MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 35, Number 3, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0006

Establishment of Mouse Monoclonal Antibody
LpMab-13 Against Human Podoplanin
Satoshi Ogasawara,1 Mika K. Kaneko,1 Ryusuke Honma,1,2 Hiroharu Oki,1,2
Yuki Fujii,1 Michiaki Takagi,2 Hiroyoshi Suzuki,3 and Yukinari Kato1

Podoplanin (PDPN)/Aggrus is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregationstimulating (PLAG) domain. The O-glycosylation on Thr52 of human PDPN (hPDPN) is critical for the interaction of hPDPN with C-type lectin-like receptor-2 (CLEC-2), resulting in platelet aggregation. Many antihPDPN monoclonal antibodies (MAbs) against PLAG domains and non-PLAG domains have been established;however, mouse anti-PLAG2/3 MAb, the epitope of which is consistent with rat anti-PLAG2/3 MAb NZ-1, has
not been established. NZ-1 inhibits the hPDPN-CLEC-2 interaction and is also useful for anti-PA tag MAb. We
recently established CasMab technology to produce MAbs against membranous proteins. Herein, we produced anovel anti-hPDPN MAb, LpMab-13, which binds to PLAG2/3 domains. LpMab-13 recognized endogenous
hPDPN of cancer cells, including glioblastoma, oral cancer, lung cancer, and malignant mesothelioma, and normal
cells such as lymphatic endothelial cells and podocytes of kidney in Western blot, flow cytometry, and immunohistochemistry. LpMab-13 recognized glycan-deficient hPDPN in flow cytometry, indicating that the interaction
between LpMab-13 and hPDPN is independent of its glycosylation. The minimum epitope of LpMab-13 was
identified as Ala42–Asp49 of hPDPN using Western blot and flow cytometry. The combination of different
epitope-possessing MAbs could be advantageous for the hPDPN-targeting diagnosis and therapy.

IntroductionP

odoplanin (PDPN), the endogenous ligand of C-type
lectin-like receptor-2 (CLEC-2) of platelet,(1,2) is highly
expressed not only in many tumors, including malignant brain
tumor, mesothelioma, oral cancer, lung cancer, esophageal
cancer, testicular tumor, and osteosarcoma (OS),(3–13) but
also in normal cells such as lymphatic endothelial cells(LEC).(14,15) ‘‘Podoplanin’’ was named after its expression in
podocytes of kidney.(14) PDPN is also called as ‘‘T1a,’’ and it
is abundant in lung type I alveolar cells.(16) We previously
named it as ‘‘Aggrus’’ because PDPN possesses a platelet
aggregation-inducing activity, which is associated with cancer metastasis.(17)The interaction of human PDPN (hPDPN) with CLEC-2 ismainly observed at Glu47 and Asp48 in the platelet
aggregation-stimulating domain-3 (PLAG3) and the a2-6
linked sialic acid residue.(18) The sequence motif is conserved among PDPN proteins of various species.(19) CLEC-2
has a recognition motif in the form ‘‘EDXXXT/S,’’ where X
is any amino acid and T (or S) contains core 1 O-glycan witha2–6 linked sialic acid + N-acetyl-d-galactosamine (GalNAc) residue. PDPN is known as a specific lymphatic en1
2
3

dothelial marker,(20) and PDPN-CLEC-2 signaling leads toplatelet aggregation, which is critical for the embryonicblood-lymphatic vascular separation.(21)In this study, we describe the development and characterization of a new anti-hPDPN monoclonal antibody (MAb),
LpMab-13, which specifically binds to PLAG2/3 domains,and might serve as a novel modality to study the function of
hPDPN.
Materials and Methods
Cell lines, animals, and tissues

LN229, HEK-293T, NCI-H226, Chinese hamster ovary(CHO)-K1, glycan-deficient CHO cell lines (Lec1, Lec2, andLec8), and P3U1 were obtained from the American Type Culture Collection (Manassas, VA). Human LEC were purchasedfrom Cambrex (Walkersville, MD). LN319 was donated by
Prof. Kazuhiko Mishima (Saitama Medical University, Saitama,
Japan).(22) HSC-2 was obtained from the Japanese Collection of
Research Bioresources ( JCRB) cell bank (Osaka, Japan). PC-10
cells were purchased from Immuno-Biological Laboratories
Co., Ltd. (Gunma, Japan). LN229, CHO-K1, Lec1, Lec2, andLec8 were transfected with hPDPN plasmids (LN229/hPDPN,

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, Yamagata, Japan.
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.

155

156

CHO/hPDPN, Lec1/hPDPN, Lec2/hPDPN, and Lec8/hPDPN)
using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.,
Waltham, MA) according to the manufacturer’s instructions.(23)
HEK-293T/hPDPN-knockout (KO) cell line (PDIS-2) and
LN319/hPDPN-KO cell line (PDIS-6) were produced bytransfecting CRISPR/Cas plasmids (Target ID: HS0000333287),
which target PDPN (Sigma-Aldrich Corp., St. Louis, MO),
using a Gene Pulser Xcell electroporation system (Bio-Rad
Laboratories, Inc., Berkeley, CA).(24–26) The amplified hPDPN
cDNA was subcloned into a pcDNA3 vector (Thermo Fisher
Scientific, Inc.) and a FLAG epitope tag was added at the Cterminus. Substitutions of amino acids to alanine, glycine,
serine, or valine in hPDPN were performed using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent
Technologies, Inc., Santa Clara, CA) with oligonucleotides
containing the desired mutations. CHO-K1 cells were transfected with the plasmids using a Gene Pulser Xcell electroporation system (Bio-Rad Laboratories). CHO-K1, Lec1,
Lec2, Lec8, CHO/hPDPN, Lec1/hPDPN, Lec2/hPDPN, Lec8/
hPDPN, CHO-K1/hPDPN point mutants, NCI-H226, PC-10,
and P3U1 were cultured in RPMI 1640 medium, including lglutamine (Nacalai Tesque, Inc., Kyoto, Japan), and LN229,
LN229/hPDPN, LN319, LN319/hPDPN-KO, HSC-2, HEK293T, and HEK-293T/hPDPN-KO were cultured in Dulbecco’s modified Eagle’s medium, including l-glutamine (Nacalai
Tesque, Inc.), supplemented with 10% heat-inactivated fetal
bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37C in ahumidified atmosphere of 5% CO2 and 95% air. The l-proline(0.04 mg/mL) was added for Lec1, Lec2, Lec8, Lec1/hPDPN,
Lec2/hPDPN, and Lec8/hPDPN. LEC was cultured in endothelial cell medium EGM-2 MV supplemented with 5% FBS(Cambrex Corp.). Antibiotics, including 100 units/mL of
penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque, Inc.), were added to all media.
Female BALB/c mice (4-weeks old) were purchased from
CLEA Japan (Tokyo, Japan).
Animals were housed under pathogen-free conditions. The
Animal Care and Use Committee of Tohoku University approved the animal experiments described herein. This study
examined cancer patients who underwent surgery at Sendai
Medical Center(27) and Yamagata University Hospital.(6,28)
Informed consent for obtaining samples and for subsequent data
analyses was obtained from patients or the patient’s guardian.
Hybridoma production

BALB/c mice were immunized by intraperitoneal (i.p.)
injection of 1 · 108 LN229/hPDPN cells together with Imject
Alum (Thermo Fisher Scientific, Inc.).(23) After several additional immunizations, a booster injection was given i.p. 2 days
before spleen cells were harvested. The spleen cells were fusedwith P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in RPMI medium
with hypoxanthine, aminopterin, and thymidine selection
medium supplement (Thermo Fisher Scientific, Inc.). The
culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA) for binding to recombinant
hPDPN purified from LN229/hPDPN cells. Proteins were
immobilized on Nunc MaxiSorp 96-well immunoplates(Thermo Fisher Scientific, Inc.) at 1 mg/mL for 30 minutes.
After blocking with 1% bovine serum albumin (BSA) in
0.05% Tween 20/phosphate-buffered saline (PBS, Nacalai

OGASAWARA ET AL.

Tesque, Inc.), the plates were incubated with culture supernatant followed by 1:2000 diluted peroxidase-conjugated antimouse IgG (Dako; Agilent Technologies, Inc.). The enzymatic
reaction was conducted with a 1-Step Ultra TMB-ELISA(Thermo Fisher Scientific, Inc.). The optical density wasmeasured at 655 nm using an iMark microplate reader (BioRad Laboratories, Inc.).
Western blot analyses

Cell lysates (5–10 mg) were boiled in SDS sample buffer(Nacalai Tesque, Inc.). The proteins were electrophoresed on
5%–20% polyacrylamide gels (Wako Pure Chemical Industries
Ltd., Osaka, Japan) and were transferred onto a PVDF membrane (EMD Millipore Corp., Billerica, MA). After blocking
with 4% skim milk (Nacalai Tesque, Inc.), the membrane wasincubated with LpMab-13 (1 mg/mL), anti-PDPN (1 mg/mL;
clone NZ-1 and clone LpMab-7),(8,28) anti-IDH1 (1 mg/mL;
clone RcMab-1 and clone RMab-3),(29) anti-FLAG (1 mg/mL;
Wako Pure Chemical Industries Ltd.), or anti-b-actin (1:10,000
diluted; clone AC-15; Sigma-Aldrich Corp.), then withperoxidase-conjugated anti-mouse or anti-rat IgG (1:1000 diluted; Dako), and developed with the ImmunoStar LD Chemiluminescence Reagent (Wako Pure Chemical Industries Ltd.)
using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Flow cytometry

Cell lines were harvested by brief exposure to 0.25%
Trypsin/1 mM EDTA (Nacalai Tesque, Inc.).(2) After washing with 0.1% BSA in PBS, the cells were treated with
LpMab-13, NZ-1, or LpMab-7 (1 mg/mL) for 30 minutes at
4C and followed by treatment with Oregon Green 488 goatanti-mouse or anti-rat IgG (Thermo Fisher Scientific, Inc.).
Fluorescence data were collected using a Cell Analyzer
EC800 (Sony Corp., Tokyo, Japan).
Determination of the binding affinity
using flow cytometry

LN319 (2 · 105 cells) was resuspended with 100 mL of seriallydiluted LpMab-13 (0.006–100 mg/mL) followed by secondaryanti-mouse IgG (Thermo Fisher Scientific, Inc.). Fluorescencedata were collected using a cell analyzer (EC800; Sony Corp.).
The dissociation constants (KD) were obtained by fitting thebinding isotherms using the built-in one-site binding models in
GraphPad PRISM 6 (GraphPad software, Inc., La Jolla, CA).
Immunohistochemical analyses

Four-micrometer-thick histologic sections were deparaffinized in xylene and rehydrated. Without antigen retrieval
procedure, sections were incubated with 5 mg/mL of LpMab13 for 1 h at room temperature followed by treatment with
EnVision+ Kit (Dako) for 30 minutes. Color was developed
using 3, 3-diaminobenzidine tetrahydrochloride (DAB; Dako)for 5 minutes and then the sections were counterstained withhematoxylin (Wako Pure Chemical Industries Ltd.).
Results
Production of a novel anti-PDPN MAb LpMab-13

Herein, we used the CasMab technology.(23–25,27, 28,30–32)
LN229/hPDPN cells were immunized into mice to develop

LPMAB-13 AGAINST HUMAN PODOPLANIN

novel anti-hPDPN MAbs. The culture supernatants werescreened using ELISA for the binding to recombinant hPDPN
purified from LN229/hPDPN cells. LpMab-13 (mouse IgG1,
kappa) was established after limiting dilution.
Characterization of LpMab-13

LpMab-13 reacted with LN229/hPDPN, not with LN229,
hPDPN-negative cell in Western blot analysis (Fig. 1A).
LpMab-13 also recognized endogenous hPDPN, which isexpressed in a glioblastoma cell line LN319 and a LEC in
Western blot analysis (Fig. 1A) and flow cytometry (Fig. 1B).
We also performed Western blot analysis using LpMab-13
against several glycan-deficient PDPN transfectants such as
Lec1/hPDPN (N-glycan deficient), Lec2/hPDPN (sialic aciddeficient), and Lec8/hPDPN (O-glycan deficient). LpMab-13
reacted with CHO-K1/hPDPN, not with CHO-K1 (Fig. 1A).
Furthermore, all transfectants of glycan-deficient PDPN were157

detected by LpMab-13 (Fig. 1A), showing that the epitope of
LpMab-13 is independent of glycan. LpMab-13 detectedendogenous hPDPN of human kidney cell line HEK-293T,
oral cancer cell line HSC-2, lung squamous cell carcinoma
cell line PC-10, and malignant mesothelioma cell line HCIH226 in flow cytometry (Fig. 1B), although hPDPN of NCIH226 and HEK293T was not detected using Western blot
analysis (Fig. 1A). LN229 and CHO-K1, which do not express hPDPN, were not detected by LpMab-13 (Fig. 1A), and
LpMab-13 did not react with HEK-293T/hPDPN-KO and
LN319/hPDPN-KO (Fig. 1B), indicating that LpMab-13
does not recognize nonrelated antigens.
Binding affinity of LpMab-13

We next performed a kinetic analysis of the interaction of
LpMab-13 with LN319 using flow cytometry. As shown in
Figure 1C, KD was determined to be 2.9 · 10-8 M, indicating

FIG. 1. Characterization of a novel anti-hPDPN monoclonal antibody (mAb), LpMab-13. (A) Western blot analysis. NZ-1
was used as positive control against hPDPN. RcMab-1 (anti-IDH1) and AC-15 (anti-b-actin) were used as internal control.
Total cell lysates (5 mg) were electrophoresed on 5%–20% polyacrylamide gels and transferred onto a PVDF membrane.
After blocking, the membrane was incubated with 1 mg/mL of mAbs and then with peroxidase-conjugated anti-mouse IgGagainst LpMab-13 and AC-15 or peroxidase-conjugated anti-rat IgG against NZ-1 and RcMab-1; the membrane was
detected using a Sayaca-Imager. (B) Flow cytometric analysis. Eight cell lines were treated with LpMab-13 (1 mg/mL; red)or control PBS (black) for 30 minutes at 4C, followed by treatment with anti-mouse IgG-Oregon green. Fluorescence data
were collected using a Cell Analyzer EC800. (C) Determination of binding affinity of LpMab-13 using flow cytometry.
hPDPN, human PDPN; PBS, phosphate-buffered saline.

158

that LpMab-13 possesses moderate affinity against endogenous hPDPN.
Immunohistochemical analysis using LpMab-13

We further investigated whether LpMab-13 is useful for
immunohistochemical analyses. The expression of hPDPN isobserved in many cancers such as malignant brain tumor,

OGASAWARA ET AL.

mesothelioma, oral cancer, lung cancer, esophageal cancer,
testicular tumor, and OS.(3,5,11,28) In this study, we picked up
several hPDPN-expressing cancers. As shown in Figure 2,
LpMab-13 stained glioblastoma (GBM; Fig. 2B, C), oral
squamous cell carcinoma (OSCC; Fig. 2E, F), esophageal
squamous cell carcinoma (Fig. 2H, I), osteosarcoma (OS;Fig. 2K, L), and seminoma (Fig. 2N, O) in a membranous/
cytoplasmic staining pattern. The expression of hPDPN is

FIG. 2. Immunohistochemical analysis by LpMab-13. Glioblastoma (GBM; A–C), oral squamous cell carcinoma (OSCC;
D–F), esophageal squamous cell carcinoma (ESCC; G–I), osteosarcoma (OS; J–L), seminoma (SE, M–O), and normal lung(P–R) were used in this study. Sections were incubated with 5 mg/mL of LpMab-13, followed by EnVision+ Kit, and color
was developed using DAB and counterstained with hematoxylin. Hematoxylin and eosin staining was performed against
each tissue (A, D, G, J, M, P). Arrowheads: microvascular endothelial cells; arrows: lymphatic endothelial cells. Scale bar:
100 mm.

LPMAB-13 AGAINST HUMAN PODOPLANIN

also observed in normal tissues such as LEC and lung type I
alveolar cells.(20) LpMab-13 reacted with LEC (arrows;
Fig. 2H, I); in contrast, it did not react with vascular endothelial cells (arrowheads; Fig. 2B, C), demonstrating that
LpMab-13 is very useful for detecting LEC. Furthermore,
LpMab-13 was also reactive with lung type I alveolar cells(Fig. 2Q, R), which are not recognized by anti-hPDPN mAbs,
NZ-1 and D2-40, in our previous study.(11)
Epitope mapping of LpMab-13

Because LpMab-13 reacted with 38–51 amino acids ofhPDPN (data not shown), we further produced several point
mutants of those residues. NZ-1 was previously produced byimmunizing rats with 38–51 amino acids of hPDPN.(8) To determine the LpMab-13 epitope, we first performed Western blot
analysis. LpMab-13 reaction was lost in several point mutations: A42G, G45A, D48A, and D49A (Fig. 3A). In contrast,
NZ-1 did not react with M43A and D49A. These results were159

confirmed by flow cytometry (Fig. 3B). Although alanine
scanning is usually performed to determine the binding epitope,
conversion to other amino acids has been utilized in some reports(33–35) when alanine might be included in the epitope.
Therefore, we further changed Ala42 and Ala46 to other aminoacids such as Ser and Val (Fig. 4). LpMab-13 lost the reaction
with A42S in the same way with A42G although it reacted with
A42V. In contrast, LpMab-13 reacted with A46S and A46V inthe same way with A46G. LpMab-7, a positive control against
hPDPN, reacted with all point mutants (Figs. 3 and 4). These
results indicate that the minimum epitope of LpMab-13 was
identified as Ala42–Asp49 of hPDPN, the epitope of which isvery similar with a rat anti-hPDPN MAb NZ-1.
Discussion

We previously produced an anti-hPDPN MAb, NZ-1.(8) NZ1 possesses not only high specificity and sensitivity but also
high-binding affinity against hPDPN to be applied for radioimmunotherapy or immunotoxin therapy.(13,36) Another study

FIG. 3. The epitope mapping of LpMab-13. (A) Western blot analysis. The epitopes between LpMab-13 and NZ-1 werecompared. LpMab-7 was used as positive control against hPDPN. 1E6 was used as anti-FLAG tag. AC-15 (anti-b-actin) andRMab-3 (anti-IDH1) were used as internal control. Total cell lysates (10 mg) were prepared from each CHO transfectant of
hPDPN mutants, electrophoresed on 5%–20% polyacrylamide gels, and transferred onto a PVDF membrane. After blocking,the membrane was incubated with 1 mg/mL of mAbs and then with peroxidase-conjugated anti-mouse IgG against LpMab-13,
LpMab-7, 1E6, AC-15, and RMab-3, and peroxidase-conjugated anti-rat IgG against NZ-1; the membrane was detected using
a Sayaca-Imager. (B) Flow cytometric analysis. Point mutants of hPDPN were treated with LpMab-13 (1 mg/mL; red), NZ-1
(1 mg/mL; red), LpMab-7 (1 mg/mL; red), or control PBS (black) for 30 minutes at 4C, followed by treatment with anti-mouseor anti-rat IgG-Oregon green. Fluorescence data were collected using a Cell Analyzer EC800. CHO, Chinese hamster ovary.

160

OGASAWARA ET AL.

FIG. 4. The epitope mapping of LpMab-13. CHO transfectants of each hPDPN mutation were treated with LpMab-13(1 mg/mL; red), NZ-1 (1 mg/mL; red), LpMab-7 (1 mg/mL; red), or control PBS (black) for 30 minutes at 4C, followed bytreatment with anti-mouse or anti-rat IgG-Oregon green. Fluorescence data were collected using a Cell Analyzer EC800.
confirmed the high-binding affinity of NZ-1.(37) NZ-1 is asuitable candidate for therapy against malignant gliomas,
because it was highly internalized into glioma cell lines, andalso well accumulated into tumors in vivo.(13) Moreover, NZ1 inhibited the tumor cell-induced platelet aggregation andtumor metastasis by its neutralizing activity.(2) Furthermore,
NZ-1 possesses antibody-dependent cellular cytotoxicity andcomplement-dependent cytotoxicity against hPDPNexpressing tumor cells.(38) Although a rat anti-hPDPN MAb
NZ-1 possesses perfect application in all experiments suchas Western blot, flow cytometry, immunoprecipitation, immunocytochemical analysis, and immunohistochemical
analysis,(8,37) the other mouse anti-hPDPN MAbs such as
D2-40(39) or 18H5(40) have been utilized more frequently.
Usually, mouse MAbs are preferred for the reason that anti-

mouse secondary antibodies are more available for almost all
experiments especially for immunohistochemical analysis.
For that reason, we recently produced anti-rat PDPN
MAbs(41) and anti-rabbit PDPN MAbs(42,43) using mice. In
this study, we tried to develop mouse anti-hPDPN MAbs, theepitope of which are similar with rat NZ-1 MAb.
We recently established the CasMab technology, which
can produce cancer-specific MAbs although one protein incancer cells and normal cells possesses the same amino acidsequence.(23) We used LN229/hPDPN cells, not purified recombinant proteins, for immunization to develop novel antiPDPN MAbs. LpMab-13 detected many PDPN-expressing
cancer cell lines such as glioblastoma, lung squamous cell
carcinoma, OSCC, and malignant mesothelioma. LpMab-13
does not need antigen retrieval procedure to detect hPDPN in

LPMAB-13 AGAINST HUMAN PODOPLANIN

immunohistochemical analysis, although LpMab-2 andLpMab-3, which were also produced by CasMab technology,
need antigen retrieval procedure.(23,30) Furthermore, LpMab9 is not useful for immunohistochemistry, whereas it is very
sensitive in flow cytometry or Western blot analysis,(25) indicating that the CasMab technology is not always helpful to
produce mAbs for immunohistochemical analysis. Although
the epitopes of D2-40 and 18H5 are very similar with those of
LpMab-13,(36) D2-40 and 18H5 also need antigen retrieval
procedure. NZ-1 is also useful for immunohistochemical
analysis without antigen retrieval; therefore, the epitope of
LpMab-13 and NZ-1 is critical for this superiority.
In conclusion, LpMab-13 could be useful for investigating
the expression and function of hPDPN in cancers and normal
tissues. Furthermore, different epitope-possessing antihPDPN MAbs should be established as powerful tools foruncovering the function of hPDPN in the future.
Acknowledgments

The authors thank Takuro Nakamura, Noriko Saidoh,
Hazuki Kanno, and Kanae Yoshida for excellent technicalassistance. This work was supported, in part, by the Platformfor Drug Discovery, Informatics, and Structural Life Science(PDIS) from Japan Agency for Medical Research and Development, AMED (Y.K.); by the Basic Science and Platform
Technology Program for Innovative Biological Medicine
from AMED (Y.K.); by the Regional Innovation Strategy
Support Program from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan (Y.K.); by
JSPS KAKENHI Grant Number 26440019 (M.K.K.), Grant
Number 25462242 (Y.K.), and Grant Number 16K10748(Y.K.); and by Takeda Science Foundation (S.O.).
Author Disclosure Statement

No competing financial interests exist.
References

1. Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima
K, Yatomi Y, Yamazaki Y, Narimatsu H, and Ozaki Y:
Involvement of the snake toxin receptor CLEC-2, in
podoplanin-mediated platelet activation, by cancer cells. J
Biol Chem 2007;282:25993–26001.
2. Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A,
Ogasawara S, Matsuura N, Hasegawa Y, Suzuki-Inoue K,
Inoue O, Ozaki Y, and Narimatsu H: Molecular analysis of
the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.
3. Shibahara J, Kashima T, Kikuchi Y, Kunita A, and Fukayama
M: Podoplanin is expressed in subsets of tumors of the central
nervous system. Virchows Arch 2006;448:493–499.
4. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
5. Martin-Villar E, Scholl FG, Gamallo C, Yurrita MM,
Munoz-Guerra M, Cruces J, and Quintanilla M: Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamouscell carcinomas. Int J Cancer 2005;113:899–910.
6. Kato Y, Sasagawa I, Kaneko M, Osawa M, Fujita N, andTsuruo T: Aggrus: A diagnostic marker that distinguishes

161

seminoma from embryonal carcinoma in testicular germ
cell tumors. Oncogene 2004;23:8552–8556.
7. Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, and Osawa
M: Enhanced expression of Aggrus (T1alpha/podoplanin),a platelet-aggregation-inducing factor in lung squamouscell carcinoma. Tumor Biol 2005;26:195–200.
8. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K,
Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H, Mishima K, and Osawa M: Inhibition of tumor cell-induced
platelet aggregation using a novel anti-podoplanin antibody
reacting with its platelet-aggregation-stimulating domain.
Biochem Biophys Res Commun 2006;349:1301–1307.
9. Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A,
Fujita N, Tsuruo T, Nishikawa R, Hirose T, and Matsutani M:
Podoplanin expression in primary central nervous system germ
cell tumors: A useful histological marker for the diagnosis
of germinoma. Acta Neuropathol (Berl) 2006;111:563–568.
10. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T, andMatsutani M: Increased expression of podoplanin in malignant
astrocytic tumors as a novel molecular marker of malignant
progression. Acta Neuropathol (Berl) 2006;111:483–488.
11. Abe S, Morita Y, Kaneko MK, Hanibuchi M, Tsujimoto Y,
Goto H, Kakiuchi S, Aono Y, Huang J, Sato S, Kishuku M,
Taniguchi Y, Azuma M, Kawazoe K, Sekido Y, Yano S,
Akiyama S, Sone S, Minakuchi K, Kato Y, and Nishioka Y:
A novel targeting therapy of malignant mesothelioma using
anti-podoplanin antibody. J Immunol 2013;190:6239–6249.
12. Kunita A, Kashima TG, Ohazama A, Grigoriadis AE, andFukayama M: Podoplanin is regulated by AP-1 and promotes platelet aggregation and cell migration in osteosarcoma. Am J Pathol 2011;179:1041–1049.
13. Kato Y, Vaidyanathan G, Kaneko MK, Mishima K, Srivastava N, Chandramohan V, Pegram C, Keir ST, Kuan
CT, Bigner DD, and Zalutsky MR: Evaluation of antipodoplanin rat monoclonal antibody NZ-1 for targeting
malignant gliomas. Nucl Med Biol 2010;37:785–794.
14. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P,
Poczewski H, Kalt R, Schaffner G, and Kerjaschki D: Podoplanin, novel 43-kd membrane protein of glomerularepithelial cells, is down-regulated in puromycin nephrosis.
Am J Pathol 1997;151:1141–1152.
15. Schacht V, Dadras SS, Johnson LA, Jackson DG, Hong
YK, and Detmar M: Up-regulation of the lymphatic marker
podoplanin, a mucin-type transmembrane glycoprotein, in
human squamous cell carcinomas and germ cell tumors.
Am J Pathol 2005;166:913–921.
16. Rishi AK, Joyce-Brady M, Fisher J, Dobbs LG, Floros J,
VanderSpek J, Brody JS, and Williams MC: Cloning,
characterization, and development expression of a rat lungalveolar type I cell gene in embryonic endodermal andneural derivatives. Dev Biol 1995;167:294–306.
17. Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M,
and Tsuruo T: Molecular identification of Aggrus/T1alphaas a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem 2003;278:51599–51605.
18. Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK, Kato
Y, and Yamaguchi Y: A platform of C-Type lectin-like receptor CLEC-2 for binding O-glycosylated podoplanin andnonglycosylated rhodocytin. Structure 2014;22:1711–1721.
19. Kaneko MK, Kato Y, Kitano T, and Osawa M: Conservationof a platelet activating domain of Aggrus/podoplanin as aplatelet aggregation-inducing factor. Gene 2006;378:52–57.
20. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R,
Amann G, Kriehuber E, Diem K, Weninger W, Tschachler162

E, Alitalo K, and Kerjaschki D: Angiosarcomas express
mixed endothelial phenotypes of blood and lymphatic
capillaries: Podoplanin as a specific marker for lymphatic
endothelium. Am J Pathol 1999;154:385–394.
21. Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z, Chen
M, Chen CY, Xu B, Lu MM, Zhou D, Sebzda E, Santore MT,
Merianos DJ, Stadtfeld M, Flake AW, Graf T, Skoda R,
Maltzman JS, Koretzky GA, and Kahn ML: Platelets regulate
lymphatic vascular development through CLEC-2-SLP-76
signaling. Blood 2010;116:661–670.
22. Hayatsu N, Ogasawara S, Kaneko MK, Kato Y, and Narimatsu H: Expression of highly sulfated keratan sulfate
synthesized in human glioblastoma cells. Biochem Biophys
Res Commun 2008;368:217–222.
23. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
24. Ogasawara S, Oki H, Kaneko MK, Hozumi Y, Liu X,
Honma R, Fujii Y, Nakamura T, Goto K, Takagi M, andKato Y: Development of monoclonal antibody LpMab-10
recognizing non-glycosylated PLAG1/2 domain including
Thr34 of human podoplanin. Monoclon Antib Immunodiagn Immunother 2015;34:318–326.
25. Kaneko MK, Oki H, Hozumi Y, Liu X, Ogasawara S, Takagi
M, Goto K, and Kato Y: Monoclonal antibody LpMab-9 recognizes O-glycosylated N-terminus of human podoplanin.
Monoclon Antib Immunodiagn Immunother 2015;34:310–317.
26. Shiina S, Ohno M, Ohka F, Kuramitsu S, Yamamichi A, Kato
A, Motomura K, Tanahashi K, Yamamoto T, Watanabe R,
Ito I, Senga T, Hamaguchi M, Wakabayashi T, Kaneko MK,
Kato Y, Chandramohan V, Bigner DD, and Natsume A: CAR
T cells targeting podoplanin reduce orthotopic glioblastomas
in mouse brains. Cancer Immunol Res 2016;4:259–68.
27. Kato Y, Ogasawara S, Oki H, Honma R, Takagi M, Fujii Y,
Nakamura T, Saidoh N, Kanno H, Umetsu M, Kamata S,
Kubo H, Yamada M, Sawa Y, Morita K, Harada H, Suzuki
H, and Kaneko MK: Novel monoclonal antibody LpMab-17
developed by CasMab technology distinguishes human
podoplanin from monkey podoplanin. Monoclon AntibImmunodiagn Immunother 2016;35:109–116.
28. Kaneko MK, Oki H, Ogasawara S, Takagi M, and Kato Y:
Anti-podoplanin monoclonal antibody LpMab-7 detects
metastatic legions of osteosarcoma. Monoclon Antib Immunodiagn Immunother 2015;34:154–161.
29. Kato Y: Specific monoclonal antibodies against IDH1/2
mutations as diagnostic tools for gliomas. Brain Tumor
Pathol 2014;32:3–11.
30. Oki H, Ogasawara S, Kaneko MK, Takagi M, Yamauchi M,
and Kato Y: Characterization of monoclonal antibody
LpMab-3 recognizing sialylated glycopeptide of podoplanin.
Monoclon Antib Immunodiagn Immunother 2015;34:44–50.
31. Oki H, Kaneko MK, Ogasawara S, Tsujimoto Y, Liu X,
Sugawara M, Takakubo Y, Takagi M, and Kato Y: Characterization of a monoclonal antibody LpMab-7 recognizing non-PLAG domain of podoplanin. Monoclon AntibImmunodiagn Immunother 2015;34:174–180.
32. Kato Y, Kunita A, Abe S, Ogasawara S, Fujii Y, Oki H, Fukayama M, Nishioka Y, and Kaneko MK: The chimeric antibody chLpMab-7 targeting human podoplanin suppresses
pulmonary metastasis via ADCC and CDC rather than via its
neutralizing activity. Oncotarget 2015;6:36003–36018.
33. Pastori C, Clivio A, Diomede L, Consonni R, De Mori GM,
Longhi R, Colombo G, and Lopalco L: Two amino acidsubstitutions within the first external loop of CCR5 induce

OGASAWARA ET AL.

human immunodeficiency virus-blocking antibodies in miceand chickens. J Virol 2008;82:4125–4134.
34. Cocco RR, Jarvinen KM, Han N, Beyer K, and Sampson HA:
Mutational analysis of immunoglobulin E-binding epitopes ofbeta-casein and beta-lactoglobulin showed a heterogeneous
pattern of critical amino acids between individual patients andpooled sera. Clin Exp Allergy 2007;37:831–838.
35. Owens GP, Ritchie A, Rossi A, Schaller K, Wemlinger S,
Schumann H, Shearer A, Verkman AS, and Bennett JL:
Mutagenesis of the aquaporin 4 extracellular domains
defines restricted binding patterns of pathogenic neuromyelitis optica IgG. J Biol Chem 2015;290:12123–12134.
36. Ogasawara S, Kaneko MK, Price JE, and Kato Y:
Characterization of anti-podoplanin monoclonal antibodies: Critical epitopes for neutralizing the interaction
between podoplanin and CLEC-2. Hybridoma 2008;27:
259–267.
37. Fujii Y, Kaneko M, Neyazaki M, Nogi T, Kato Y, and Takagi
J: PA tag: A versatile protein tagging system using a superhigh affinity antibody against a dodecapeptide derived fromhuman podoplanin. Protein Expr Purif 2014;95:240–247.
38. Kaneko MK, Kunita A, Abe S, Tsujimoto Y, Fukayama M,
Goto K, Sawa Y, Nishioka Y, and Kato Y: Chimeric antipodoplanin antibody suppresses tumor metastasis throughneutralization and antibody-dependent cellular cytotoxicity.
Cancer Sci 2012;103:1913–1919.
39. Marks A, Sutherland DR, Bailey D, Iglesias J, Law J, Lei M,
Yeger H, Banerjee D, and Baumal R: Characterization anddistribution of an oncofetal antigen (M2A antigen) expressedon testicular germ cell tumours. Br J Cancer 1999;80:569–
578.
40. Zimmer G, Oeffner F, Von Messling V, Tschernig T,
Groness HJ, Klenk HD, and Herrler G: Cloning and characterization of gp36, a human mucin-type glycoprotein
preferentially expressed in vascular endothelium. Biochem J
1999;341:277–284.
41. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Kaneko MK,
Takagi M, and Kato Y: Development of a sensitive monoclonal antibody PMab-2 against rat podoplanin. Monoclon
Antib Immunodiagn Immunother 2015;34:396–403.
42. Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura T,
Kaneko MK, Takagi M, and Kato Y: Establishment of a novel
monoclonal antibody PMab-32 against rabbit podoplanin.
Monoclon Antib Immunodiagn Immunother 2016;35:41–47.
43. Honma R, Fujii Y, Ogasawara S, Oki H, Konnai S, Kagawa
Y, Takagi M, Kaneko MK, and Kato Y: Critical epitope ofanti-rabbit podoplanin monoclonal antibodies for immunohistochemical analysis monoclon. Antib Immunodiagn
Immunother 2016;35:65–72.

Address correspondence to:
Dr. Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: January 26, 2016
Accepted: March 2, 2016

This article has been cited by:
1. Honma Ryusuke, Ogasawara Satoshi, Kaneko Mika K., Fujii Yuki, Oki Hiroharu, Nakamura Takuro, Takagi Michiaki, Konnai
Satoru, Kato Yukinari. PMab-44 Detects Bovine Podoplanin in Immunohistochemistry. Monoclonal Antibodies in Immunodiagnosis
and Immunotherapy, ahead of print. [Abstract] [Full Text HTML] [Full Text PDF] [Full Text PDF with Links]