MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 3, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0004

Epitope Mapping of PMab-241, a Lymphatic Endothelial
Cell-Specific Anti-Bear Podoplanin Monoclonal Antibody

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Yusuke Sayama,1 Masato Sano,1 Teizo Asano,1 Yoshikazu Furusawa,1 Junko Takei,1Takuro Nakamura,1 Miyuki Yanaka,1 Saki Okamoto,1 Saori Handa,1 Yu Komatsu,1
Yoshimi Nakamura,1 Mikiko Yanagawa,1 Mika K. Kaneko,1 and Yukinari Kato1,2

Anti-bear podoplanin (bPDPN) monoclonal antibodies (mAbs), including PMab-247 and PMab-241, have beenpreviously established. Although PMab-247 has shown positive immunostaining for lymphatic endothelial cells(LECs), type I alveolar cells of the lung, and podocytes of the kidney, PMab-241 stains LECs but does notreact with lung type I alveolar cells. PDPN possesses three platelet aggregation-stimulating (PLAG) domains(PLAG1, PLAG2, and PLAG3) and the PLAG-like domain (PLD). The binding epitope of PMab-247 was previously determined to include bPDPN residues Asp76, Arg78, Glu80, and Arg82. Among these, Glu80 and Arg82
are included in PLD of bPDPN. The purpose of this study is to determine the binding epitope of PMab-241 and toclarify the difference between these two anti-bPDPN mAbs. Analysis of bPDPN deletion mutants revealed that the
N-terminus of the PMab-241 epitope exists between amino acids (aa) 75 and 80 of bPDPN. In addition, analysis ofbPDPN point mutants demonstrated that the critical epitope of PMab-241 includes Thr75, Asp76, and Arg78 ofbPDPN. The binding epitopes of PMab-241 and PMab-247 seem to overlap, but this slight difference may be sufficient to provide the specificity of PMab-241 to discriminate LECs from type I alveolar cells of the lung.
Keywords: bear podoplanin, monoclonal antibody, epitope, PMab-241

known as Aggrus,(14) a platelet aggregation-inducing factor that comprises three platelet aggregation-stimulating(PLAG) domains (PLAG1, PLAG2, and PLAG3)(15) at the
N-terminus and PLAG-like domains (PLDs)(3,13,16–18) in the
middle of the PDPN protein. Among these, Glu80 and Arg82
are included in PLD of bPDPN. However, the PMab-241
epitope has not been clarified.
The aim of this study was to determine the binding epitopeof PMab-241 and to uncover the difference between the twoanti-bPDPN mAbs.

Introduction

S

ensitive and specific monoclonal antibodies (mAbs)
against podoplanin (PDPN) of various species, including
human,(1) experimental animals,(2) and wild animals,(3) arein high demand to analyze the expression and pathophysiological function of PDPN. PDPN is used for discriminating
lymphatic endothelial cells (LECs) from vascular endothelial
cells(4) or type I alveolar cells of the lung from type II alveolar
cells of the lung.(5) We recently developed anti-bear podoplanin (bPDPN) mAbs, including PMab-247(6) and PMab241,(7) using the Cell-Based Immunization and Screening(CBIS) method.(8–10) Similar to other typical anti-PDPN mAbs,
PMab-247 demonstrated positive immunoreaction(11) for
LECs,(4) type I alveolar cells of the lung,(5) and podocytes of
the kidney.(12) By contrast, PMab-241 stained LECs but did notreact with type I alveolar cells of the lung,(7) suggesting thatthe binding epitope of PMab-247 is different from that ofPMab-241.
To identify the binding epitope of PMab-247, we previously produced deletion mutants and point mutants ofbPDPN and clearly showed that the PMab-247 epitope is
Asp76, Arg78, Glu80, and Arg82 of bPDPN.(13) PDPN is also
12

Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 cells were obtained
from the American Type Culture Collection (Manassas, VA).
The bPDPN mutation plasmids containing bovine Aggrus/
podoplanin (BAP) tag(6) were transfected into CHO-K1 cells
using Lipofectamine LTX (Thermo Fisher Scientific, Inc.,
Waltham, MA). The BAP tag comprises 16 amino acids (aa)(EKTTLGVEDYTTTPAA), and is detected by PMab-44
mAb.(19) Cells transiently transfected with deletion mutantsor point mutants were cultured in Roswell Park Memorial

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

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Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto,
Japan), supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Thermo Fisher Scientific, Inc.), 100 U/mL of
penicillin, 100 lg/mL of streptomycin, and 25 lg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere of 5% CO2 and 95% air.

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Production of bPDPN mutants

Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan)
encoding bPDPN (Accession No. XM_008694703.1) was
subcloned into the pCAG vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan), and a BAP tag wasadded at the N-terminus. Deletion mutants of the bPDPN
sequence were produced using a HotStar HiFidelity Polymerase Kit (Qiagen, Inc., Hilden, Germany) with oligonucleotides. Substitutions of amino acids to alanine in thebPDPN sequence were conducted by QuikChange Lightning
Site-Directed Mutagenesis Kits (Agilent Technologies, Inc.,
Santa Clara, CA). Polymerase chain reaction fragments
bearing the desired mutations were inserted into the pCAG
vector using the In-Fusion HD Cloning Kit (Takara Bio, Inc.,
Shiga, Japan).
Flow cytometry

Transiently transfected CHO-K1 cells were detached by
0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA;

FIG. 1. Epitope mapping of PMab-241 using deletion mutants of bPDPN. Deletion mutants of bPDPN were analyzed
using flow cytometry. Mutants were incubated with PMab-44(anti-BAP tag; red line, A), PMab-241 (anti-bPDPN mAb; redline, B), or buffer control (black line, A, B) for 30 minutes at 4C,
followed by secondary antibodies. BAP, bovine Aggrus/podoplanin; bPDPN, bear podoplanin; mAbs, monoclonal antibodies.

SAYAMA ET AL.

Nacalai Tesque, Inc.) and were collected using 10% FBS in
RPMI 1640 medium. After washing with 0.1% bovine serum
albumin/phosphate buffered saline, the cells were incubatedwith an anti-bPDPN antibody (PMab-241; 1 lg/mL) or ananti-BAP tag antibody (PMab-44; 1 lg/mL) for 30 minutes at
4C. Alexa Fluor 488-conjugated anti-mouse IgG (1:2000;
Cell Signaling Technology, Inc., Danvers, MA) for detectionof PMab-241 and PMab-44 was added to each cell and was
incubated for 30 minutes at 4C. Fluorescence data werecollected and analyzed using a Cell Analyzer EC800 (SonyCorp., Tokyo, Japan).
Results

We generated seven deletion mutants of bPDPN in CHOK1 cells, namely dN30 (corresponding to 30–160 aa), dN40
(corresponding to 40–160 aa), dN50 (corresponding to 50–
160 aa), dN60 (corresponding to 60–160 aa), dN70 (corresponding to 70–160 aa), dN75 (corresponding to 75–160 aa),and dN80 (corresponding to 80–160 aa) or wild type (WT)bPDPN (corresponding to 27–160 aa). All deletion mutants
and WT of bPDPN containing the N-terminal BAP tag weredetected by PMab-44 (an anti-BAP tag mAb), indicating thatthe expression level of each construct is very high (Fig. 1A).
Although PMab-241 detected dN30, dN40, dN50, dN60,
dN70, and dN75, it did not react with dN80 (Fig. 1B),

FIG. 2. Epitope mapping of PMab-241 using point mutants of bPDPN. Transient point mutants expressing T75A,
D76A, L77A, R78A, L79A, E80A, D81A, R82A, T83A,
T84A, E85A, or S86A of bPDPN were incubated with
PMab-44 (red line, A), PMab-241 (red line, B), or buffer
control (black line, A, B) for 30 minutes at 4C, followed bysecondary antibodies.

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EPITOPE OF PMAB-241

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FIG. 3. Schematic illustration of the epitope recognized by PMab-241. Illustration of WT bPDPN and its seven deletionmutants: dN30, dN40, dN50, dN60, dN70, dN75, and dN80. Black bars: positive reactions of PMab-241. White bar:negative reactions of PMab-241. Red amino acids indicate a critical epitope of PMab-241. PLAG, platelet aggregationstimulating domain; PLD, PLAG-like domain; WT, wild type.

suggesting that the N-terminus of the PMab-241 epitope
exists between bPDPN aa 75 and 80.
Next, to determine the binding epitope of PMab-241, we
produced the following series of point mutants of bPDPN:
T75A, D76A, L77A, R78A, L79A, E80A, D81A, R82A,
T83A, T84A, E85A, and S86A. PMab-44 reacted with all
point mutants (Fig. 2A). By contrast, PMab-241 did not reactwith T75A, D76A, and R78A (Fig. 2B). These results indicated that Thr75, Asp76, and Arg78 of bPDPN are essential
for PMab-241 binding to bPDPN (Fig. 3).
Discussion

Highly sensitive and specific mAbs against not only human
but also experimental animals and wild animals are necessary
to analyze the expression and pathophysiological function of
PDPN. Thus, we have developed mAbs against human,(20)mouse,(21) rat,(22) rabbit,(23) dog,(24) cat,(25) bovine,(26) pig,(27,28)
Tasmanian devil,(29) alpaca,(30) tiger,(31) whale,(32) goat,(33,34)
horse,(35,36) and bear(7,11) PDPNs using the CBIS method.(8–10) Among these antibodies, we have shown thatanti-bPDPN mAb PMab-247 is very useful for immunohistochemical analyses for detecting LECs and type I alveolar
cells of the bear lung.(6) We sometimes need to discriminate
LECs from vascular endothelial cells in each organ; PDPN isused for the specific marker for LECs. However, it is very
difficult to detect LECs in lung because PDPN is also highly
expressed in type I alveolar cells. PMab-247 reacts with both
LECs and type I alveolar cells(11); therefore, a specific mAb
for LECs, not reacting with type I alveolar cells is advantageous in pathological diagnosis.

After screening of many anti-bPDPN mAbs, we finallyestablished a LEC-specific anti-bPDPN mAb, PMab-241.(7)In this study, we investigated the binding epitope of PMab241 and compared it with that of PMab-247. We previously
demonstrated that the PMab-247 epitope is Asp76, Arg78,
Glu80, and Arg82 of bPDPN.(13) Because Glu80 and Arg82
are included in the PLD of bPDPN, PMab-247 may inhibit platelet aggregation by bPDPN. We herein clarified thatThr75, Asp76, and Arg78 of bPDPN are critical epitope
components for PMab-241, and these residues are not locatedin PLAG and/or PLD. We likewise hope to establish in future studies LEC-specific anti-PDPN mAbs against otherspecies for the pathophysiological analysis of LECs in lungs
or other organs.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by AMED under grant
numbers P20am0401013 (Y.K.), JP20am0101078 (Y.K.),
and JP20ae0101028 (Y.K.), and JSPS KAKENHI grant
numbers 17K07299 (M.K.K.) and 19K07705 (Y.K.).
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2–1 Seiryo-machi
Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: February 14, 2020
Accepted: March 5, 2020