MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 35, Number 2, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2015.0077

Novel Monoclonal Antibody LpMab-17 Developed
by CasMab Technology Distinguishes Human
Podoplanin from Monkey Podoplanin
Yukinari Kato,1 Satoshi Ogasawara,1 Hiroharu Oki,1,2 Ryusuke Honma,1,2 Michiaki Takagi,2Yuki Fujii,1 Takuro Nakamura,1 Noriko Saidoh,1 Hazuki Kanno,1,3 Mitsuo Umetsu,3 Satoshi Kamata,4,5
Hiroshi Kubo,4 Mitsuhiro Yamada,6 Yoshihiko Sawa,7 Kei-ichi Morita,8 Hiroyuki Harada,8Hiroyoshi Suzuki,9 and Mika Kato Kaneko1

Podoplanin (PDPN) is a type-I transmembrane sialoglycoprotein, which possesses a platelet aggregationstimulating (PLAG) domain in its N-terminus. Among the three PLAG domains, O-glycan on Thr52 of PLAG3
is critical for the binding with C-type lectin-like receptor-2 (CLEC-2) and is essential for platelet-aggregating
activity of PDPN. Although many anti-PDPN monoclonal antibodies (mAbs) have been established, almost all
mAbs bind to PLAG domains. We recently established CasMab technology to produce mAbs against membranous proteins. Using CasMab technology, we produced a novel anti-PDPN mAb, LpMab-17, which binds to
non-PLAG domains. LpMab-17 clearly detected endogenous PDPN of cancer cells and normal cells in
Western-blot, flow cytometry, and immunohistochemistry. LpMab-17 recognized glycan-deficient PDPN inflow cytometry, indicating that the interaction between LpMab-17 and PDPN is independent of its glycosylation. The minimum epitope of LpMab-17 was identified as Gly77–Asp82 of PDPN using enzyme-linked
immunosorbent assay. Of interest, LpMab-17 did not bind to monkey PDPN, whereas the homology is 94%
between human PDPN and monkey PDPN, indicating that the epitope of LpMab-17 is unique compared withthe other anti-PDPN mAbs. The combination of different epitope-possessing mAbs could be advantageous for
the PDPN-targeting diagnosis or therapy.

IntroductionP

odoplanin (PDPN) is highly expressed not only inmany cancers but also in normal cells such as lymphatic
endothelial cells (LECs) and podocytes.(1,2) PDPN is also
called as ‘‘Aggrus’’ because PDPN possesses a platelet
aggregation-inducing activity, which is associated with cancer metastasis.(3) Another name is ‘‘T1a’’; its expression
is observed in lung type I alveolar cells.(4) PDPN binds toC-type lectin-like receptor-2 (CLEC-2)(5,6) and rearranges
the actin cytoskeleton in dendritic cells to promote efficientmotility along stromal surfaces.(7) The physical elasticity oflymph nodes is maintained by PDPN of stromal fibroblastic

reticular cells.(8) The signaling from CLEC-2 to PDPN controls the contractility of fibroblastic reticular cells and lymph
node microarchitecture.(9) PDPN is known as a specificlymphatic endothelial marker,(10) and PDPN–CLEC-2 interaction is critical for the embryonic blood–lymphatic vascular separation.(11) The development of ectopic lymphoidfollicles is dependent on Th17-expressing PDPN.(12)The expression of PDPN has been reported in many tumors,
including oral cancer, lung cancer, esophageal cancer, malignant brain tumors, mesotheliomas, testicular tumors, and osteosarcoma.(3,13–25) To study these complicated functions of
PDPN, different epitope-possessing anti-PDPN monoclonal
antibodies (mAbs) are necessary. Using conventional methods

1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Orthopaedic Surgery, Faculty of Medicine, Yamagata University, Yamagata, Japan.
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
4
Department of Advanced Preventive Medicine for Infectious Disease, Tohoku University Graduate School of Medicine, Sendai, Japan.
5
Department of Thoracic Surgery, Institute of Development, Aging, and Cancer, Tohoku University Graduate School of Medicine,
Sendai, Japan.
6
Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan.
7
Department of Morphological Biology, Fukuoka Dental College, Fukuoka, Japan.
8Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
9Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
2
3

109

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of antibody production, almost all anti-PDPN mAbs react withplatelet aggregation-stimulating (PLAG) domain of human
PDPN.(18,24,26,27) Rabbit polyclonal antibodies, which were
produced by Matsui et al., also recognized the PLAG domain
because the PLAG domain was shown to be immunodominantantigenic sites.(28) We have further produced many mAbs
against mouse, rat, and rabbit PDPNs.(29–31)

KATO ET AL.

Recently, we have established the CasMab technology to
produce cancer-specific mAbs or antiglycopeptide mAbs.
Using CasMab technology, we established different epitopepossessing anti-PDPN mAbs such as LpMab-2, LpMab-3,
LpMab-7, LpMab-9, and LpMab-10.(32–38) Interestingly,
LpMab-2, LpMab-3, and LpMab-9 detect the site-specific Oglycosylation: LpMab-2 against Thr55/Ser56, LpMab-3

FIG. 1. Characterization of a novel anti-PDPN mAb, LpMab-17. (A) Western-blot analysis by LpMab-17. AC-15 (anti-bactin) was used as internal control. Total cell lysates were electrophoresed on 5%–20% polyacrylamide gels, and transferred
onto a PVDF membrane. After blocking, the membrane was incubated with 1 mg/mL of LpMab-17 and then withperoxidase-conjugated antimouse IgG; the membrane was detected using a Sayaca-Imager. (B, C) Flow cytometric analysis
by LpMab-17. (B) Four cell lines were treated with LpMab-17 (1 mg/mL; red) or control PBS (black) for 30 minutes at 4C,
followed by treatment with antimouse IgG-Oregon green. Fluorescence data were collected using a Cell Analyzer EC800.
(C) HEK-293T and HEK-293T/hPDPN-KO cells were treated with LpMab-17 (1 mg/mL; red) or control PBS (black) for 30
minutes at 4C, followed by treatment with antimouse IgG-Oregon green. Fluorescence data were collected using a Cell
Analyzer EC800. (D) Determination of binding affinity of LpMab-17 using flow cytometry. (E, F) Immunohistochemical
analysis by LpMab-17 against lymphatic endothelial cells (E) and cancer cells (F) of oral squamous cell carcinomas.
Sections were incubated with 1 mg/mL of LpMab-17, followed by Envision+ kit, and color was developed using DAB andcounterstained with hematoxylin. Scale bar: 100 mm. DAB, 3,3-diaminobenzidine tetrahydrochloride; KO, knock out; mAb,
monoclonal antibody; PBS, phosphate-buffered saline; PDPN, podoplanin; PVDF, polyvinylidene fluoride.

LPMAB-17 AGAINST HUMAN PODOPLANIN

against Thr76, and LpMab-9 against Thr25. Here, we developed and characterized a novel anti-PDPN mAb, LpMab17, which possesses a novel epitope of PDPN.
Materials and Methods
Cell lines, animals, and tissues

LN229, HEK-293T, Chinese hamster ovary (CHO)-K1,
glycan-deficient CHO cell lines (Lec1, Lec2, and Lec8),
COS-7, and P3U1 were obtained from the American Type
Culture Collection (ATCC, Manassas, VA). Human LECs
were purchased from Cambrex (Walkersville, MD). The
human glioblastoma cell line, LN319, was donated by
Dr. Kazuhiko Mishima (Saitama Medical University, Saitama, Japan) and Dr. Webster K. Cavenee (Ludwig Institute forCancer Research, San Diego, CA). LN229, CHO-K1, Lec1,
Lec2, and Lec8 were transfected with human PDPN plasmids(LN229/hPDPN, CHO/hPDPN, Lec1/hPDPN, Lec2/hPDPN,
and Lec8/hPDPN) using Lipofectamine 2000 (Thermo Fisher
Scientific, Inc., Waltham, MA) according to the manufacturer’s instructions.(32) HEK-293T/hPDPN-knock out cells(HEK-293T/hPDPN-KO; PDIS-2) were produced by transfecting 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., Philadelphia, PA). CHO-K1, Lec1, Lec2,
Lec8, and P3U1 were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), and LN229, LN319, and
COS-7 were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Inc.), supplemented with 10% heatinactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) at 37C in a humidified atmosphere of 5% CO2 and
95% air. l-Proline (0.04 mg/mL) was added for Lec1, Lec2,and Lec8. LEC was cultured in endothelial cell medium
EGM-2MV supplemented with 5% FBS (Cambrex). Female
BALB/c mice (4 weeks old) were purchased from CLEA

111

Japan (Tokyo, Japan). Animals were housed under pathogenfree conditions. The Animal Care and Use Committee of
Tohoku University approved the animal experiments described herein. This study examined two oral cancer patients
who underwent surgery at Sendai Medical Center. 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.). After several additional
immunizations, a booster injection was given i.p. 2 days before
spleen cells were harvested. The spleen cells were fused with
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 human PDPN
purified from LN229/hPDPN cells.
Enzyme-linked immunosorbent assay

Seven peptides of human PDPN(26) and 20 synthetic peptides, which include a point mutation among 75–94 aminoacids of human PDPN,(34) were synthesized previously.
Peptides 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 in
0.05% Tween20/phosphate-buffered saline (PBS; Nacalai
Tesque, Inc.), the plates were incubated with culture supernatant or purified mAbs (1 mg/mL) followed by 1:1000 diluted peroxidase-conjugated antimouse IgG or antirat IgG(Dako, Agilent Technologies, Inc., Santa Clara, CA). The
enzymatic reaction was conducted with a 1-Step Ultra TMB-

FIG. 2. Flow cytometric analysis by LpMab-17. (A) CHO-K1 and CHO-K1/hPDPN cells were treated with LpMab-17(1 mg/mL; red) or control PBS (black) for 30 minutes at 4C, followed by treatment with antimouse IgG-Oregon green.
Fluorescence data were collected using a Cell Analyzer EC800. (B) Glycan-deficient PDPN-expressing cells (Lec1/hPDPN,
Lec2/hPDPN, Lec8/hPDPN) were analyzed in the same way. CHO, Chinese hamster ovary.

112

ELISA (Thermo Fisher Scientific, Inc.). The optical density
was measured at 655 nm using an iMark microplate reader(Bio-Rad Laboratories, Inc.).
Western-blot analyses

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate(SDS) sample buffer (Nacalai Tesque, Inc.). The proteins
were electrophoresed on 5%–20% polyacrylamide gels
(Wako Pure Chemical Industries Ltd., Osaka, Japan) andwere transferred onto a polyvinylidene fluoride (PVDF)
membrane (EMD Millipore Corp., Billerica, MA). After
blocking with 4% skimmed milk (Nacalai Tesque, Inc.), themembrane was incubated with LpMab-17 or anti-b-actin(clone AC-15; Sigma-Aldrich Corp.) and then withperoxidase-conjugated antimouse IgG (1:1000 diluted; Dako,
Agilent Technologies, Inc.), 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.).(6) After washingwith PBS, the cells were treated with LpMab-17 or LpMab-7(1 mg/mL) for 30 minutes at 4C, followed by treatment with
Oregon Green 488 goat antimouse 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) were resuspended with 100 mL ofserially diluted LpMab-17 (0.0061–50 mg/mL) followed bysecondary antimouse IgG (Thermo Fisher Scientific, Inc.).
Fluorescence data were collected using a cell analyzer(EC800; Sony Corp.). The dissociation constants (KD) wereobtained by fitting the binding isotherms using the built-in
one-site binding models in GraphPad PRISM 6 (GraphPad
Software, Inc., La Jolla, CA).
Immunohistochemical analyses

Four-micrometer-thick histologic sections were deparaffinized in xylene and rehydrated. Without antigen retrieval
procedure, sections were incubated with 1 mg/mL of LpMab17 for 1 hour at room temperature followed by treatment withEnvision+ kit (Dako, Agilent Technologies, Inc.) for 30
minutes. Color was developed using 3,3-diaminobenzidine
tetrahydrochloride (DAB; Dako, Agilent Technologies, Inc.)for 5 minutes, and then the sections were counterstained withhematoxylin (Wako Pure Chemical Industries Ltd.).
Results
Production of a novel anti-PDPN mAb LpMab-17

In this study, we employed the CasMab technology. In
brief, we immunized mice with LN229/hPDPN cells to develop novel anti-PDPN mAbs. Using ELISA, the culture
supernatants were screened for binding to recombinanthuman PDPN purified from LN229/hPDPN cells. After thelimiting dilution, LpMab-17 (mouse IgG1, kappa) was es-

KATO ET AL.

Table 1. Determination of NZ-1 and LpMab-17
Epitopes by Enzyme-Linked Immunosorbent Assay
Mutants
hpp23–36
hpp29–47
hpp38–51
hpp49–68
hpp69–88
hpp89–108
hpp109–128

LpMab-17

LpMab-7

NZ-1

+++
-

+++
-

+++
-

+++, OD450 ‡ 2.0; -, OD450 < 0.1; hpp, human podoplanin
peptide.

tablished. LpMab-17 reacted with LN229/hPDPN, not with
LN229, a PDPN-negative cell in Western-blot analysis(Fig. 1A) and flow cytometry (Fig. 1B). LpMab-17 also
recognized endogenous PDPN, which is expressed in a
glioblastoma cell line LN319 and a LEC in Western-blot
analysis (Fig. 1A) and flow cytometry (Fig. 1B). LN229,
which does not express PDPN, was not detected by LpMab17, indicating that LpMab-17 does not recognize nonrelated
antigens (Fig. 1A, B). The LpMab-17 reaction against HEK293T was lost by knocking out of human PDPN using
CRISPR/Cas (Fig. 1C).
We next performed a kinetic analysis of the interaction of
LpMab-17 with LN319 using flow cytometry. As shown in
Figure 1D, KD was determined to be 6.5 · 10-9 M, indicatingthat LpMab-17 possesses high affinity against human PDPN.
We further investigated whether LpMab-17 is useful for
immunohistochemical analyses. LpMab-17 stained LECs
(Fig. 1E) and oral squamous cell carcinoma cells (Fig. 1F) ina membranous/cytoplasmic staining pattern, demonstratingthat LpMab-17 is very useful in immunohistochemical
analysis.

Table 2. Determination of LpMab-17 Epitopeby Enzyme-Linked Immunosorbent Assay
Mutation

Sequence

LpMab-17 LpMab-7

Wild
type
V75A
T76A
G77A
I78A
R79A
I80A
E81A
D82A
L83A
P84A
T85A
S86A
E87A
S88A
T89A
V90A
H91A
Q93A
E94A

VTGIRIEDLPTSESTVHAQE

++

+++

ATGIRIEDLPTSESTVHAQEVAGIRIEDLPTSESTVHAQEVTAIRIEDLPTSESTVHAQEVTGARIEDLPTSESTVHAQEVTGIAIEDLPTSESTVHAQEVTGIRAEDLPTSESTVHAQEVTGIRIADLPTSESTVHAQEVTGIRIEALPTSESTVHAQEVTGIRIEDAPTSESTVHAQEVTGIRIEDLATSESTVHAQEVTGIRIEDLPASESTVHAQEVTGIRIEDLPTAESTVHAQEVTGIRIEDLPTSASTVHAQEVTGIRIEDLPTSEATVHAQEVTGIRIEDLPTSESAVHAQEVTGIRIEDLPTSESTAHAQEVTGIRIEDLPTSESTVAAQEVTGIRIEDLPTSESTVHAAEVTGIRIEDLPTSESTVHAQA

+++
+++
–
+
+
++
+
++
+
+
++
+
++
+

+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

+++, OD450 ‡ 2.0; ++, 1.0£OD450 < 2.0; +, 0.2£OD450 < 1.0; –,0.1<OD450 < 0.2; -, OD450 < 0.1.

LPMAB-17 AGAINST HUMAN PODOPLANIN

We also performed flow cytometric analysis using LpMab17 against several glycan-deficient PDPN transfectants suchas Lec1/hPDPN (N-glycan deficient), Lec2/hPDPN (sialic
acid deficient), and Lec8/hPDPN (O-glycan deficient).
LpMab-17 reacted with CHO-K1/hPDPN, not with CHO-K1(Fig. 2A). Furthermore, all transfectants of glycan-deficient
PDPN were detected by LpMab-17 (Fig. 2B), showing thatthe epitope of LpMab-17 is independent of glycan.
Epitope mapping using ELISA

To investigate whether LpMab-17 reacts with synthetic
peptides, we performed ELISA using several synthetic pep-113

tides of human PDPN (Table 1). A rat anti-PDPN mAb NZ-1
reacted with PLAG2/3 domain that corresponds to 38–51
amino acids of human PDPN (hpp38-51). In contrast, LpMab17 and LpMab-7 strongly recognized non-PLAG domain that
corresponds to 69–88 amino acids of human PDPN (hpp6988). We further performed ELISA using 20 peptides that
corresponds to 75–94 amino acids of human PDPN to determine the LpMab-17 epitope in detail (Table 2). The results
showed that LpMab-17 reaction was lost in point mutationsof 77–82 amino acids. LpMab-17 did not react with COS-7,
whereas LpMab-7 did (Fig. 3A). Sequence comparison
between human PDPN and monkey PDPN revealed thatthe epitope of LpMab-17 is 77-GIRIED-82, whereas the

FIG. 3. The epitope difference between LpMab-17 and LpMab-7. (A) Flow cytometric analysis. COS-7 cells were treatedwith LpMab-17 (1 mg/mL; red), LpMab-7 (1 mg/mL; blue), or control PBS (black) for 30 minutes at 4C, followed bytreatment with antimouse IgG-Oregon green. Fluorescence data were collected using a Cell Analyzer EC800. (B) The
sequence comparison between human PDPN and monkey PDPN. (C) Comparison of epitopes of anti-PDPN mAbs.

114

corresponding sequence of monkey PDPN is 77-DIHIED-82;
the amino acids consistency is only 67% (Fig. 3B).
Discussion

We recently established the CasMab technology, which
can produce cancer-specific mAb, although one protein incancer cells and normal cells possesses the same amid acidsequence.(32) Using the CasMab technology, we are able totry to develop antibody drugs against many targets, whichwere excluded from antibody-drug candidates. The posttranslational difference such as glycans should be utilized to
produce cancer-specific mAbs.
In this study, we used LN229/hPDPN cells, not purified
recombinant proteins, for immunization to develop novelanti-PDPN mAbs. LpMab-17 recognized not only exogenous
PDPN but also endogenous PDPN, which is expressed in a
glioblastoma cell line LN319 and a LEC in Western-blot
analysis and flow cytometry. LpMab-17 also detected manyother PDPN-expressing cancer cell lines such as glioblastoma, lung squamous cell carcinoma, oral squamous cell
carcinoma, and malignant mesothelioma (data not shown).
LpMab-17 is very useful in immunohistochemical analysis. Importantly, LpMab-17 does not need antigen retrieval
procedure to detect human PDPN in immunohistochemical
analysis, although LpMab-2 and LpMab-3, which were also
produced by CasMab technology, need antigen retrieval
procedure.(32,35) Furthermore, LpMab-9 is not useful for
immunohistochemistry, whereas it is very sensitive in flow
cytometry or Western-blot analysis,(36) indicating that the
CasMab technology is not always helpful to produce mAbs
for immunohistochemical analysis. The antigen retrieval
procedure often causes nonspecific staining; therefore,
LpMab-17 is advantageous for not only reducing costs andsaving time but also showing specific immunohistochemical
staining against human PDPN.
Because human and monkey PDPN shows 94% homology,
almost all anti-PDPN mAbs react with PDPN of a monkey renal
epithelial cell, COS-7. LpMab-17 did not detect mouse, rat,
rabbit, dog, and bovine PDPNs (data not shown); therefore,
LpMab-17 is an absolutely specific antihuman PDPN mAb. As
shown in Figure 3C, LpMab-17 epitope is independent of previously established anti-PDPN mAbs such as LpMab-2,
LpMab-3, LpMab-7, LpMab-9, LpMab-10, and NZ-1. The
epitope of D2-40, the most prevalent anti-PDPN mAb, is also
different from that of LpMab-17.(26) The epitopes of LpMab-17
and LpMab-7 are very similar; however, the critical difference
has been observed in immunohistochemical analysis against one
respiratory disease (data not shown). The combination of different epitope-possessing mAbs could be advantageous for the
PDPN-targeting diagnosis or therapy; therefore, we should
further characterize the activity of LpMab-17 and compare thedifference among anti-PDPN mAbs.
In conclusion, LpMab-17 could be advantageous for investigating the expression and function of PDPN in cancers
and normal tissues. Further different epitope-possessing antiPDPN mAbs should be established as powerful tools foruncovering the function of PDPN in the future.
Acknowledgments

We thank Kanae Yoshida for excellent technical assistance. This work was supported, in part, by the Platform for

KATO ET AL.

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., M.U.); 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.) and grant number 25462242 (Y.K.); andby Takeda Science Foundation (S.O.).
Author Disclosure Statement

No competing financial interests exist.
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M: Enhanced expression of Aggrus (T1alpha/podoplanin),a platelet-aggregation-inducing factor in lung squamouscell carcinoma. Tumor Biol 2005;26:195–200.
17. Yuan P, Temam S, El-Naggar A, Zhou X, Liu D, Lee J, and
Mao L: Overexpression of podoplanin in oral cancer and its
association with poor clinical outcome. Cancer 2006;107:563–569.
18. 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.
19. 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 2006;111:563–
568.
20. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T,
and Matsutani M: Increased expression of podoplanin inmalignant astrocytic tumors as a novel molecular marker ofmalignant progression. Acta Neuropathol 2006;111:483–
488.

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21. 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.
22. 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.
23. Chandramohan V, Bao X, Kato Kaneko M, Kato Y, Keir
ST, Szafranski SE, Kuan CT, Pastan IH, and Bigner DD:
Recombinant anti-podoplanin (NZ-1) immunotoxin for thetreatment of malignant brain tumors. Int J Cancer 2013;
132:2339–2348.
24. 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.
25. Kunita A, Kashima TG, Morishita Y, Fukayama M, Kato
Y, Tsuruo T, and Fujita N: The platelet aggregationinducing factor aggrus/podoplanin promotes pulmonary
metastasis. Am J Pathol 2007;170:1337–1347.
26. 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.
27. Chaipan C, Steffen I, Tsegaye TS, Bertram S, Glowacka I,
Kato Y, Schmokel J, Munch J, Simmons G, GerardySchahn R, and Pohlmann S: Incorporation of podoplanin
into HIV released from HEK-293T cells, but not PBMC, isrequired for efficient binding to the attachment factor
CLEC-2. Retrovirology 2010;7:47.
28. Matsui K, Breiteneder-Geleff S, and Kerjaschki D:
Epitope-specific antibodies to the 43-kD glomerular membrane protein podoplanin cause proteinuria and rapid flattening of podocytes. J Am Soc Nephrol 1998;9:2013–2026.
29. Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, and Sawa
Y: Immunohistochemical examination of novel rat monoclonal antibodies against mouse and human podoplanin.
Acta Histochem Cytochem 2012;45:227–237.
30. 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.
31. Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura
T, Kaneko MK, Takagi M, and Kato Y: Establishment of anovel monoclonal antibody PMab-32 against rabbit podoplanin. Monoclon Antib Immunodiagn Immunother 2016;35:41–47.
32. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
33. 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.
34. 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.

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35. 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.
36. 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 Immunother2015;34:310–317.
37. 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.
38. Kato Y, Kunita A, Abe S, Ogasawara S, Fujii Y, Oki H,
Fukayama M, Nishioka Y, and Kaneko MK: The chimericantibody chLpMab-7 targeting human podoplanin sup-

KATO ET AL.

presses pulmonary metastasis via ADCC and CDC rather
than via its neutralizing activity. Oncotarget 2015;6:36003–
36018.

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: December 9, 2015
Accepted: January 19, 2016