MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 35, Number 1, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2015.0066

Establishment of Novel Monoclonal Antibody
PMab-32 Against Rabbit Podoplanin
Ryusuke Honma,1,2* Yuki Fujii,1* Satoshi Ogasawara,1* Hiroharu Oki,1,2 Xing Liu,1,2
Takuro Nakamura,1 Mika K. Kaneko,1 Michiaki Takagi,2 and Yukinari Kato1

Podoplanin (PDPN) is a type I transmembrane O-glycoprotein, which is known as a specific lymphatic marker.
PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. PDPN is also
expressed in several normal tissues, including podocytes and type I alveolar cells. Although many monoclonal
antibodies (MAbs) against human PDPN (hPDPN), mouse PDPN (mPDPN), and rat PDPN (rPDPN) have been
established, useful antibodies against rabbit PDPN (rabPDPN) have not been developed. In this study, we immunized mice with the recombinant proteins of rabPDPN, and developed a novel anti-rabPDPN MAb, named PMab32. PMab-32 could detect endogenous and exogenous rabPDPN in flow cytometry and Western blot analysis. The
KD of PMab-32 was determined to be 6.2 · 10-8 M by flow cytometry. Immunohistochemical analysis showed that
PMab-32 is useful for detecting podocytes, type I alveolar cells, and lymphatic endothelial cells in normal rabbit
tissues. PMab-32 is expected to be useful for various rabbit experiments.
Introduction

Materials and Methods
Cell lines

P

odoplanin (PDPN/Aggrus/E11 antigen) is a type I
transmembrane O-glycoprotein, which is known as a
specific lymphatic marker.(1–4) PDPN is also expressed inseveral normal tissues, including glomerular epithelial cells(podocytes), type I alveolar cells, osteocytes, and chondrocytes.(1,5) PDPN activates platelet aggregation by binding
to C-type lectin-like receptor-2 (CLEC-2) on platelet.(6,7) The
interaction with CLEC-2 was mainly observed at Glu47 andAsp48 in the platelet aggregation-stimulating (PLAG) domain
and the a2,6-linked sialic acid at Thr52 of human PDPN.(8)PDPN possesses many important functions, which are associated with blood/lymphatic vessel separation,(9) rearrangementof the actin cytoskeleton in dendritic cells,(10) development ofectopic lymphoid follicles,(11) the contractility of fibroblastic
reticular cells and lymph node microarchitecture,(12) and thephysical elasticity of lymph nodes.(13)Expression of PDPN has been reported in many malignant
tumors such as malignant brain tumors,(14–17) oral squamouscell carcinomas,(18) lung cancers,(19) esophageal cancers,(20)malignant mesotheliomas,(21,22) testicular tumors,(23) osteosarcomas,(5,24,25) and chondrosarcomas.(5) PDPN is also associated with tumor invasion and metastasis.(26) Moreover,
PDPN expression in cancer-associated fibroblasts (CAFs) is
involved in poor prognosis of several malignant tumors.(27)In this study, we immunized mice with the recombinant
proteins of rabbit PDPN (rabPDPN) and developed a novelanti-rabPDPN monoclonal antibody (MAb).

Chinese hamster ovary (CHO)-K1 and P3U1 were purchased from American Type Culture Collection (ATCC,
Manassas, VA). VX2 (rabbit skin squamous cell carcinoma)
was obtained from Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer (Tohoku
University, Sendai, Japan). CHO-K1 was previously transfected with human PDPN (hPDPN)-FLAG tag, mouse PDPN
(mPDPN)-FLAG tag, or rat PDPN (rPDPN)-V5-His tag
plasmids (CHO/hPDPN, CHO/mPDPN, CHO/rPDPN) using
Lipofectamine LTX (Thermo Fisher Scientific, Waltham,MA). CHO-K1 cells were transfected with the rabbit PDPN
(rabPDPN)-PA tag plasmid using a Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Philadelphia, PA).
CHO-K1, CHO/rabPDPN, CHO/hPDPN, CHO/mPDPN,
CHO/rPDPN, VX2, and P3U1 were cultured in RPMI 1640
medium including l-glutamine (Nacalai Tesque, Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine
serum (FBS, Thermo Fisher Scientific), 100 U/mL penicillin,
100 mg/mL streptomycin, and 25 mg/mL amphotericin B (Nacalai Tesque) at 37C in a humidified atmosphere of 5% CO2
and 95% air.
Animals

Female BALB/c mice (4 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Japan.
Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, Yamagata, Japan.
*These authors contributed equally to this work.

2

41

42

pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved the animal experiments described herein.
Expression and purification of recombinant rabPDPN

The rabPDPN cDNA (GenBank accession no. XM_0082
75367) was obtained by polymerase chain reaction (PCR)
using total RNA extracted from a normal rabbit lung. The
amplified cDNAs were subcloned into a pCR4-TOPO vector(Thermo Fisher Scientific). Expression construct for
rabPDPN was cloned into XhoI-NotI site of pCAG-Ble PA
tag-N vector (Wako Pure Chemical Industries, Osaka, Japan).
This construct was verified by DNA sequencing. For the
purification of rabPDPN from cell membrane, CHO/rab
PDPN was plated in 100 10-cm culture dishes. Cells weredetached from the plates using phosphate buffered saline(PBS) containing 1 mM EDTA. The pelleted cells were washed using PBS and solubilized by adding 10 mL of PBS containing 1% (w/v) Triton X-100 and protease inhibitor cocktail(Wako Pure Chemical Industries). The solubilized cell lysates
were incubated at 4C for 30 min and further centrifuged 15 min
at 13,000 g at 4C. The pelleted cells were solubilized by 5 mLof the same solution and the same process was repeated twice.
The cleared lysates were mixed with NZ-1-Sepharose (3 mL
bed volume) and incubated at 4C for 2 h under gentle agitation.(28) The beads were then transferred to a column and washed with 60 mL of PBS. The bound protein was eluted with
PBS containing 0.1mg/mL epitope peptide (EGGVAMPGAEDDVV). The elution was conducted at room temperature
in a step-wise manner (3 mL ·10), where dissociation time of
5 min was given for each elution step.

HONMA ET AL.

20% polyacrylamide gels (Wako Pure Chemical Industries) and were transferred onto a PVDF membrane(EMD Millipore, Billerica, MA). After blocking with 4%
skim milk (Nacalai Tesque), the membrane was incubatedwith PMab-32, LpMab-7, PMab-1, PMab-2, and anti-bactin (clone AC-15, Sigma-Aldrich, St. Louis, MO) andthen with peroxidase-conjugated secondary antibodies (1:1000
diluted; Dako, Agilent Technologies), and developed with the
ImmunoStar LD Chemiluminescence Reagent (Wako Pure
Chemical Industries) using a Sayaca-Imager (DRC Co., Tokyo, Japan).
Flow cytometry

Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque). After washing with
0.1% BSA/PBS, the cells were treated with primary MAbs
(1 mg/mL) for 30 min at 4C followed by treatment with
Oregon green-conjugated anti-mouse IgG or anti-rat IgG(1:1000 diluted; Thermo Fisher Scientific). Fluorescencedata were collected using a Cell Analyzer EC800 (Sony,Tokyo, Japan).
Determination of binding affinity by flow cytometry

Binding affinity was determined as described previously.(17,29) Briefly, cells (2 · 105 cells) were resuspended at 100 mLof serially diluted antibodies (600 pg/mL-10 ug/mL), followed
by secondary antibodies (Thermo Fisher Scientific). Fluorescence data were collected using a Cell Analyzer EC800. The
dissociation constants (KD) were obtained by fitting the binding
isotherms using the built-in one-site binding models in GraphPad PRISM 6 (GraphPad Software, La Jolla, CA).

Hybridoma production

BALB/c mice were immunized by intraperitoneal (i.p.)
injection of 100 mg of recombinant rabPDPN together with
Imject Alum (Thermo Fisher Scientific). After several additional immunizations of 50 mg, a booster injection of 50 mg
was given i.p. 2 days before spleen cells were harvested. The
spleen cells were fused with P3U1 cells using GenomONECF (Ishihara Sangyo Kaisha, Osaka, Japan). The hybridomas
were grown in RPMI medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo
Fisher Scientific). The culture supernatants were screenedusing enzyme-linked immunosorbent assay (ELISA) for
binding to the recombinant proteins of rabPDPN.
ELISA

Recombinant proteins of rabPDPN were immobilized on
Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific) at 1 mg/mL for 30 min. After blocking with 1% BSA/PBS,
the plates were incubated with culture supernatant followed by
1:3000 diluted peroxidase-conjugated anti-mouse IgG (Dako,
Agilent Technologies, Santa Clara, CA). The enzymatic reaction was conducted with a 1-Step Ultra TMB-ELISA (Thermo
Fisher Scientific). The optical density was measured at 655 nm
using an iMark microplate reader (Bio-Rad Laboratories).
Western blot analysis

Cell lysates (10 mg) were boiled in SDS sample buffer(Nacalai Tesque). The proteins were electrophoresed on 5–

Determination of binding affinity by surface
plasmon resonanceTo determine the affinity, biotinylated recombinant
rabPDPN was immobilized on the SA sensor chip for
analysis using the BIAcore X100 system (GE Healthcare,
Piscataway, NJ). The running buffer was PBS containing
0.005% Tween-20 (PBST). The PMab-32 was diluted in
PBST (6.25, 12.5, 25, 50, 100 nM), and was passed over thebiosensor chip. The binding was monitored by the singlecycle kinetics method. The binding curves were analyzed by
nonlinear curve-fitting using the bivalent analyte model ofthe BIAcore X100 Evaluation Software (BIAcore). The
affinity constant (KA1) at equilibrium was calculated as
KA1 = kassoc1/kdiss1, and the dissociate constant (KD1) was
determined as 1/KA1.
Immunohistochemical analyses

Four-mm-thick histologic sections were deparaffinized inxylene and rehydrated. Then, they were autoclaved in citratebuffer (pH 6.0; Dako, Agilent Technologies) for 20 min. Sections were incubated with 1 mg/mL of PMab-32 for 1 h at room
temperature, followed by treatment with Envision+ kit for
30 min (Dako, Agilent Technologies). Color was developed
using 3, 3-diaminobenzidine tetrahydrochloride (DAB; Dako,
Agilent Technologies) for 1 min, and then the sections werecounterstained with hematoxylin (Wako Pure Chemical
Industries).

ESTABLISHMENT OF ANTI-RABBIT PDPN MAb
Results
Production of monoclonal antibody against
rabbit podoplanin

We first immunized mice with the recombinant protein of
CHO/rabPDPN, and the ELISA screening was performed.
Among 127 ELISA-positive wells, 55 wells reacted with CHO/
rabPDPN in flow cytometry. Twenty-four of these 55 wells
reacted with VX2 rabbit skin squamous cell carcinoma cell line,

43

which expresses the endogenous rabPDPN in flow cytometry.
Seventeen of 24 wells of IgG1 subclass were cloned by limiting
dilution. Then, PMab-32 (mouse IgG1, kappa) was established.
PMab-32 reacted with CHO/rabPDPN, but not with CHO/
hPDPN, CHO/mPDPN, CHO/rPDPN, and CHO cells in flow
cytometry (Fig. 1). LpMab-7,(24,30,31) PMab-1,(32,33) and PMab2(34) specifically reacted with CHO/hPDPN, CHO/mPDPN, andCHO/rPDPN, respectively. PMab-32 also recognized endogenous rabPDPN, which is expressed in VX2.

FIG. 1. Flow cytometric analysis by anti-PDPN MAbs. CHO/rabPDPN, CHO/hPDPN, CHO/mPDPN, CHO/rPDPN,
CHO, and VX2 were treated with PMab-32, LpMab-7, PMab-1, and PMab-2, followed by treatment with Oregon greenconjugated anti-mouse IgG or anti-rat IgG. Fluorescence data were collected using a Cell Analyzer EC800. Red line, PMab32, LpMab-7, PMab-1, or PMab-2; black line, negative control.

44

HONMA ET AL.

FIG. 3. Determination of binding affinity using flow cytometry and surface plasmon resonance. (A) Flow cytometry. CHO/rabPDPN (2 · 105 cells) were resuspended at
100 mL of serially diluted PMab-32 (600 pg/mL-10 ug/mL).
Fluorescence data were collected using a Cell Analyzer
EC800. KD was obtained by fitting the binding isotherms
using the built-in one-site binding models in Prism software.
(B) Surface plasmon resonance. Biotinylated recombinant
rabPDPN was immobilized on the SA sensor chip. PMab-32
was diluted (6.25, 12.5, 25, 50, and 100 nM) and was passed
over the biosensor chip. The binding was monitored by thesingle cycle kinetics method and analyzed by nonlinear
curve-fitting using the bivalent analyte model of the BIAcore X100 Evaluation Software.
FIG. 2. Western blot analysis by anti-PDPN MAbs. Cell
lysates (10 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 (PMab-32, LpMab-7, PMab-1, PMab-2, and anti-bactin), and then with peroxidase-conjugated secondary antibodies; the membrane was developed with ImmunoStar LD
Chemiluminescence Reagent and detected using a SayacaImager.
PMab-32 detected about a 40 kDa band of CHO/rabPDPN,
but not with CHO/hPDPN, CHO/mPDPN, CHO/rPDPN, andCHO cells in Western blot analysis (Fig. 2). In contrast,
LpMab-7, PMab-1, and PMab-2 detected about 40 kDa bandsof CHO/hPDPN, CHO/mPDPN, and CHO/rPDPN, respectively. PMab-32 also detected endogenous rabPDPN, whichis expressed in VX2 rabbit skin squamous cell carcinoma cellline, indicating that PMab-32 is also useful in Western
blot analyses to detect rabPDPN. The molecular weight ofendogenous rabPDPN is smaller than that of PA-tagged
rabPDPN.
We further performed a kinetic analysis of the interactionof PMab-32 with CHO/rabPDPN using flow cytometry. As
shown in Figure 3A, KD of PMab-32 was determined to be

6.2 · 10-8 M using flow cytometry against CHO/rabPDPN.
BIAcore analysis showed that KD1 of PMab-32 was determined to be 1.1 · 10-7 M (Fig. 3B).
Immunohistochemical analysis using PMab-32

We investigated whether PMab-32 can detect rabPDPN of
normal tissues such as kidney, lung, and small intestine. As
shown in Figure 4, PMab-32 reacted with podocytes of kidney, type I alveolar cells of lung, and lymphatic endothelial
cells of small intestine. Lymphatic endothelial cells of othertissues were also stained by PMab-32 (data not shown). The
antigen retrieval procedure is essential for staining of type I
alveolar cells of lung and lymphatic endothelial cells,
whereas podocytes were stained by PMab-32 without antigen
retrieval (data not shown). These data show that PMab-32 isuseful for immunohistochemistry using paraffin-embedded
tissues; however, antigen retrieval is necessary for using alymphatic endothelial cell marker.
Discussion

PDPN is expressed in human fetal rib and chondrocytes ofthe proliferative and hypertrophic regions of the growthplate.(35) Binding of the cytoplasmic tail of PDPN to the ERM

ESTABLISHMENT OF ANTI-RABBIT PDPN MAb

45

strong background staining in immunohistochemistry (datanot shown). Twenty of 24 clones (83%) were determined tobe IgG class; therefore, amounts and time of immunization
were enough for affinity maturation. Then, we changed themethods in subsequent experiments. Firstly, purified membranous rabPDPN from CHO/rabPDPN cells was used for
immunization. Secondly, we used an endogenous rabPDPNexpressing cell line VX2 for second screening of flow cytometry. The binding affinity of PMab-1, PMab-2, and NZ-1,
which was determined using flow cytometry, was 3.2 · 10-9
M, 5.9 · 10-10 M, and 2.9 · 10-9 M, respectively.(34) Although the binding affinity of PMab-32 (6.2 · 10-8 M using
flow cytometry) was lower than PMab-1, PMab-2, and NZ-1;
PMab-32 is highly sensitive in Western blot, flow cytometry,
and immunohistochemistry. Taken together, PMab-32 reacts
with rabPDPN-expressing normal cells; therefore, it could be
useful for uncovering the PDPN function in the rabbit model.
Author Disclosure Statement

The authors have no financial interests to disclose.
Acknowledgments

FIG. 4. Immunohistochemical analysis by PMab-32. (A)Hematoxylin and eosin staining. Sections of rabbit kidney,lung, and small intestine were incubated with 1 mg/mL of
PMab-32 (B) or isotype control PMab-2 (C), followed by
EnVision+ kit. Color was developed using DAB and counterstained with hematoxylin. Arrow heads, glomerulus; arrows, lymphatic vessels; scale bar, 100 mm.

We thank Noriko Saidoh, Hazuki Kanno, and Kanae
Yoshida for their excellent technical assistance. We also
thank Satoru Konnai, Masahiro Maruyama, and Akiko Kunita for preparing paraffin block of rabbit tissues. This work
was supported in part by the Platform for Drug Discovery,
Informatics, and Structural Life Science (PDIS) from Japan
Agency for Medical Research and development, AMED
(Y.K.); the Basic Science and Platform Technology Programfor Innovative Biological Medicine from AMED (Y.K.); theRegional Innovation Strategy Support Program from the
Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Y.K.); and JSPS KAKENHI (grantnos. 26440019 [M.K.K.] and 25462242 [Y.K.]).
References

proteins may change cytoskeletal organization, which alters
the phenotype of PDPN expression cells. This may contributeto morphological changes in the rudiment cartilages, whichleads to the establishment of primary ossification centers
during the endochondral ossification.
Rabbits are one of the most commonly used animals inorthopedic research such as osteoarthritis, osteochondritis
dissecans, bone formation, bone healing, and cartilage repair.(36–40) Several specific MAbs against osteonectin, collagen types I and II, and osteocalcin were used in thosestudies. Although many MAbs against hPDPN,(17,24,31,41–46)
mPDPN,(32) and rPDPN(34) have been established, useful
antibodies against rabbit PDPN, especially in immunohistochemical analysis, have not been developed. Many clones,
which can be used only in Western blot and flow cytometry,
but not in immunohistochemistry, have been produced before
PMab-32 was established (data not shown). Because we established specific MAbs against hPDPN, mPDPN, andrPDPN by immunizing mice or rats with synthetic peptides of
PLAG domains, we applied the same method in several trials;
synthetic peptides including the PLAG domain of rabPDPN
were used for production of specific MAbs. However, established 24 clones (PMab-8 through PMab-31) showed

1. Wetterwald A, Hoffstetter W, Cecchini MG, Lanske B,
Wagner C, Fleisch H, and Atkinson M: Characterizationand cloning of the E11 antigen, a marker expressed by rat
osteoblasts and osteocytes. Bone 1996;18:125–132.
2. 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.
3. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R,
Amann G, Kriehuber E, Diem K, Weninger W, Tschachler
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.
4. Kaneko MK, Kato Y, Kitano T, and Osawa M: Conservation of a platelet activating domain of Aggrus/podoplanin
as a platelet aggregation-inducing factor. Gene 2006;378:
52–57.
5. Ariizumi T, Ogose A, Kawashima H, Hotta T, Li G, Xu Y,
Umezu H, Sugai M, and Endo N: Expression of podoplaninin human bone and bone tumors: new marker of osteogenic
and chondrogenic bone tumors. Pathol Int 2010;60:193–
202.

46

6. 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.
7. 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.
8. Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK,
Kato Y, and Yamaguchi Y: A platform of C-type lectin-likereceptor CLEC-2 for binding O-glycosylated podoplanin
and nonglycosylated rhodocytin. Structure 2014;22:1711–
1721.
9. 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.
10. 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, and Turley SJ: Podoplanin-richstromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2. Immunity 2012;37:276–289.
11. Peters A, Pitcher LA, Sullivan JM, Mitsdoerffer M, Acton
SE, Franz B, Wucherpfennig K, Turley S, Carroll MC,
Sobel RA, Bettelli E, and Kuchroo VK: Th17 cells induce
ectopic lymphoid follicles in central nervous system tissue
inflammation. Immunity 2011;35:986–996.
12. Astarita JL, Cremasco V, Fu J, Darnell MC, Peck JR,
Nieves-Bonilla JM, Song K, Kondo Y, Woodruff MC,
Gogineni A, Onder L, Ludewig B, Weimer RM, Carroll
MC, Mooney DJ, Xia L, and Turley SJ: The CLEC-2podoplanin axis controls the contractility of fibroblastic
reticular cells and lymph node microarchitecture. Nat Immunol 2015;16:75–84.
13. Acton SE, Farrugia AJ, Astarita JL, Mourao-Sa D, Jenkins
RP, Nye E, Hooper S, van Blijswijk J, Rogers NC, Snelgrove KJ, Rosewell I, Moita LF, Stamp G, Turley SJ, Sahai
E, and Reis e Sousa C: Dendritic cells control fibroblastic
reticular network tension and lymph node expansion. Nature 2014;514:498–502.
14. 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 (Berl) 2006;111:
483–488.
15. 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.
16. 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 anti-

HONMA ET AL.

podoplanin rat monoclonal antibody NZ-1 for targeting
malignant gliomas. Nucl Med Biol 2010;37:785–794.
17. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
18. Ochoa-Alvarez JA, Krishnan H, Pastorino JG, Nevel E,
Kephart D, Lee JJ, Retzbach EP, Shen Y, Fatahzadeh M,
Baredes S, Kalyoussef E, Honma M, Adelson ME, Kaneko
MK, Kato Y, Young MA, Deluca-Rapone L, Shienbaum
AJ, Yin K, Jensen LD, and Goldberg GS: Antibody andlectin target podoplanin to inhibit oral squamous carcinoma
cell migration and viability by distinct mechanisms. Oncotarget 2015;6:9045–9060.
19. 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.
20. Schoppmann SF, Jesch B, Riegler MF, Maroske F,
Schwameis K, Jomrich G, and Birner P: Podoplanin expressing cancer associated fibroblasts are associated withunfavourable prognosis in adenocarcinoma of the esophagus. Clin Exp Metastasis 2013;30:441–446.
21. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
22. 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.
23. Kato Y, Sasagawa I, Kaneko M, Osawa M, Fujita N, andTsuruo T: Aggrus: a diagnostic marker that distinguishes
seminoma from embryonal carcinoma in testicular germ
cell tumors. Oncogene 2004;23:8552–8556.
24. 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.
25. 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.
26. 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.
27. Kawase A, Ishii G, Nagai K, Ito T, Nagano T, Murata Y,
Hishida T, Nishimura M, Yoshida J, Suzuki K, and Ochiai
A: Podoplanin expression by cancer associated fibroblasts
predicts poor prognosis of lung adenocarcinoma. Int J
Cancer 2008;123:1053–1059.
28. Fujii Y, Kaneko M, Neyazaki M, Nogi T, Kato Y, andTakagi 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.
29. Stein RA, Wilkinson JC, Guyer CA, and Staros JV: An
analytical approach to the measurement of equilibrium
binding constants: application to EGF binding to EGF receptors in intact cells measured by flow cytometry. Biochemistry 2001;40:6142–6154.
30. 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-

ESTABLISHMENT OF ANTI-RABBIT PDPN MAb

presses pulmonary metastasis via ADCC and CDC rather
than via its neutralizing activity. Oncotarget 2015;DOI:
10.18632/oncotarget.5339.
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. 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.
33. Maruyama Y, Maruyama K, Kato Y, Kajiya K, Moritoh S,
Yamamoto K, Matsumoto Y, Sawane M, Kerjaschki D,
Nakazawa T, and Kinoshita S: The effect of podoplanininhibition on lymphangiogenesis under pathological conditions. Invest Ophthalmol Vis Sci 2014;55:4813–4822.
34. 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.
35. Smith SM, and Melrose J: Podoplanin is expressed by a
sub-population of human foetal rib and knee joint rudimentchondrocytes. Tissue Cell 2011;43:39–44.
36. Akasaki Y, Matsuda S, Nakayama K, Fukagawa S, MiuraH, and Iwamoto Y: Mevastatin reduces cartilage degradation in rabbit experimental osteoarthritis through inhibitionof synovial inflammation. Osteoarthritis Cartilage 2009;17:
235–243.
37. Stevens MM, Marini RP, Schaefer D, Aronson J, Langer R,
and Shastri VP: In vivo engineering of organs: the bonebioreactor. Proc Natl Acad Sci USA 2005;102:11450–
11455.
38. Murali S, Rai B, Dombrowski C, Lee JL, Lim ZX, Bramono DS, Ling L, Bell T, Hinkley S, Nathan SS, Hui JH,
Wong HK, Nurcombe V, and Cool SM: Affinity-selected
heparan sulfate for bone repair. Biomaterials 2013;34:
5594–5605.
39. Pulkkinen HJ, Tiitu V, Valonen P, Jurvelin JS, Rieppo L,
Toyras J, Silvast TS, Lammi MJ, and Kiviranta I: Repair ofosteochondral defects with recombinant human type II
collagen gel and autologous chondrocytes in rabbit. Osteoarthritis Cartilage 2013;21:481–490.
40. Lyon R, Liu XC, Kubin M, and Schwab J: Does extracorporeal shock wave therapy enhance healing of os-

47

teochondritis dissecans of the rabbit knee?: a pilot study.
Clin Orthop Relat Res 2013;471:1159–1165.
41. 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.
42. Ogasawara S, Oki H, Kaneko MK, Hozumi Y, Liu X,
Honma R, Fujii Y, Nakamura T, Goto K, Takagi M, andKato Y: Development of a monoclonal antibody LpMab-10
recognizing non-glycosylated PLAG1/2 domain including
Thr34 of human podoplanin. Monoclon Antib Immunodiagn Immunother 2015;34:318–326.
43. Kaneko MK, Oki H, Hozumi Y, Liu X, Ogasawara S,
Takagi M, Goto K, and Kato Y: A monoclonal antibody
LpMab-9 recognizes O-glycosylated N-terminus of human
podoplanin. Monoclon Antib Immunodiagn Immunother2015;34:310–317
44. 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.
45. 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.
46. Kaneko M, Kato Y, Kunita A, Fujita N, Tsuruo T, andOsawa M: Functional sialylated O-glycan to platelet aggregation on Aggrus (T1alpha/podoplanin) molecules expressed in Chinese hamster ovary cells. J Biol Chem 2004;
279:38838–38843.

Address correspondence to:
Dr. Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: October 16, 2015
Accepted: November 13, 2015