Characterization of a monoclonal antibody LpMab-7 recognizing
non-PLAG domain of podoplanin
Hiroharu Oki1,2,3, Mika K. Kaneko1,3, Satoshi Ogasawara1,3, Yuta Tsujimoto1, Xing Liu1,2, Masato Sugawara2,
Yuya Takakubo2, Michiaki Takagi2, Yukinari Kato1
1

Department 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
Corresponding author: Yukinari Kato
Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku,
Sendai, Miyagi 980-8575, Japan
E-mail: yukinari-k@bea.hi-ho.ne.jp; or, yukinarikato@med.tohoku.ac.jp
TEL/FAX: +81-22-717-8207
3
Theses authors contributed equally to this work.
Key words: monoclonal antibody, podoplanin, CasMab, epitope, PLAG domain

Podoplanin (PDPN/Aggrus/T1α), a platelet aggregation-inducing type Ⅰ transmembrane sialoglycoprotein, isinvolved in tumor invasion and metastasis. Furthermore, podoplanin expression was reported to be involved inpoor prognosis of several cancers. Although many anti-podoplanin mAbs such as NZ-1 and D2-40 have been
established, those epitopes are limited to platelet aggregation-stimulating (PLAG) domain of podoplanin. In
this study, we developed and characterized a novel anti-podoplanin mAb, LpMab-7, which is more sensitive
than NZ-1 in immunohistochemistry. We identified the minimum epitope of LpMab-7 as Arg79–Leu83 ofhuman podoplanin, which is different from PLAG domain, using ELISA, Western-blot, and flow cytometry.
Because LpMab-7 has high sensitivity against podoplanin, LpMab-7 is expected to be useful for moleculartargeting therapy for podoplanin-expressing cancers.

Introduction

Many anti-podoplanin mAbs have been produced,
and those mAbs are useful for detecting podoplanin,
which functions physiologically in normal tissues.
Almost all anti-podoplanin mAbs react with platelet
aggregation-inducing (PLAG) domain of human
podoplanin.(7, 26-30) Matsui et al. previously reported thatrabbit polyclonal antibodies produced by immunizing
recombinant rat podoplanin also recognize PLAG
domains, which were shown to be immunodominantantigenic sites.(31) Recently, we established the platformto produce cancer-specific mAbs (CasMabs).(32) Using
CasMab methods, we could produce several
anti-podoplanin mAbs against a non-PLAG domain. In
this study, we characterized a novel anti-podoplaninmAb, LpMab-7, which possesses high sensitivity
against podoplanin.

Podoplanin is a platelet aggregation-inducing type Ⅰ
transmembrane sialoglycoprotein, which is involved intumor invasion and metastasis.(1, 2) Expression of
podoplanin has been reported in many tumors including
malignant brain tumors, mesotheliomas, lung cancer,
esophageal cancer, testicular tumors, bladder cancer,
and osteosarcoma.(1, 3-14) Until now, several
physiological functions of podoplanin have beenreported. The podoplanin-CLEC-2 interaction isimportant for platelet aggregation and embryonicblood-lymphatic vascular separation.(1, 2, 15-19)
Podoplanin is also very useful for detecting lymphatic
endothelial cells.(20-22) The development of ectopic
lymphoid follicles is dependent on Th17-expressing
podoplanin.(23) Local sphingosine-1-phosphate release
after podoplanin-CLEC-2-mediated platelet activationis critical for high endothelial venule integrity during
immune responses.(24) Furthermore, the activation of
CLEC-2 by podoplanin rearranges the actin
cytoskeleton in dendritic cells to promote efficientmotility along stromal surfaces.(25)Materials and Methods
Cell lines and tissues
Chinese hamster ovary (CHO)-K1, SaOS2, and U-2
OS cell lines were obtained from the American Type
Culture Collection (ATCC, Manassas, VA). OST cell1line was obtained from the RIKEN BioResource Center(Ibaraki, Japan).(33) HuO9 cell line was obtained fromthe Japanese Cancer Research Resources Bank (Tokyo,
Japan).(33) NOS-1 cell line was donated by Dr. Teiichi
Motoyama.(34) CHO-K1 cells were cultured in RPMI
1640 medium (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan) supplemented with 10% heat-inactivatedfetal bovine serum (FBS; Life Technologies Corp.,
Carlsbad, CA), 2 mM L-glutamine (Life Technologies
Corp.), 100 units/ml of penicillin, and 100 μg/ml of
streptomycin (Life Technologies Corp.) at 37°C in ahumidified atmosphere of 5% CO2 and 95% air. SaOS2,
HuO9, U-2 OS, OST, and NOS-1 cells were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM; Wako
Pure Chemical Industries Ltd.) supplemented with 10%
heat-inactivated FBS, 2 mM L-glutamine, 100 units/mlof penicillin, and 100 μg/ml of streptomycin. This
study examined osteosarcoma patients who underwent
surgery at Yamagata University Hospital.(35) The ethicalcommittee of the Yamagata University Faculty of
Medicine approved our study. Informed consent for
obtaining samples and for subsequent data analyses was
obtained from each patient or the patient’s guardian.

specimens. Briefly, 4-μm-thick histologic sections weredeparaffinized in xylene and rehydrated. Then, they
were autoclaved in citrate buffer (pH 6.0; Dako) for 20
min. Sections were incubated with 5 μg/ml of primary
antibodies overnight at 4 °C followed by treatment withan LSAB+ kit or Envision+ kit (Dako). Color wasdevelopedusing
3,
3-diaminobenzidine
tetrahydrochloride (DAB; Dako) for 10 min, and thenthe sections were counterstained with hematoxylin(Wako Pure Chemical Industries Ltd.).
Production of podoplanin mutants
The amplified human podoplanin cDNA was
subcloned into a pcDNA3 vector (Life Technologies
Corp.) and a FLAG epitope tag was added at the
C-terminus. Substitution of amino acids to alanine inpodoplanin was performed using a QuikChangeLightning site-directed mutagenesis kit (Agilent
Technologies Inc., Santa Clara, CA).(32, 37) CHO-K1
cells were transfected with the plasmids using a Gene
Pulser Xcell electroporation system (Bio-Rad
Laboratories Inc., Philadelphia, PA).
Enzyme-linked immunosorbent assay (ELISA)
Podoplanin peptides were immobilized on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher
Scientific Inc.) at 1 μg/ml for 30 min.(16) After blocking
with SuperBlock T20 (PBS) Blocking Buffer, the plates
were incubated with culture supernatant or purified
mAbs (1 μg/ml) followed by 1:1,000 diluted
peroxidase-conjugated anti-mouse IgG (Dako). The
enzymatic reaction was conducted with a 1-Step Ultra
TMB-ELISA (Thermo Fisher Scientific Inc.), and was
stopped using 1M H2SO4. The optical density wasmeasured at 450 nm using an iMark microplate reader(Bio-Rad Laboratories Inc.). These reactions were
performed with a volume of 50 μl at 37°C.

Flow cytometry
Cell lines were harvested by brief exposure to 0.25%
Trypsin/1 mM EDTA (Wako Pure Chemical Industries
Ltd.).(16) After washing with phosphate buffered saline(PBS), the cells were treated with primary antibodies (1μg/ml) for 30 min at 4°C, followed by treatment with
Alexa Fluor 488 conjugated anti-mouse IgG or anti-rat
IgG (Cell Signaling Technology, Inc., Danvers, MA).
Fluorescence data were collected using a Cell Analyzer
EC800 (Sony Corp., Tokyo, Japan).
Western-blot analyses
Cell lysates (10 μg) were boiled in SDS sample
buffer (Nacalai Tesque, Inc., Kyoto, Japan).(36) The
proteins
were
electrophoresedon
5–20%
polyacrylamide gels (Wako Pure Chemical Industries
Ltd.) and were transferred onto a PVDF membrane(EMD Millipore Corp., Billerica, MA). After blocking
with SuperBlock T20 (PBS) Blocking Buffer (Thermo
Fisher Scientific Inc., Waltham, MA), the membrane
was incubated with primary antibodies (1 μg/ml), thenwith peroxidase-conjugated secondary antibodies(Dako, Glostrup, Denmark; 1/1,000 diluted), anddeveloped with the ECL-plus reagent (Thermo Fisher
Scientific Inc.) using a Sayaca-Imager (DRC Co. Ltd.,Tokyo, Japan).

Results
Characterization of a novel anti-podoplanin
monoclonal antibody (mAb), LpMab-7
We recently immunized mice with human
podoplanin (PDPN)-expressing LN229 glioma cells(LN229/PDPN), which possess cancer-type glycan
patterns including highly sulfated polylactosamine andaberrant sialylation, and developed a novelanti-podoplanin mAb, LpMab-7 (IgG1, kappa).(32) First,
podoplanin expression of 5 osteosarcoma cell lines(SaOS2, HuO9, U-2 OS, OST, and NOS-1) wasinvestigated by flow cytometry and Western-blot
analysis. An anti-podoplanin mAb NZ-1 (rat IgG2a,
lambda) was used for positive control of detecting
human podoplanin.(7) As shown in Fig. 1a and 2b, both

Immunohistochemical analyses
Podoplanin protein expression was detected
immunohistochemically in paraffin-embedded tumor2

NZ-1 and LpMab-7 recognized podoplanin of U-2 OS.
LpMab-7 detected 35-kDa band, which was notdetected by NZ-1 (Fig. 1b). This 35-kDa band was also
detected in CHO/PDPN or LN229/PDPN in ourprevious reports.(32) Immunohistochemical analysis
showed that LpMab-7 detected podoplanin moresensitively
than
NZ-1
(Fig.
1c).
The
membranous/cytosolic staining pattern was observed.
These results indicated that a novel anti-podoplaninmAb, LpMab-7 is much more useful inimmunohistochemistry than previously establishedanti-podoplanin mAbs.

by its neutralizing activity, indicating that NZ-1 is acandidate of anti-metastatic neutralizing mAb.(7, 16, 36)
Furthermore, NZ-1 was highly internalized into glioma
cell lines and also accumulated efficiently into tumors
in vivo.(14) NZ-1 and its rat-human chimericanti-podoplaninantibody(NZ-8)
possess
antibody-dependent cellular cytotoxicity (ADCC) andcomplement-dependent cytotoxicity (CDC) against
podoplanin-expressing glioblastoma or malignant
mesothelioma cell lines.(10, 36) Recently, we havedeveloped the novel platform, the CasMab method, toproduce mAbs against membranous glycoproteins.(32)
Using the CasMab method, we can obtain not only
cancer-specificmAbs(CasMabs)but
also
non-CasMabs. Intriguingly, non-CasMabs also include
the glycan within those epitopes or detects specificconformation of membranous glycoproteins expressedin cancers.(32) LpMab-7 recognized the usual 40-kDa
band and the additional 35-kDa band, which has notbeen detected by previously reported anti-podoplaninmAbs. This 35-kDa band might be a low glycosylated
form. Taken together, the ability to detect 35-kDa bandmight lead to the high sensitivity of LpMab-7 in
immunohistochemistry against podoplanin-expressing
tumors. In this report, we compared the reactivity of
NZ-1 (rat IgG2a, lambda) and LpMab-7 (mouse IgG1,
kappa). To remove the possibility that the difference ofsensitivity in immunohistochemistry occurred bysecondary antibodies, we also examined the differenceof sensitivity between D2-40 (mouse IgG1, kappa) and
LpMab-7. D2-40 is commercially available and themost used mAb against human podoplanin in pathology
or histology because D2-40 is also known as alymphatic endothelial marker.(26) However, the intensityof LpMab-7 is much higher than that of D2-40 in
immunohistochemistry of osteosarcomas (data notshown), demonstrating that LpMab-7 sensitivity is notdependent on the species and the subclass. Indeed, there
are many immunohistochemical methods suitable foreach mAb; therefore, we should further consider theother methods for comparing those anti-podoplaninmAbs. Although ADCC and CDC activities are very
important for an antibody-based molecular targetingtherapy, we could not investigate those activities
because the subclass of LpMab-7 is IgG1. The
conversion of subclass into human IgG1 or mouse IgG2ais necessary to demonstrate ADCC/CDC activities. In
conclusion, LpMab-7 is expected to be useful formolecular targeting therapy for podoplanin-expressing
cancers.

Epitope mapping by ELISA, flow cytometry, andWestern-blot analysis
To determine the LpMab-7 epitope, we first
performed ELISA using several deletion mutants ofhuman podoplanin (Table 1). NZ-1 reacted with 38-51
amino acids of human podoplanin (hpp38-51) that is
platelet aggregation-inducing (PLAG) domain. In
contrast, LpMab-7 strongly recognized 69-88 aminoacids of human podoplanin (hpp69-88), indicating that
LpMab-7 epitope does not include O-glycan and is notdifferent from PLAG domain. We next performed an
additional ELISA assay using 20 synthetic peptides,
which include a point mutation among 75-94 aminoacids of human podoplanin (Table 2). LpMab-7 did notreact with R79A, I80A, E81A, D82A, and L83A,
indicating that the critical amino acid sequence for
LpMab-7 is RIEDL. To confirm this result, we
performed flow cytometry and Western-blot analysis.
Flow cytometric analysis revealed that LpMab-7 did
not react with I80A, E81A, and L83A, and very weakly
recognized R79A and D82A, indicating that Ilu80,
Glu81, and Leu83 are much more important for
LpMab-7 (Fig. 2a). Western-blot analysis also showedthat LpMab-7 reaction was lost in point mutations of
79-83 amino acids (Fig. 2b). Taken together, thepodoplanin epitope of LpMab-7 is Arg79-Leu83, andIlu80, Glu81, and Leu83 are the most important aminoacids.

Discussion
Until now, other groups and we have developed
many anti-podoplanin mAbs. To neutralize platelet
aggregation activity by blocking the association
between podoplanin and CLEC-2, we produced NZ-1
mAb by immunizing rats with synthetic peptides of
podoplanin PLAG domain.(7) NZ-1 possesses very highbinding-affinity, which was clarified by several
methods including BIAcore, ELISA, flow cytometry,and Scatchard analysis.(14, 38) NZ-1 inhibited tumor
cell-induced platelet aggregation and tumor metastasis

Acknowledgements
We thank Takuro Nakamura, Noriko Saidoh, andKanae Yoshida for their excellent technical assistance.

3

This work was supported in part by the Platform for
Drug Discovery, Informatics, and Structural Life
Science (PDIS) from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of
Japan (Y.K.); by the Basic Science and Platform
Technology Program for Innovative Biological
Medicine from MEXT of Japan (Y.K.); by the Regional
Innovation Strategy Support Program from MEXT of
Japan (Y.K.); and by a Grant-in-Aid for Scientific
Research (C) (M.K.K., Y.K.) and a Grant-in-Aid forYoung Scientists (B) (S.O.) from MEXT of Japan.

8.

Author Disclosure Statement
The authors have no financial interests to disclose.

9.

Reference
1.

2.

3.

4.

5.

6.

7.

Kato Y, Fujita N, Kunita A, Sato S, Kaneko
M, Osawa M, Tsuruo T: Molecular
identification of Aggrus/T1alpha as a platelet
aggregation-inducing factor expressed incolorectal
tumors.
J.
Biol.
Chem.
2003;278:51599-51605.
Kaneko MK, Kato Y, Kitano T, Osawa M:
Conservation of a platelet activating domainof Aggrus/podoplanin as a platelet
aggregation-inducing
factor.
Gene.
2006;378:52-57.
Kato Y, Sasagawa I, Kaneko M, Osawa M,
Fujita N, Tsuruo T: Aggrus: A diagnostic
marker that distinguishes seminoma fromembryonal carcinoma in testicular germ celltumors. Oncogene. 2004;23:8552-8556.
Martin-Villar E, Scholl FG, Gamallo C,
Yurrita MM, Munoz-Guerra M, Cruces J,
Quintanilla M: Characterization of human
PA2.26 antigen (T1alpha-2, podoplanin), a
small membrane mucin induced in oral
squamous cell carcinomas. Int. J. Cancer.
2005;113:899-910.
Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo
T, Osawa M: Enhanced expression of Aggrus(T1alpha/podoplanin),aplatelet-aggregation-inducing factor in lungsquamous cell carcinoma. Tumor Biol.
2005;26:195-200.
Yuan P, Temam S, El-Naggar A, Zhou X, Liu
D, Lee J, Mao L: Overexpression of
podoplanin in oral cancer and its association
with poor clinical outcome. Cancer.
2006;107:563-569.
Kato Y, Kaneko MK, Kuno A, Uchiyama N,
Amano K, Chiba Y, Hasegawa Y,
Hirabayashi J, Narimatsu H, Mishima K,

10.

11.

12.

13.

14.

15.

4

Osawa M: Inhibition of tumor cell-induced
plateletaggregation
using
a
novel
anti-podoplanin antibody reacting with its
platelet-aggregation-stimulating
domain.
Biochem.
Biophys.
Res.
Commun.
2006;349:1301-1307.
Mishima K, Kato Y, Kaneko MK, Nakazawa
Y, Kunita A, Fujita N, Tsuruo T, Nishikawa
R, Hirose T, Matsutani M: Podoplanin
expression in primary central nervous systemgerm cell tumors: a useful histologicalmarker for the diagnosis of germinoma. Acta
Neuropathol. (Berl). 2006;111:563-568.
Mishima K, Kato Y, Kaneko MK, Nishikawa
R, Hirose T, Matsutani M: Increased
expression of podoplanin in malignant
astrocytic tumors as a novel molecularmarker of malignant progression. Acta
Neuropathol. (Berl). 2006;111:483-488.
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,
Nishioka Y: A novel targeting therapy ofmalignantmesothelioma
usinganti-podoplanin antibody. J. Immunol.
2013;190:6239-6249.
Takagi S, Oh-hara T, Sato S, Gong B,
Takami M, Fujita N: Expression of
Aggrus/podoplanin in bladder cancer and its
role in pulmonary metastasis. Int. J. Cancer.
2014;134:2605-2614.
Kunita A, Kashima TG, Ohazama A,
Grigoriadis AE, Fukayama M: Podoplanin isregulated by AP-1 and promotes platelet
aggregationandcell
migrationinosteosarcoma.
Am.
J.
Pathol.
2011;179:1041-1049.
Chandramohan V, Bao X, Kato Kaneko M,
Kato Y, Keir ST, Szafranski SE, Kuan CT,
Pastan IH, Bigner DD: Recombinantanti-podoplanin (NZ-1) immunotoxin for thetreatment of malignant brain tumors. Int. J.
Cancer. 2013;132:2339-2348.
Kato Y, Vaidyanathan G, Kaneko MK,
Mishima K, Srivastava N, Chandramohan V,
Pegram C, Keir ST, Kuan CT, Bigner DD,
Zalutsky MR: Evaluation of anti-podoplanin
rat monoclonal antibody NZ-1 for targeting
malignant gliomas. Nucl. Med. Biol.
2010;37:785-794.
Kaneko M, Kato Y, Kunita A, Fujita N,
Tsuruo T, Osawa M: Functional sialylated

16.

17.

18.

19.

20.

21.

22.

O-glycan to platelet aggregation on Aggrus(T1alpha/podoplanin) molecules expressed in
Chinese Hamster Ovary cells. J. Biol. Chem.
2004;279:38838-38843.
Kato Y, Kaneko MK, Kunita A, Ito H,
Kameyama A, Ogasawara S, Matsuura N,
Hasegawa Y, Suzuki-Inoue K, Inoue O,
Ozaki Y, Narimatsu H: Molecular analysis of
the pathophysiological binding of the platelet
aggregation-inducing factor podoplanin to theC-type lectin-like receptor CLEC-2. Cancer
Sci. 2008;99:54-61.
Kaneko MK, Kato Y, Kameyama A, Ito H,
Kuno A, Hirabayashi J, Kubota T, Amano K,
Chiba Y, Hasegawa Y, Sasagawa I, Mishima
K, Narimatsu H: Functional glycosylation ofhuman podoplanin: glycan structure ofplatelet aggregation-inducing factor. FEBS
Lett. 2007;581:331-336.
Suzuki-Inoue K, Kato Y, Inoue O, Kaneko
MK, Mishima K, Yatomi Y, Yamazaki Y,
Narimatsu H, Ozaki Y: Involvement of the
snake
toxin
receptor
CLEC-2,inpodoplanin-mediated platelet activation, by
cancercells.
J.
Biol.
Chem.
2007;282:25993-26001.
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, Kahn
ML: Platelets regulate lymphatic vasculardevelopment
through
CLEC-2-SLP-76
signaling. Blood. 2010;116:661-670.
Breiteneder-Geleff S, Soleiman A, Kowalski
H, Horvat R, Amann G, Kriehuber E, Diem
K, Weninger W, Tschachler E, Alitalo K,
Kerjaschki D: Angiosarcomas express mixed
endothelial phenotypes of blood andlymphatic capillaries: podoplanin as a
specific marker for lymphatic endothelium.
Am. J. Pathol. 1999;154:385-394.
Kaji C, Tsujimoto Y, Kato Kaneko M, Kato
Y,
Sawa
Y:
Immunohistochemical
Examination of Novel Rat Monoclonal
Antibodies against Mouse and Human
Podoplanin. Acta histochem. cytochem.
2012;45:227-237.
Maruyama Y, Maruyama K, Kato Y, Kajiya
K, Moritoh S, Yamamoto K, Matsumoto Y,
Sawane M, Kerjaschki D, Nakazawa T,
Kinoshita S: The effect of podoplanininhibition on lymphangiogenesis underpathological conditions. Invest Ophthalmol

23.

24.

25.

26.

27.

28.

29.

30.

5

Vis Sci. 2014;55:4813-4822.
Peters A, Pitcher LA, Sullivan JM,
Mitsdoerffer M, Acton SE, Franz B,
Wucherpfennig K, Turley S, Carroll MC,
Sobel RA, Bettelli E, Kuchroo VK: Th17
cells induce ectopic lymphoid follicles incentral nervous system tissue inflammation.
Immunity. 2011;35:986-996.
Herzog BH, Fu J, Wilson SJ, Hess PR, Sen A,
McDaniel JM, Pan Y, Sheng M, Yago T,
Silasi-Mansat R, McGee S, May F,
Nieswandt B, Morris AJ, Lupu F, Coughlin
SR, McEver RP, Chen H, Kahn ML, Xia L:
Podoplanin maintains high endothelial venule
integrity by interacting with platelet CLEC-2.
Nature. 2013;502:105-109.
Acton SE, Astarita JL, Malhotra D,
Lukacs-Kornek V, Franz B, Hess PR, Jakus Z,
Kuligowski M, Fletcher AL, Elpek KG,
Bellemare-Pelletier A, Sceats L, Reynoso ED,
Gonzalez SF, Graham DB, Chang J, Peters A,
Woodruff M, Kim YA, Swat W, Morita T,
Kuchroo V, Carroll MC, Kahn ML,
Wucherpfennig
KW,
TurleySJ:Podoplanin-rich stromal networks induce
dendritic cell motility via activation of the
C-type lectin receptor CLEC-2. Immunity.
2012;37:276-289.
Ogasawara S, Kaneko MK, Price JE, Kato Y:
Characterizationofanti-podoplanin
monoclonal antibodies: critical epitopes forneutralizingtheinteraction
betweenpodoplanin and CLEC-2. Hybridoma.
2008;27:259-267.
Takagi S, Sato S, Oh-hara T, Takami M,
Koike S, Mishima Y, Hatake K, Fujita N:
Platelets promote tumor growth andmetastasis via direct interaction between
Aggrus/podoplanin and CLEC-2. PLoS One.
2013;8:e73609.
Nakazawa Y, Takagi S, Sato S, Oh-hara T,
Koike S, Takami M, Arai H, Fujita N:
Prevention of hematogenous metastasis byneutralizing mice and its chimericanti-Aggrus/podoplanin antibodies. Cancer
Sci. 2011;102:2051-2057.
Marks A, Sutherland DR, Bailey D, Iglesias J,
Law J, Lei M, Yeger H, Banerjee D, Baumal
R: Characterization and distribution of an
oncofetal antigen (M2A antigen) expressedon testicular germ cell tumours. Br. J. Cancer.
1999;80:569-578.
Kono T, Shimoda M, Takahashi M,
Matsumoto K, Yoshimoto T, Mizutani M,

31.

32.

33.

34.

35.

36.

37.

38.

Tabata C, Okoshi K, Wada H, Kubo H:
Immunohistochemical detection of thelymphatic marker podoplanin in diverse typesof human cancer cells using a novel antibody.
Int. J. Oncol. 2007;31:501-508.
Matsui K, Breiteneder-Geleff S, Kerjaschki
D: Epitope-specific antibodies to the 43-kD
glomerular membrane protein podoplanincause proteinuria and rapid flattening of
podocytes.
J
Am
Soc
Nephrol.
1998;9:2013-2026.
Kato Y, Kaneko MK: A cancer-specificmonoclonal
antibody
recognizes
theaberrantly glycosylated podoplanin. Sci Rep.
2014;4:5924.
Ogose A, Motoyama T, Hotta T, Watanabe H,
Takahashi HE: Bone formation in vitro and innude mice by human osteosarcoma cells.
Virchows Arch. 1995;426:117-125.
Hotta T, Motoyama T, Watanabe H: Threehuman osteosarcoma cell lines exhibiting
different phenotypic expressions. Acta Pathol
Jpn. 1992;42:595-603.
Liu X, Kato Y, Kaneko MK, Sugawara M,
Ogasawara S, Tsujimoto Y, Naganuma Y,
Yamakawa M, Tsuchiya T, Takagi M:
Isocitrate dehydrogenase 2 mutation is afrequent event in osteosarcoma detected by amulti-specificmonoclonal
antibody
MsMab-1. Cancer Med. 2013;2:803-814.
Kaneko MK, Kunita A, Abe S, Tsujimoto Y,
Fukayama M, Goto K, Sawa Y, Nishioka Y,
Kato Y: Chimeric anti-podoplanin antibody
suppresses
tumor
metastasis
throughneutralization
andantibody-dependent
cellularcytotoxicity.
CancerSci.
2012;103:1913-1919.
Kaneko MK, Tian W, Takano S, Suzuki H,
Sawa Y, Hozumi Y, Goto K, Yamazaki K,
Kitanaka C, Kato Y: Establishment of a novel
monoclonal antibody SMab-1 specific for
IDH1-R132S mutation. Biochem. Biophys.
Res. Commun. 2011;406:608-613.
Fujii Y, Kaneko M, Neyazaki M, Nogi T,
Kato Y, Takagi J: PA tag: a versatile protein
tagging system using a super high affinity
antibody against a dodecapeptide derived
from human podoplanin. Protein Expr Purif
2014;95:240-247.

6

Fig. 2. Epitope mapping of LpMab-7 by ELISA,
flow cytometry, and Western-blot analysis. (a) Point
mutants of human podoplanin were treated with NZ-1
and LpMab-7 (1 μg/ml) for 30 min at 4°C, followed bytreatment with Alexa Fluor 488 conjugated anti-rat IgG
and anti-mouse IgG, respectively. Fluorescence data
were collected using a Cell Analyzer EC800. Red line,
NZ-1 or LpMab-7; black line, negative control. (b)Total cell lysate of point mutants of human podoplanin
were electrophoresed on 5-20% polyacrylamide gels,and transferred onto a PVDF membrane. After blocking,the membrane was incubated with 1μg/ml of primary
antibodies and then with peroxidase-conjugatedsecondary antibodies; the membrane was detected using
a Sayaca-Imager.

Fig. 1. Characterization of a novel anti-podoplaninmAb, LpMab-7. (a) Flow cytometric analysis by NZ-1
and LpMab-7 against osteosarcoma cell lines. Five celllines were treated with NZ-1 and LpMab-7 (1 μg/ml)
for 30 min at 4°C, followed by treatment with Alexa
Fluor 488 conjugated anti-rat IgG and anti-mouse IgG,
respectively. Fluorescence data were collected using a
Cell Analyzer EC800. Red line, NZ-1 or LpMab-7;
black line, negative control. (b) Western-blot analysis
by NZ-1 and LpMab-7 against osteosarcoma cell lines.
AC-15 (α-β-actin), RcMab-1 (α-IDH1), and RMab-3(α-IDH1) were used as positive control. Total celllysate of osteosarcoma cell lines (SaOS2, HuO9, U-2
OS, OST, NOS-1) were electrophoresed on 5-20%
polyacrylamide gels, and transferred onto a PVDF
membrane. After blocking, the membrane wasincubated with 1μg/ml of primary antibodies and thenwith peroxidase-conjugated secondary antibodies; themembrane was detected using a Sayaca-Imager. (c)Immunohistochemical analysis by NZ-1 and LpMab-7
against osteosarcoma tissues. Serial sections wereincubated with 5 μg/ml of NZ-1 and LpMab-7,
followed by biotin-labeled anti-rat IgG and anti-mouse
IgG, respectively. Then, LSAB+ kit was used, andcolor was developed using DAB and counterstainedwith hematoxylin. Original magnification: ×200. Scalebar: 100 µm.

7