MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 2, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0001

Epitope Mapping of the Antihorse Podoplanin
Monoclonal Antibody PMab-202

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Mika K. Kaneko,1 Yoshikazu Furusawa,1–3 Masato Sano,1 Shunsuke Itai,1,4 Junko Takei,1,4 Hiroyuki Harada,4Masato Fukui,3 Shinji Yamada,1 and Yukinari Kato1,2

Horse podoplanin (horPDPN), a type I transmembrane sialoglycoprotein, is expressed on the podocytes of thekidneys, alveolar type I cells of the lungs, and lymphatic endothelial cells. PDPN is a platelet aggregationinducing factor, and it primarily possesses three platelet aggregation-stimulating (PLAG) domains, that is,
PLAG1, PLAG2, and PLAG3, at the N-terminus and several PLAG-like domains. In a previous study, we
reported on a mouse anti-horPDPN monoclonal antibody (mAb) clone, PMab-202. Although the effectivenessof PMab-202 in flow cytometry and Western blotting is known, its exact binding epitope remains unknown todate. In this study, enzyme-linked immunosorbent assay and flow cytometry were used to identify the epitope ofPMab-202. We found that the critical epitopes of PMab-202 include Lys64, Thr66, and Phe70 of horPDPN. We
believe that our findings can be applied in the production of more functional anti-horPDPN mAbs.
Keywords: podoplanin, PDPN, PMab-202, epitope mapping

been reported to be present in the middle of PDPN.(35) Almost all mAbs against PDPNs have been reported to reactwith the PLAG domains or PLDs.(35,36)
In this study, we determined the epitope responsible forthe binding of PMab-202 to horPDPN using enzyme-linked
immunosorbent assay (ELISA) and flow cytometry.

IntroductionP

odoplanin (PDPN) induces platelet aggregation bybinding to C-type lectin-like receptor-2 (CLEC-2).(1–8)
PDPN is a type I transmembrane sialoglycoprotein expressed in normal tissues, including renal corpuscles, alveolar type I cells of the lungs, and lymphatic endothelial
cells.(3,9) The interaction between the PDPN of lymphatic
endothelial cells and CLEC-2 of platelets facilitates embryonic blood/lymphatic vessel separation.(10) Expressionof human PDPN, a protein associated with malignant progression and cancer metastasis,(5,11,12) has been reported inseveral malignant tumors, such as brain tumors,(11,13–15) mesotheliomas,(16,17) oral squamous cell carcinomas,(18) esophageal cancers,(19) lung cancers,(20) osteosarcomas,(21–23) andtesticular tumors.(24)
In a previous study, we developed monoclonal antibodies(mAbs) not only against human,(25) mouse,(25) and rat(26)PDPNs but also against rabbit,(27) dog,(28) cat,(29) bovine,(30)pig,(31) and horse(32–34) PDPNs (horPDPNs). PDPN primarily
possesses three platelet aggregation-stimulating (PLAG)
domains: PLAG1, PLAG2, and PLAG3; these domains arepresent at the N-terminus of PDPN.(2) One PLAG-like domain (PLD), important for PDPN–CLEC-2 interaction, has

Materials and Methods
Cell line

Chinese hamster ovary (CHO)-K1 was obtained from
American Type Culture Collection (ATCC, Manassas, VA).
The horse kidney cell line FHK-Tcl3.1 was prepared at Yamaguchi University.(37) horPDPN bearing an N-terminal
PA16 tag (PA16-horPDPN) was inserted into a pCAG-Ble
vector (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan).(33) The PA16 tag comprised 16 amino acids(GLEGGVAMPGAEDDVV).(38) Furthermore, the CHO-K1
cells were transfected with pCAG-Ble/PA16-horPDPN using
Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants were selected by limiting dilution and cultivating in a medium
containing 0.5 mg/mL Zeocin (InvivoGen, San Diego, CA).
The CHO/horPDPN cells were cultured in Roswell Park1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
3
ZENOAQ Resource Co., Ltd., Koriyama, Japan.
4
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
2

79

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KANEKO ET AL.

Table 1. Determination of PMab-202 Epitopeby Enzyme-Linked Immunosorbent Assay
PeptideSequence

PMab-202

Mutation

Sequence

PMab-202

pp23-42
pp33-52
pp43-62
pp53-72
pp63-82
pp73-92
pp83-102
pp93-112
pp103-118

ASTLGPEDNIMTPGVEDGMVMTPGVEDGMVTPGGSEDSESTPGGSEDSESTGSPALVPRSTGSPALVPRSTKSTGGDFEDTKSTGGDFEDRSTLGNTVHTRSTLGNTVHTPGESQSTRTP
PGESQSTRTPSVLTGHPTEKSVLTGHPTEKTDGNTKATVETDGNTKATVEKDGLST

+++
+++
-

T53A
G54A
S55A
P56A
A57G
L58A
V59A
P60A
R61A
S62A
T63A
K64A
S65A
T66A
G67A
G68A
D69A
F70A
E71A
D72A

AGSPALVPRSTKSTGGDFEDTASPALVPRSTKSTGGDFEDTGAPALVPRSTKSTGGDFEDTGSAALVPRSTKSTGGDFEDTGSPGLVPRSTKSTGGDFEDTGSPAAVPRSTKSTGGDFEDTGSPALAPRSTKSTGGDFEDTGSPALVARSTKSTGGDFEDTGSPALVPASTKSTGGDFEDTGSPALVPRATKSTGGDFEDTGSPALVPRSAKSTGGDFEDTGSPALVPRSTASTGGDFEDTGSPALVPRSTKATGGDFEDTGSPALVPRSTKSAGGDFEDTGSPALVPRSTKSTAGDFEDTGSPALVPRSTKSTGADFEDTGSPALVPRSTKSTGGAFEDTGSPALVPRSTKSTGGDAEDTGSPALVPRSTKSTGGDFADTGSPALVPRSTKSTGGDFEA

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

+++, OD655 S 0.6; -, OD655 < 0.1.

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Table 2. Determination of PMab-202 Epitopeby Enzyme-Linked Immunosorbent Assay

Memorial Institute (RPMI) 1640 medium (Nacalai Tesque,Inc., Kyoto, Japan), whereas FHK-Tcl3.1 was cultured in
Dulbecco’s modified Eagle’s medium (DMEM; Nacalai
Tesque, Inc.).(33) RPMI 1640 and DMEM were supplemented
with 10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific, Inc.), 100 units/mL penicillin, 100 mg/mL streptomycin, and 25 mg/mL amphotericin B (Nacalai Tesque,Inc.). The cells were grown at 37C in a humidified environment under a 5% CO2 atmosphere.
Enzyme-linked immunosorbent assay

The horPDPN peptides synthesized using PEPScreen(Sigma-Aldrich Corp., St. Louis, MO) were immobilized on
Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific, Inc.) at concentrations of 1 mg/mL for 30 minutes at
37C. After blocking with SuperBlock T20 (PBS) Blocking
Buffer (Thermo Fisher Scientific, Inc.), the plates were incubated with 1 mg/mL purified PMab-202, followed by
1:2000 dilution of peroxidase-conjugated antimouse IgG(Agilent Technologies, Inc., Santa Clara, CA). The enzymatic reaction was performed using 1-Step Ultra TMBELISA (Thermo Fisher Scientific, Inc.). Optical density wasmeasured at 655 nm using an iMark microplate reader (BioRad Laboratories, Inc., Berkeley, CA). These reactions were
performed at 37C using a total sample volume of 50–100 mL.
Flow cytometry

CHO/horPDPN or FHK-Tcl3.1 cells were harvested after abrief exposure to 0.25% trypsin/1 mM EDTA (Nacalai Tesque, Inc.), and were washed with 0.1% bovine serum albumin/phosphate-buffered saline. CHO/horPDPN cells weretreated with 0.1 mg/mL PMab-202 or 0.1 mg/mL PMab-202
plus 50 mg/mL peptides for 30 minutes at 4C. FHK-Tcl3.1
cells were treated with 1 mg/mL PMab-202 or 1 mg/mL
PMab-202 plus 10 mg/mL peptides for 30 minutes at 4C.
Thereafter, the cells were treated with Alexa Fluor 488conjugated antimouse IgG (1:2000; Cell Signaling Tech-+++, OD655 S 0.6; +, 0.1 & OD655 < 0.4; -, OD655 < 0.1.

nology, Inc., Danvers, MA). Fluorescence data were collected using SA3800 Cell Analyzers (Sony Corp., Tokyo,
Japan).
Results and Discussion

In a previous study, we immunized mice with a synthesized peptide (horPP6279) corresponding to the amino acids
62–79 of horPDPN.(33) ELISA screening indicated positive
signals in 21 of 960 (2.2%) wells. Among these 21 wells, 6(29%) tested positive against CHO/horPDPN in flow cytometry. One clone, that is, PMab-202 (IgG1, kappa), among
the six positive clones was established by limiting dilution.
PMab-202 could detect endogenous horPDPN expressed in
FHK-Tcl3.1, a horse kidney cell line, in flow cytometry andWestern blotting. However, PMab-202 was not advantageousfor immunohistochemical analysis.
We first synthesized a series of peptides of horPDPN(Table 1). Using ELISA, PMab-202 detected 53–72 and 63–
82 corresponding to the amino acids 53–72 and 63–82 of
horPDPN, respectively. Next, we synthesized the point mutants of 53–72 peptides (Table 2). Using ELISA, PMab-202
detected the following antigens: T53A, G54A, S55A, P56A,
A57G, L58A, V59A, P60A, R61A, S62A, T63A, S65A,
G67A, G68A, D69A, E71A, and D72A. However, F70A was
not recognized, and it weakly reacted with K64A and T66A,
indicating that Lys64, Thr66, and Phe70 are the critical epitopes of PMab-202.

‰
FIG. 1. Flow cytometry using PMab-202 and point mutants of horPDPN. CHO/horPDPN cells were treated with
0.1 mg/mL PMab-202 or 0.1 mg/mL PMab-202 plus 50 mg/mL peptides for 30 minutes at 4C. FHK-Tcl3.1 cells were treatedwith 1 mg/mL PMab-202 or 1 mg/mL PMab-202 plus 10 mg/mL peptides for 30 minutes at 4C. Thereafter, the cells weretreated with Alexa Fluor 488-conjugated antimouse IgG. Solid line with gray shade, control (second Ab only); dotted line,
PMab-202 or PMab-202 plus peptides. CHO, Chinese hamster ovary; horPDPN, horse podoplanin.

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KANEKO ET AL.

FIG. 2. Schematic illustration of the epitope recognized by PMab-202. Underlined amino acids are the critical epitope
PMab-202. PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain.
Next, we performed a blocking assay using flow cytometry.
We found that PMab-202 reacted with the CHO/horPDPN cellline (Fig. 1). This reaction was partially neutralized by T53A.
However, K64A, T66A, and F70A did not block the reactionof PMab-202 with CHO/horPDPN. Similarly, PMab-202
reacted with the FHK-Tcl3.1 cell line (Fig. 1). Notably, thisreaction was completely neutralized by T53A. In contrast,
K64A, T66A, and F70A did not block the reaction of PMab202 with FHK-Tcl3.1, thereby confirming that Lys64, Thr66,
and Phe70 of horPDPN are critical for PMab-202 detection.
As shown in Figure 2, Lys64, Thr66, and Phe70 are included
between PLAG3 and PLD.
Taken together, the study findings indicate that the critical
epitopes of PMab-202 are Lys64, Thr66, and Phe70 of
horPDPN. We believe that these findings will be beneficial in
the production of more functional anti-horPDPN mAbs.
Acknowledgments

We thank Takuro Nakamura, Miyuki Yanaka, Saori Handa,
Kayo Hisamatsu, and Yoshimi Nakamura for excellent technical assistance. FHK-Tcl3.1 was kindly provided by Ken
Maeda (Yamaguchi University). This research was supported,in part, by AMED under Grant Nos.: JP18am0101078 (Y.K.),
JP18am0301010 (Y.K.), and JP18ae0101028 (Y.K.), and by
JSPS KAKENHI Grant No. 17K07299 (M.K.K.) and Grant
No. 16K10748 (Y.K.).
Author Disclosure Statement

Y.K. received research funding from ZENOAQ ResourceCo., Ltd. The other authors have no conflict of interest.
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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: yukinarikato@med.tohoku.ac.jp
Received: January 16, 2019
Accepted: March 4, 2019