MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 6, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0032

Epitope Mapping of Monoclonal Antibody PMab-233
Against Tasmanian Devil Podoplanin

Downloaded by Yukinari Kato from www.liebertpub.com at 12/12/19. For personal use only.

Masato Sano,1 Mika K. Kaneko,1 and Yukinari Kato1,2

The highly O-glycosylated membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in several
malignant cancers, such as oral cancer, lung cancer, germinal neoplasia, mesothelioma, and brain tumor. The
expression of PDPN is strongly associated with cancer progression and poor prognosis. PDPN possesses threetandem repeats of platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2, and PLAG3) and PLAGlike domain (PLD), and binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediatedplatelet aggregation. We have previously established a novel anti-Tasmanian devil PDPN (tasPDPN) monoclonal
antibody (mAb), PMab-233, which specifically detects tasPDPN using flow cytometry, Western blot, and immunohistochemical analyses. However, the specific binding epitope of tasPDPN for PMab-233 remains to beclarified. Herein, a series of deletion or point mutants of tasPDPN were utilized for investigating the binding
epitopes of PMab-233 using flow cytometry. The findings of this study demonstrated that Asp30, Thr33, and Thr34
of tasPDPN, which are located in PLAG1, are responsible for the binding of PMab-233 to tasPDPN.
Keywords: Tasmanian devil podoplanin, PLAG domain, monoclonal antibody, epitope

Introduction

Podoplanin (PDPN)/Aggrus/T1a is a highly Oglycosylated transmembrane glycoprotein, and is extensively distributed in normal tissues, such as podocytes of
the kidneys, type I alveolar cells of the lungs, lymphatic
endothelial cells of all organs, myofibroblasts, mesothelial
cells, and central nervous system.(1–4) Many physiological
functions of PDPN have been reported to play crucial roles inblood/lymphatic vessel separation,(5,6) embryonic cardiac
development,(7,8) and high endothelial venule integrity.(9)The PDPN overexpression has also been observed in several
malignant tumors, such as brain tumors,(10,11) oral cancers,(12)lung cancers,(13) melanomas,(14) mesotheliomas,(15) breast cancers,(16,17) and osteosarcomas.(18–20) Clinical data demonstrated
that PDPN expression is associated with poor prognosis andtumor malignancy in lung carcinomas, oral squamous cell carcinomas, and breast cancers.(8,21–24) PDPN facilitates hematogenous metastasis by eliciting tumor cell-induced platelet
aggregation response through its interaction with platelet C-type
lectin-like receptor 2 (CLEC-2).(25–28) These pieces of evidence
imply the importance of developing anti-PDPN monoclonal
antibodies (mAbs) for cancer therapeutic treatment.
We have recently established a novel PMab-233 (IgG1,kappa), which specifically detects Tasmanian devil PDPN
(tasPDPN) using Cell-Based Immunization and Screening
12(CBIS) method.(29) PMab-233 detected Chinese hamster
ovary (CHO)/tasPDPN cells by flow cytometry and recognized tasPDPN protein by Western blotting. Furthermore,
PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233may be useful as a lymphatic endothelial cell marker of theTasmanian devil. However, the specific binding region oftasPDPN for PMab-233 remains to be elucidated. In thisstudy, we investigated the binding epitopes of PMab-233 byanalyzing a series of deletion or point mutants of tasPDPN
using flow cytometry.
Materials and Methods
Cell lines

CHO-K1 was purchased from the American Type Culture
Collection (ATCC, Manassas, VA). The tasPDPN mutation
plasmids containing PA16 tag were transfected into CHO-K1
cells using Lipofectamine LTX (Thermo Fisher Scientific,Inc., Waltham, MA). Transiently transfected cells withdeletion/point mutants were cultured in RPMI 1640 medium(Nacalai Tesque, Inc., Kyoto, Japan), supplemented with
10% heat-inactivated fetal bovine serum (FBS; ThermoFisher Scientific, Inc.), 100 U/mL of penicillin, 100 mg/mL of
streptomycin, and 25 mg/mL of amphotericin B (Nacalai

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

261

SANO ET AL.

Downloaded by Yukinari Kato from www.liebertpub.com at 12/12/19. For personal use only.

262

FIG. 1. Epitope mapping of PMab-233 using deletion mutants of tasPDPN. (A) Illustration of six tasPDPN deletion mutantsof dN30, dN40, dN50, dN60, dN70, and dN80. (B, C) Deletion mutants of tasPDPN were analyzed using flow cytometry.
Deletion mutants were expressed on CHO-K1 cells and were then incubated with NZ-1 (B; anti-PA16 tag, red line), PMab-233(C; anti-tasPDPN mAb; red line), or buffer control (black line) for 30 minutes at 4C, followed by treatment with corresponding secondary antibodies. PLAG1, platelet aggregation-stimulating; PLD, PLAG-like domain.

Tesque, Inc.) at 37C in a humidified atmosphere of 5% CO2
and 95% air.
Production of tasPDPN mutants

The synthesized DNA of tasPDPN was subcloned into apCAG vector (FUJIFILM Wako Pure Chemical Corporation,Osaka, Japan), and PA16 tag (GLEGGVAMPGAEDDVV)

was added at the N-terminus. The deletion mutants oftasPDPN produced using PCR were subcloned into pCAG
vector with PA16 tag using the In-Fusion HD Cloning Kit(Takara Bio, Inc., Shiga, Japan). Substitutions of amino acids
to alanine in tasPDPN sequence were conducted using theQuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA). These constructs
were verified using direct DNA sequencing.

Downloaded by Yukinari Kato from www.liebertpub.com at 12/12/19. For personal use only.

FIG. 2. Epitope mapping of PMab-233 using point mutants of tasPDPN. (A, B) Flow cytometry. Transient point mutants
expressing L27A, P28A, E29A, D30A, A31G, A32G, T33A, T34A, I35A, and D36A of tasPDPN were incubated with NZ-1(A; red line), PMab-233 (B; red line), or buffer control (black line) for 30 minutes at 4C, followed by treatment withcorresponding secondary antibodies. (C) Schematic illustration of the epitope recognized by PMab-233. Underlined aminoacids are the critical epitope of PMab-233.

263

264
Flow cytometry

Transiently transfected CHO-K1 cells were detached by
0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA;Nacalai Tesque, Inc.). After washing with 0.1% bovine serumalbumin (BSA)/phosphate buffered saline (PBS), the cells
were incubated with anti-tasPDPN antibody (PMab-233;1 mg/mL) or control anti-PA16 tag antibody (NZ-1; 1 mg/mL)
for 30 minutes at 4C followed by treatment with Alexa Fluor
488-conjugated antimouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA) and Oregon Green-conjugatedantirat IgG (1:1000; Thermo Fisher Scientific, Inc.), respectively. Fluorescence data were collected using a Cell Analyzer
EC800 (Sony Corp., Tokyo, Japan).

Downloaded by Yukinari Kato from www.liebertpub.com at 12/12/19. For personal use only.

Results and Discussion

In our previous study, we established a novel anti-tasPDPN
mAb, PMab-233, which can be efficiently utilized for flow
cytometry, Western blotting, and immunohistochemical detection of tasPDPN.(29) Unfortunately, we could not investigate the PDPN expression using normal tissues of Tasmaniandevil; therefore, we used formalin-fixed paraffin-embedded
CHO/tasPDPN cells. PMab-233 did not react with human,
mouse, rat, rabbit, dog, bovine, cat, pig, horse, tiger, alpaca,bear, goat, sheep, or whale PDPNs, which indicates that
PMab-233 is specific to tasPDPN.(29) On the basis of theseresults, the epitope mapping of PMab-233 could be beneficial
in uncovering the pathophysiological function of tasPDPN.
Although three tandem repeats of platelet aggregationstimulating (PLAG) domain (PLAG1, PLAG2, PLAG3) areobserved in human or mouse PDPNs,(30) PLAG2 and PLAG3
do not exist in tasPDPN (Fig. 1A). In contrast, two PLAGlike domains were observed. We first constructed six deletionmutants of tasPDPN (Fig. 1A). Transient transfections oftasPDPN-mutant clones were produced using CHO-K1 cells,including dN30 (corresponding to 30–143 amino acids [aa]);dN40 (corresponding to 40–143 aa); dN50 (corresponding to50–143 aa); dN60 (corresponding to 60–143 aa); dN70
(corresponding to 70–143 aa); and dN80 (corresponding to80–143 aa). All deletion mutants of tasPDPN containN-terminal PA16 tag and were analyzed using flow cytometry for epitope mapping of PMab-233. NZ-1 (anti-PA16 tagmAb) detected all deletion mutants of tasPDPN, including
dN30, dN40, dN50, dN60, dN70, and dN80 (Fig. 1B). In
contrast, PMab-233 lost the reaction with dN30, dN40, dN50,dN60, dN70, and dN80 (Fig. 1C). These results imply that
N-terminus of the epitope-binding region of PMab-233 is
located between the 27th and 30th amino acids.
Next, we investigated the epitope-binding region of PMab233 by producing 10 point mutants of tasPDPN, including L27A,
P28A, E29A, D30A, A31G, A32G, T33A, T34A, I35A, andD36A. All these mutants can be recognized by NZ-1 (Fig. 2A).
We observed that PMab-233 reacted with L27A, P28A, E29A,
A31G, A32G, I35A, and D36A mutants, and weakly reactedwith D30A using flow cytometry (Fig. 2B). In contrast, PMab233 did not react with T33A and T34A (Fig. 2B).
In conclusion, we demonstrated that Asp30, Thr33, andThr34 of tasPDPN are critical for PMab-233-specific binding
to tasPDPN (Fig. 2C). Asp30, Thr33, and Thr34 are located in
PLAG1 domain of tasPDPN. PMab-233 can be a useful toolin elucidating the pathophysiological function of tasPDPN.

SANO ET AL.
Author Disclosure Statement

The authors have no conflict of interest.
Funding Information

This research was supported, in part, by AMED under Grant
Numbers JP19am0401013 (Y.K.), JP19am0101078 (Y.K.), andJP19ae0101028 (Y.K.), and by JSPS KAKENHI Grant Number 17K07299 (M.K.K.) and Grant Number 19K07705 (Y.K.).
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. Retzbach EP, Sheehan SA, Nevel EM, Batra A, Phi T,Nguyen ATP, Kato Y, Baredes S, Fatahzadeh M, Shienbaum AJ, and Goldberg GS: Podoplanin emerges as afunctionally relevant oral cancer biomarker and therapeutictarget. Oral Oncol 2018;78:126–136.
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. 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.
5. 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.
6. Hess PR, Rawnsley DR, Jakus Z, Yang Y, Sweet DT, Fu J,Herzog B, Lu M, Nieswandt B, Oliver G, Makinen T, Xia
L, and Kahn ML: Platelets mediate lymphovenous hemostasis to maintain blood-lymphatic separation throughout
life. J Clin Invest 2014;124:273–284.
7. Mahtab EA, Wijffels MC, Van Den Akker NM, Hahurij
ND, Lie-Venema H, Wisse LJ, Deruiter MC, Uhrin P,Zaujec J, Binder BR, Schalij MJ, Poelmann RE, andGittenberger-De Groot AC: Cardiac malformations andmyocardial abnormalities in podoplanin knockout mouseembryos: Correlation with abnormal epicardial development. Dev Dyn 2008;237:847–857.
8. Astarita JL, Acton SE, and Turley SJ: Podoplanin: Emerging functions in development, the immune system, andcancer. Front Immunol 2012;3:283.
9. 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, and Xia L: Podoplaninmaintains high endothelial venule integrity by interactingwith platelet CLEC-2. Nature 2013;502:105–109.
10. 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 12/12/19. For personal use only.

EPITOPE OF PMAB-233

11. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
12. 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.
13. 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. Tumour Biol 2005;26:195–200.
14. Abe S, Kaneko MK, Tsuchihashi Y, Izumi T, Ogasawara S,Okada N, Sato C, Tobiume M, Otsuka K, Miyamoto L,
Tsuchiya K, Kawazoe K, Kato Y, and Nishioka Y: Antitumor effect of novel anti-podoplanin antibody NZ-12against malignant pleural mesothelioma in an orthotopic
xenograft model. Cancer Sci 2016;107:1198–1205.
15. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
16. Wahal SP, Goel MM, and Mehrotra R: Lymphatic vessel
assessment by podoplanin (D2-40) immunohistochemistryin breast cancer. J Cancer Res Ther 2015;11:798–804.
17. Schoppmann SF, Berghoff A, Dinhof C, Jakesz R, Gnant
M, Dubsky P, Jesch B, Heinzl H, and Birner P: Podoplaninexpressing cancer-associated fibroblasts are associated withpoor prognosis in invasive breast cancer. Breast Cancer ResTreat 2012;134:237–244.
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. Ariizumi T, Ogose A, Kawashima H, Hotta T, Li G, Xu Y,Umezu H, Sugai M, and Endo N: Expression of podoplanin inhuman bone and bone tumors: New marker of osteogenic andchondrogenic bone tumors. Pathol Int 2010;60:193–202.
20. Kaneko MK, Oki H, Ogasawara S, Takagi M, and Kato Y:
Anti-podoplanin monoclonal antibody LpMab-7 detects
metastatic lesions of osteosarcoma. Monoclon Antib Immunodiagn Immunother 2015;34:154–161.
21. Xu Y, Ogose A, Kawashima H, Hotta T, Ariizumi T, Li G,Umezu H, and Endo N: High-level expression of podoplanin in benign and malignant soft tissue tumors: Immunohistochemical and quantitative real-time RT-PCR
analysis. Oncol Rep 2011;25:599–607.
22. Yuan P, Temam S, El-Naggar A, Zhou X, Liu DD, Lee JJ,and Mao L: Overexpression of podoplanin in oral cancer
and its association with poor clinical outcome. Cancer
2006;107:563–569.
23. Yurugi Y, Wakahara M, Matsuoka Y, Sakabe T, Kubouchi
Y, Haruki T, Nosaka K, Miwa K, Araki K, Taniguchi Y,

265

Shiomi T, Nakamura H, and Umekita Y: Podoplanin Expression in Cancer-associated Fibroblasts Predicts Poor
Prognosis in Patients with Squamous Cell Carcinoma of theLung. Anticancer Res 2017;37:207–213.
24. Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, Wojnar A, Witkiewicz W, Rys J, UgorskiM, Dziegiel P, and Podhorska-Okolow M: Podoplanin expression by cancer-associated fibroblasts predicts pooroutcome in invasive ductal breast carcinoma. Histopathology 2011;59:1249–1260.
25. 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.
26. Chang YW, Hsieh PW, Chang YT, Lu MH, Huang TF,Chong KY, Liao HR, Cheng JC, and Tseng CP: Identification of a novel platelet antagonist that binds to CLEC-2
and suppresses podoplanin-induced platelet aggregationand cancer metastasis. Oncotarget 2015;6:42733–42748.
27. 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.
28. 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.
29. Furusawa Y, Yamada S, Itai S, Nakamura T, Takei J, Sano
M, Harada H, Fukui M, Kaneko MK, and Kato Y: Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil
podoplanin. Biochem Biophys Rep 2019;18:100631.
30. 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.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: July 9, 2019
Accepted: September 20, 2019