomas, oral cancers, lung cancers, esophageal
cancers, testicular cancers, and osteosarcoma [1, 9, 32].
Flow cytometry using LpMab-­
21 detected endogenous
expression of PDPN by the human cancer cell lines asfollows: mesothelioma, NCI-­
H226; oral squamous cell
386

carcinoma, HSC-­2 and HSC-­4; squamous cell carcinomaof the lung, PC-­
10; and human osteosarcoma, U-­
2 OS
and MG-­63 (Fig. 4A). LpMab-­17 detected PDPN expression by all cell lines (Fig. 4B).
Immunohistochemical analysis (with or without antigen
retrieval), using LpMab-­21 detected membrane-­associated
PDPN expression in the human tumor tissues as follows:
glioblastoma (Fig. 5A), oral squamous cell carcinoma(OSCC) (Fig. 5B), and a seminoma (Fig. 5C). LpMab-­21
reacted with lymphatic endothelial cells in OSCC tissues(Fig. 5D), but not with vascular endothelial cells (Fig. 5D),
demonstrating that LpMab-­21 is useful for detecting lymphatic endothelial cells in cancer tissues.

Characterization of LpMab-­21 using glycan-­deficient cell lines
Human PDPN is O-­glycosylated, but not N-­glycosylated[6, 24, 46–48]. In this study, we generated a© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

M. K. Kaneko et al.

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

Figure 4. Flow cytometric analysis using LpMab-­21 to detect PDPN expression in human cancer cells. Human cell lines analyzed were as follows:
mesothelioma, NCI-­H226; oral squamous cell carcinomas, HSC-­2 and HSC-­4; lung squamous cell carcinoma, PC-­10; and osteosarcomas U-­2 OS andMG-­63. Cells were treated reacted with LpMab-­21 (A) 1 μg/mL; red), LpMab-­17 (B, 1 μg/mL; red), or PBS (A and B, black) for 30 min at 4°C, followed
by treatment with anti-­mouse IgG-­Oregon green. Fluorescence data were acquired using a Cell Analyzer EC800. Geometric Mean was described.

GnT-­1-­knockout (KO) cell line (CHO-­S/GnT-­1-­KO,
PDIS-­
9)
and
a
CMP-­
sialicacidtransporter(SLC35A1)-­KO cell line (CHO-­S/SLC35A1-­KO, PDIS-­14)by transfecting them with TALEN and CRISPR/Cas9
plasmids, respectively (Table 1). PDIS-­
9 and PDIS-­
14
cells were screened using leukoagglutinin from Phaseolus
vulgaris (L-­
PHA) and MAL-­
II, respectively. When weused the hPDPN expression vector to transfect PDIS-­9and PDIS-­
14 cells, we found that LpMab-­
21 reactedwith CHO-­S/hPDPN and PDIS-­9/hPDPN cells but notwith PDIS-­14/hPDPN cells (Fig. 6A and B). We further
generated a GnT-­1-KO cell line (HEK-­293T/GnT-­1-­KO,
PDIS-­1 or PDIS-­12) and a CMP-­sialic acid transporter(SLC35A1)-­KO cell line (HEK-­293T/SLC35A1-­KO,© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

PDIS-­22) by transfecting them with TALEN and CRISPR/
Cas9 plasmids, respectively (Table 1). PDIS-­1 and PDIS-­
12 cells were screened using L-­PHA. PDIS-­22 cells werescreened using MAL-­II. We found that LpMab-­21 reactedwith HEK-­293T, PDIS-­1, and PDIS-­12 cells but not with
PDIS-­22 cells (Fig. S1). These results indicate that thehPDPN epitope recognized by LpMab-­
21 includes a
peptide sequence linked to sialic acid.

Epitope mapping of LpMab-­21
We expressed hPDPN deletion mutants in CHO-­K1 cells(Fig. 7A). LpMab-­21 detected dN23, dN37, dN46, dN55,
and dN64. In contrast, LpMab-­
21 did not react with387

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

M. K. Kaneko et al.

A

E

B

F

C

G

D

H

Figure 5. Immunohistochemical analysis using LpMab-­21 to detect PDPN expression in human cancer tissues. Tissue sections were prepared from thehuman cancer tissues as follows: glioblastoma (GBM, A and E); oral squamous cell carcinoma (OSCC; B, D, F, and H); seminoma (SE, C and G).
Sections were incubated (antigen retrieval omitted) with 1 μg/mL of LpMab-­21 (A–D), reacted with the Envision+ kit, color was developed using DAB,
and samples were then counterstained with hematoxylin. Sections were stained with hematoxylin and eosin as well (E–H). Arrowheads: lymphatic
endothelial cells. Scale bar = 100 μm. DAB, 3-­diaminobenzidine tetrahydrochloride

dN75, dN85, dN95, or dN105, indicating that theN-­terminus of the epitope recognized by LpMab-­21 resides
between hPDPN Thr65 and Val75 (Fig. 7B). All deletionmutants were detected, using the anti-MAP-tag mAb,
PMab-1 (Fig. 7C).
Next, we generated stable CHO-­S cell lines expressing
the hPDPN point mutants T65A, T66A, T70A, S71A,
S74A, T76A, T85A, S86A, and S88A (Fig. 8A). These
388

mutants were chosen, because the epitope of LpMab-­21
includes a sialylated O-­glycan (Fig. 6) and starts between
residues Thr65 and Val75 (Fig. 7). LpMab-­21 reactedwith all transfectants, except for T76A (Fig. 8B). All
point mutants targeting Ser/Thr residues were detectedby LpMab-­17 (Fig. 8C). Together, these data support
the conclusion that the epitope recognized by LpMab-­21
includes sialic acid linked to PDPN Thr76.

© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

M. K. Kaneko et al.

Table 1. Characterization of glycan-­deficient or PDPN-­knock out cells.
Cell name

Parental cells

Targeted genes

Genom editing

Deficient

PDIS-­1
PDIS-­2
PDIS-­6
PDIS-­9
PDIS-­12
PDIS-­14
PDIS-­22

HEK-­293T
HEK-­293T
LN319
CHO-­S
HEK-­293T
CHO-­S
HEK-­293T

hsMgat1/GnT-­1
PDPN
PDPN
hsMgat1/GnT-­1
hsMgat1/GnT-­1
SLC35A1
SLC35A1TALEN
CRISPR/Cas9
CRISPR/Cas9
TALEN
TALEN
CRISPR/Cas9
CRISPR/Cas9N-­glycan
PDPN
PDPN
N-­glycan
N-­glycan
Sialic acid
Sialic acid

Figure 6. Flow cytometric analysis using LpMab-­21 to detect hPDPN expression in sialic acid-­deficient cells. CHO-­S, CHO-­S/hPDPN, PDIS-­9/hPDPN, and
PDIS-­14/hPDPN cells were reacted with LpMab-­21 (A, 1 μg/mL; red), LpMab-­21 (B, 10 μg/mL; red), or LpMab-­17 (C, 1 μg/mL; red), or PBS (A, B, andC; black) for 30 min at 4°C, followed by treatment with anti-­mouse IgG-­Oregon green. Fluorescence data were acquired using a Cell Analyzer EC800.
Geometric Mean was described.

We used alanine scanning to localize the epitope recognized by LpMab-­
21 near hPDPN Thr76. Thus, nine
hPDPN point mutants (Ser74–Thr85) were transiently
expressed in CHO-­
K1 cells. LpMab-­
21 did not detect
T76A, G77A, I78A, or R79A. In contrast, LpMab-­12, which© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

recognizes an epitope comprising hPDPN Thr52, detectedeach point mutant (Fig. 9B), indicating that Thr76–Arg79
is the minimum epitope recognized by LpMab-­
21. Wesummarized and compared LpMab-­
21 with the several
anti-­hPDPN mAbs (Table 2).
389

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

M. K. Kaneko et al.

Figure 7. Epitope mapping of LpMab-­21 using deletion mutants of hPDPN. (A) Structures of hPDPN deletion dN23, dN37, dN46, dN55, dN64, dN75,
dN85, dN95, dN105. (B, C) Each hPDPN deletion mutant was reacted with LpMab-­21 (B, 1 μg/mL; red), PMab-­1 (C, 1 μg/mL; red), or PBS (B and C,
black) for 30 min at 4°C, followed by treatment with anti-­mouse IgG-­Oregon green (B) or anti-­rat IgG-­Oregon green (C). Fluorescence data wereacquired using a Cell Analyzer EC800. Geometric Mean was described.

390© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

M. K. Kaneko et al.

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

Figure 8. Epitope mapping of LpMab-­21 using hPDPN-­Ser/Thr point mutants. (A) Amino acid sequence of hPDPN encompassing Thr76. (B, C) Stable
CHO-­S transfectants expressing hPDPN point mutants T65A, T66A, T70A, S71A, S74A, T76A, T85A, S86A, and S88A were reacted with LpMab-­21(B, 1 μg/mL; red), LpMab-­17 (C, 1 μg/mL; red), or PBS (B and C; black) for 30 min at 4°C, followed by treatment with anti-­mouse IgG-­Oregon green.
Fluorescence data were acquired using a Cell Analyzer EC800. Geometric Mean was described.

© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

M. K. Kaneko et al.

Figure 9. Epitope mapping of LpMab-­21, using point mutants of hPDPN. Nine hPDPN point Ser74–Thr85 point mutants were transiently expressedin CHO-­K1 cells. Cells were reacted with LpMab-­21 (A, 1 μg/mL; red), LpMab-­12 (B, 1 μg/mL; red), or control PBS (A and B, black) for 30 min at 4°C,
followed by treatment with anti-­mouse IgG-­Oregon green. Fluorescence data were acquired using a Cell Analyzer EC800. (C) Illustration of the
epitope recognized by anti-­hPDPN mAbs. Geometric Mean was described. PBS, phosphate-­buffered saline.

Discussion
The anti-­hPDPN mAb (NZ-­1) detects hPDPN with highspecificity and sensitivity [6, 10, 25]. Moreover NZ-­
1,392

which is also useful for detecting the podoplanin/aggrus(PA) epitope tag [49, 50], is efficiently internalized byglioma cell lines, accumulates in tumors in vivo, and is© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

M. K. Kaneko et al.

Table 2. Characterization of anti-­PDPN glycopeptide mAbs.
Anti-­glycopeptide mAb

LpMab-­21
LpMab-­2
LpMab-­3
LpMab-­9
LpMab-­12
LpMab-­19

Subclass

IgG2a, kappa
IgG1, kappa
IgG1, kappa
IgG1, kappa
IgG1, kappa
IgG2b, kappa

EpitopeO-­glycan

PepitdeThr76
Thr55/Ser56
Thr76
Thr25
Thr52
Thr76

Thr76–Arg79
Thr55–Leu64
Thr76–Glu81
Thr25–Asp30
Asp49–Pro53
Thr76–Arg79

CasMab/non-­CasMab

HC (LEC, T1a, POD)

Non-­CasMab
CasMabNon-­CasMab
Non-­CasMab
Non-­CasMab
Non-­CasMab

positive for only LEC
negative
positive for LEC, T1a, POD
not applicable
positive for LEC, T1a, POD
positive for LEC, T1a, POD

CasMab, cancer-specific mAb; IHC, immunohistochemical analysis; T1a, type I alveolar cells; POD, podocyte.

therefore a suitable candidate for therapy for malignant
gliomas [5, 10]. Further, NZ-­1 inhibits tumor cell-­induced
platelet aggregation and tumor metastasis [23]. NZ-­
1
mediates antibody-­dependent cellular cytotoxicity (ADCC)
and complement-­
dependent cytotoxicity (CDC) against
tumor cells that express hPDPN [51]. Moreover, NZ-­
1
is suitable for western blotting, flow cytometry, immunohistochemistry,andimmunoprecipitation[51].
However, NZ-­1 was produced using synthetic peptide [6];therefore, further mAbs against hPDPN, especially anti-­
glycopeptide mAbs are necessary to investigate the structureand function of PDPN.
Previously, we developed the original technology to
produce cancer-­specific mAbs that detect cell type-­specificposttranslational modifications of the same protein [30].
We used LN229/hPDPN cells as the immunogen to elicit
novel anti-­PDPN mAbs. We produced several clones including LpMab-­2, LpMab-­3, and LpMab-­9 as anti-­glycopeptide
mAbs [30]. Recently, we further immunized mice with
LN229/hPDPN cells to develop further anti-­
glycopeptide
mAbs against human PDPN, and characterized several
clones, including LpMab-­12 and LpMab-­19. In this study,
we characterized another clone LpMab-­21, which detects
many human cancer cell lines that express PDPN, suchas those derived from glioblastomas, lung squamous cell
carcinomas, oral squamous cell carcinomas, osteosarcomas,
and malignant mesotheliomas. The isotypes of previously
established anti-­PDPN mAbs are IgG1 (seven clones) andIgG3 (one clone). However, the applications of mouse IgG3
mAbs are limited because they often aggregate [52].
Moreover, mouse IgG1 and IgG3 isotypes do not induce
ADCC or CDC. Therefore, we required chimeric mAbs
using human IgG1 to investigate these activities [33].
LpMab-­21 (IgG2a subclass) could be used to investigate
the function of anti-­tumor activities in xenograft models
because LpMab-­
21 induced ADCC and CDC (data notshown).
Furthermore, we need several glycan-­deficient cell lines
such as sialic acid-­deficient or N-­glycan deficient cell lines
to characterize those mAbs. In this study, we successfully© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

produced several glycan-­deficient cell lines such as sialic
acid deficient (PDIS-­14 and PDIS-­22) or N-­glycan deficient
cell lines (PDIS-­1, PDIS-­9, and PDIS-­12), using CRISPR/
Cas9 and TALEN systems. Using those cell lines, we
determined that the epitope of LpMab-­21 includes sialic
acids, indicating that we can also investigate whether the
epitope of novel mAbs against the other membrane proteins possesses sialic acids or N-­glycans.
We showed here that LpMab-­21 detected glioblastomas,
oral cancers, and seminomas (Fig. 5) as well as normal
cells such as lymphatic endothelial cells, basal epithelial cellsof the esophagus, and myoepithelial cells of breast glands(Fig. 3). In contrast, LpMab-­21 did not react with the renal
glomerulus or with type I alveolar cells of lung (Fig. 3),
indicating that sialylation of hPDPN is tissue-specific.
In conclusion, LpMab-­21 shows promise for investigating the expression and function of hPDPN in cancers
and normal tissues. Further, mAbs that recognize different
epitopes of hPDPN should serve as powerful tools for
identifying the function of hPDPN.

Acknowledgments
We thank Noriko Saidoh and Kanae Yoshida for their
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 the Platform for Drug Discovery, Informatics,
and Structural Life Science (PDIS) from AMED (Y.K.),
by the Practical Research for Innovative Cancer Control
from AMED (Y.K.); by JSPS KAKENHI Grant Number
26440019 (M.K.K.), 25462242 (Y.K.), and 16K10748 (Y.K.),
and by the Regional Innovation Strategy Support Programfrom the Ministry of Education, Culture, Sports, Scienceand Technology (MEXT) of Japan (Y.K.). This work was
performed in part under the Cooperative Research Programof Institute for Protein Research, Osaka University, CR-­
15-­
05 and CR-­
16-­
05. The authors thank Enago (www.
enago.jp) for the English language review.

393

Anti-­glycopeptide mAb LpMab-­21 against Podoplanin

Conflict of Interest
No declared.
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Supporting Information
Additional supporting information may be found in the
online version of this article:
Figure S1. Flow cytometric analysis, using LpMab-­21
to detect hPDPN expression in sialic acid-­deficient cells.
HEK-­
293T, PDIS-­
1, PDIS-­12, and PDIS-­
22 cells werereacted with LpMab-­21 (A, 1 μg/mL; red), or LpMab-­17(B, 1 μg/mL; red), or PBS (A and B; black) for 30 min
at 4°C, followed by treatment with anti-­mouse IgG-­Oregon
green. Fluorescence data were acquired using a Cell Analyzer
EC800.

© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.