MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 5, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0030

Establishment of Monoclonal Antibody PMab-202
Against Horse Podoplanin

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Yoshikazu Furusawa,1–3 Shinji Yamada,1 Shunsuke Itai,1,4 Masato Sano,1 Takuro Nakamura,1Miyuki Yanaka,1 Saori Handa,1 Takuya Mizuno,5 Ken Maeda,6 Masato Fukui,3 Hiroyuki Harada,4Mika K. Kaneko,1 and Yukinari Kato1,2

Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in several body tissues, including
podocytes of renal glomerulus, type I alveolar cells of lung, and lymphatic endothelial cells. PDPN activates
platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) presented on platelets. Monoclonal
antibodies (mAbs) against human-, mouse-, rat-, rabbit-, dog-, bovine-, and cat-PDPN have already been
established. However, anti-horse PDPN mAbs have not yet been developed. In this study, we immunized micewith synthetic horse PDPN peptides and developed anti-horse PDPN mAbs. One of the established mAbs,
PMab-202 (IgG1, kappa), was specifically able to detect horse PDPN in Chinese hamster ovary/horse PDPN
(CHO/horPDPN) cells in flow cytometry experiments. PMab-202 was also able to detect endogenous horse
PDPN expressed in and a horse kidney cell line, FHK-Tcl3.1, in flow cytometry and Western blot analyses.
PMab-202 is expected to prove useful in investigating the function of horse PDPN.
Keywords: horse podoplanin, PDPN, PMab-202

nized mice with synthesized peptides of horse PDPN anddeveloped anti-horse PDPN mAbs.

IntroductionP

odoplanin (PDPN)/T1a/Aggrus, a type I transmembrane glycoprotein, induces platelet aggregation bybinding to the C-type lectin-like receptor-2 (CLEC-2)(1–8)
The interaction between PDPN and CLEC-2 facilitates blood/
lymphatic vessel separation.(9) PDPN is also expressed in human
fetal rib and chondrocytes of the proliferative and hypertrophic regions of the growth plate.(10) The expression of human
PDPN has been reported in several malignant tumors, such aslung cancers,(11) esophageal cancers,(12) malignant mesotheliomas,(13,14) testicular tumors,(15) osteosarcomas,(16–18) chondrosarcomas,(17) oral squamous cell carcinomas,(19) andmalignant brain tumors.(20–23) PDPN expression is also associated with malignant progression and cancer metastasis.(5,20,24)
Previously we established monoclonal antibodies (mAbs)
against human,(25) mouse,(26) rat,(27) rabbit,(28) dog,(29) bovine,(30) and cat PDPN.(31) However, anti-horse PDPN mAbs
have not been developed yet. To investigate the expression
and function of horse PDPN, sensitive and specific mAbs
against horse PDPN are necessary. In this study, we immu-

Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 and P3U1 were obtained from the American Type Culture Collection (ATCC,
Manassas, VA). The horse kidney cell line, FHK-Tcl3.1,(32)was established in Yamaguchi University. Horse PDPN
(horPDPN) bearing an N-terminal PA16 tag (PA16-horPDPN)
was inserted into pCAG-Ble vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan). The PA16 tag consistsof 16 amino acids (GLEGGVAMPGAEDDVV).(33) CHO-K1
was transfected with pCAG-Ble/PA16-horPDPN using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific,
Inc., Waltham, MA). Stable transfectants were selected bylimiting dilution and cultivated in a medium containing
0.5 mg/mL of zeocin (InvivoGen, San Diego, CA). CHO-K1,
CHO/horPDPN, and P3U1 were cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) and FHK-Tcl3.1

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan.
ZENOAQ RESOURCE CO., LTD., Koriyama, Fukushima, Japan.
4
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
Laboratories of 5Molecular Diagnostics and Therapeutics and 6Veterinary Microbiology, Joint Faculty of Veterinary Medicine,
Yamaguchi University, Yamaguchi, Japan.
2
3

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was cultured in DMEM medium (Nacalai Tesque, Inc.) supplemented with 10% heat-inactivated fetal bovine serum(FBS; Thermo Fisher Scientific, Inc), 100 units/mL of penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere of 5% CO2 and 95% air.

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Hybridoma production

Female BALB/c mice (4 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under
specific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved the animal experiments described herein. BALB/c mice were immunizedagainst the synthesized horse PDPN peptides (horPP6279)
corresponding to amino acids 62–79 of horse PDPN, whichwere administered intraperitoneally (i.p.) together with Imject Alum (Thermo Fisher Scientific, Inc.). After several
additional immunizations, a booster injection was administered 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 weregrown in RPMI medium supplemented with hypoxanthine,aminopterin, and thymidine for selection (Thermo Fisher
Scientific, Inc.). The culture supernatants were screenedusing enzyme-linked immunosorbent assay (ELISA) for
assessing binding to horPP6279.
Enzyme-linked immunosorbent assay

The culture supernatants were screened using ELISA for
analyzing their binding to horPP6279, which was immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at 1 mg/mL for 30 minutes. After
blocking nonspecific binding sites with SuperBlock T20(phosphate-buffered saline [PBS]) Blocking Buffer (Thermo
Fisher Scientific, Inc.), the plates were incubated with culture
supernatant followed by incubation with 1:1000 diluted
peroxidase-conjugated anti-mouse IgG (Agilent Technologies, Inc., Santa Clara, CA). The enzymatic reaction was
conducted with a 1-Step Ultra TMB-ELISA instrument(Thermo Fisher Scientific, Inc.). Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad
Laboratories, Inc., Berkeley, CA).
Flow cytometry

Cells were harvested after a brief exposure to 0.25%
trypsin/1 mM EDTA (Nacalai Tesque, Inc.). After washingwith 0.1% BSA/PBS, the cells were treated with a primarymAb for 30 minutes at 4C followed by treatment with Alexa
Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA). For the peptide blocking assay, cells were treated with PMab-202 (10 mg/mL) plus
peptides (1 mg/mL) for 30 minutes at 4C, followed by secondary antibodies. Fluorescence data were collected using
SA3800 Cell Analyzers (Sony Corporation, Tokyo, Japan).
Western blot analysis

Cell lysates (10 mg) were boiled in SDS sample buffer(Nacalai Tesque, Inc.). The proteins were electrophoresed on
5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and separated proteins were transferred

FURUSAWA ET AL.

onto a polyvinylidene difluoride membrane (Merck KGaA,
Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.), the membrane was incubated with
1 mg/mL of PMab-202, and subsequently with peroxidaseconjugated anti-mouse IgG (1:1000 diluted; Agilent Technologies, Inc.). The immune reaction was observed with
ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Determination of the binding affinity using flow
cytometry

CHO/horPDPN or FHK-Tcl3.1 (2 · 105 cells) was suspended in 100 mL of serially diluted PMab-202, followed byaddition of Alexa Fluor 488-conjugated anti-mouse IgG(1:200; Cell Signaling Technology, Inc.). Fluorescence data
were collected using EC800 Cell Analyzer (Sony Corporation). The dissociation constants (KD) were obtained by fitting the binding isotherms using built-in one-site binding
models in GraphPad PRISM 6 (GraphPad Software, Inc., La
Jolla, CA).
Results and Discussion

Mice were immunized against horPP6279, corresponding
to amino acids 62–79 of horse PDPN (NCBI Reference Sequence: XM_014737713). ELISA screening indicated positive signals in 21 out of 960 wells (2.2%). Among the 21
wells, 6 wells (29%) tested positive against CHO/horPDPN
in flow cytometry analysis. One clone among the six positive
clones, namely, PMab-202 (IgG1, kappa), was established bylimiting dilution.
PMab-202 recognized horse PDPN in CHO/horPDPN and
did not react with CHO-K1 cells in flow cytometry analysis(Fig. 1A). Furthermore, PMab-202 reacted with the horsekidney cell line FHK-Tcl3.1, indicating the ability of PMab202 to recognize endogenous horse PDPN. PMab-202 reacted with FHK-Tcl3.1 cells in a dose-dependent manner(Fig. 1B). Flow cytometry was used to perform a kinetic
analysis of the interaction of PMab-202 with CHO/horPDPN
or FHK-Tcl3.1 cells. The dissociation constant (KD) of
PMab-202 against CHO/horPDPN and FHK-Tcl3.1 was determined to be 8.7 · 10-9 M and 2.1 · 10-6 M, respectively, indicating a moderate affinity of PMab-202 toward CHO/horPDPN
and a very low affinity of PMab-202 toward FHK-Tcl3.1.
PMab-202 was able to identify horse PDPN as a 35 kDaband in FHK-Tcl3.1 cells in Western blot analysis (Fig. 1C).
This signal was blocked by horPP6279, but not by
horPP2336, which is corresponding to amino acids 23–36 of
horse PDPN (data not shown), indicating that PMab-202
specifically recognized horse PDPN. Furthermore, several
high-molecular bands were observed at weak intensities(Fig. 1C). These bands were also blocked by horPP6279, but
not by horPP2336, suggesting that these bands might be aggregation products of horse PDPN proteins.
In flow cytometry, the PMab-202 reaction with FHKTcl3.1 was blocked by horPP6279, but not by horPP2336(Fig. 2), also indicating that PMab-202 specifically recognized horse PDPN.
Taken together, PMab-202 could prove useful for elucidating the pathophysiological functions of horse PDPN.
Unfortunately, PMab-202 was not found useful forDownloaded by Yukinari Kato from www.liebertpub.com at 10/27/18. For personal use only.

FIG. 1. Detection of horse PDPN by flow cytometry using PMab-202. (A) CHO-K1, CHO/horPDPN, and FHK-Tcl3.1
cells were treated with PMab-202 at a concentration of 1 mg/mL (dashed line) or control (gray shade) for 30 minutes at 4Cfollowed by the addition of secondary antibodies. (B) FHK-Tcl3.1 cells were treated with PMab-202 at a concentration of
1 mg/mL (dotted line), 10 mg/mL (solid line), and 50 mg/mL (dashed line) for 30 minutes at 4C followed by the addition of
secondary antibodies. (C) Cell lysates of FHK-Tcl3.1 (10 mg) were electrophoresed and transferred onto a PVDF membrane.
The membrane was incubated with 1 mg/mL of PMab-202 and subsequently with peroxidase-conjugated anti-mouse IgG.
CHO, Chinese hamster ovary; PDPN, podoplanin; PVDF, polyvinylidene difluoride.

FIG. 2. Blocking assay of PMab-202 by horse PDPN peptide. PMab-202 (10 mg/mL) and horse peptides (1 mg/mL) weremixed (dashed line) and allowed to react with FHK-Tcl3.1 cells for 30 minutes at 4C, followed by addition of secondary
antibodies. Gray shade, control (without a primary mAb).
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immunohistochemical analysis within this study probably
due to a very low affinity of PMab-202 toward endogenous
horse PDPN. Anti-horse PDPN mAbs, which can be utilized
for immunohistochemical analysis, should be developed using other current mAb-producing methods such as Cell-Based
Immunization and Screening (CBIS) method33 in future study.
Acknowledgments

We thank Kayo Hisamatsu and Yoshimi Nakamura forexcellent technical assistance. This research was supported inpart by AMED under grant numbers JP18am0101078 (Y.K.),
JP18am0301010 (Y.K.), and JP18ae0101028 (Y.K.), and by
JSPS KAKENHI grant numbers 17K07299 (M.K.K.) and
16K10748 (Y.K.).
Author Disclosure Statement

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No competing financial interests exist.
References

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
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:

FURUSAWA ET AL.

Platelets regulate lymphatic vascular development through
CLEC-2-SLP-76 signaling. Blood 2010;116:661–670.
10. 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.
11. 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.
12. 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.
13. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
14. 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.
15. 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.
16. 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.
17. 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.
18. 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.
19. 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.
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 (Berl) 2006;111:
483–488.
21. 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.
22. 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.

Downloaded by Yukinari Kato from www.liebertpub.com at 10/27/18. For personal use only.

PMAB202 AGAINST HORSE PDPN4

23. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
24. 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.
25. 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.
26. 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.
27. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Takagi M,
Kaneko MK, and Kato Y: Development of sensitive
monoclonal antibody PMab-2 against rat podoplanin.
Monoclon Antib Immunodiagn Immunother 2015;34:396–
403.
28. 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.
29. Honma R, Kaneko MK, Ogasawara S, Fujii Y, Konnai S,
Takagi M, and Kato Y: Specific detection of dog podoplanin
expressed in renal glomerulus by a novel monoclonal antibody PMab-38 in immunohistochemistry. Monoclon AntibImmunodiagn Immunother 2016;35:212–216.

237

30. Honma R, Ogasawara S, Kaneko M, Fujii Y, Oki H, Nakamura T, Takagi M, Konnai S, and Kato Y: PMab-44 detects
bovine podoplanin in immunohistochemistry. Monoclon
Antib Immunodiagn Immunother 2016;35:186–190.
31. Yamada S, Itai S, Nakamura T, Yanaka M, Saidoh N,
Chang YW, Handa S, Harada H, Kagawa Y, Ichii O,
Konnai S, Kaneko MK, and Kato Y: PMab-52: Specific andsensitive monoclonal antibody against cat podoplanin for
immunohistochemistry. Monoclon Antib Immunodiagn
Immunother 2017;36:224–230.
32. Andoh K, Kai K, Matsumura T, and Maeda K: Further
development of an equine cell line that can be propagatedover 100 times. J Equine Sci 2009;20:11–14.
33. Yamada S, Itai S, Nakamura T, Yanaka M, Kaneko MK,
and Kato Y: Detection of high CD44 expression in oral
cancers using the novel monoclonal antibody, C44Mab-5.
Biochem Biophys Rep 2018;14:64–68.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi
Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: August 20, 2018
Accepted: September 22, 2018