MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 6, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0048

Epitope Mapping of Monoclonal Antibody
PMab-38 Against Dog Podoplanin

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Yao-Wen Chang,1 Shinji Yamada,1 Mika K. Kaneko,1 and Yukinari Kato1,2

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is extensively expressed by normal lymphatic
endothelial cells, renal podocytes, and pulmonary type I alveolar cells. Nevertheless, increased expression ofPDPN in malignant tumors not only associates with poor prognosis but also facilitates hematogenous metastasis
through interaction with C-type lectin-like receptor-2 presented on platelets, followed by PDPN-mediatedplatelet activation. We previously reported a novel PMab-38 antibody, an anti-dog PDPN (dPDPN) monoclonal
antibody, which specifically recognizes PDPN in squamous cell carcinomas melanomas and cancer-associatedfibroblasts in canine cancer tissues. However, the specific binding with the epitope of PMab-38 remains undefined. In this study, flow cytometry was utilized to investigate the epitope of PMab-38, which was determinedusing a series of deletion or point mutants of dPDPN. The results revealed that the critical epitope of PMab-38
is Tyr67 and Glu68 of dPDPN.
Keywords: podoplanin, epitope, dog PDPN

Introduction

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is also known as Aggrus/T1a-2/gp36.(1–3) The
first characterized dog PDPN (dPDPN) is reported to be
gp40.(4) Notably, PDPN is expressed in normal kidney podocytes, pulmonary type I alveolar cells, lymphatic endothelial cells (LECs), chondrocytes, myofibroblasts, andmesothelial cells.(5–10) However, higher expression of PDPN
has also been observed in different tumor types, including
squamous cell carcinoma (SCC),(11) melanoma,(12) central
nervous system tumors,(13) glioblastoma,(14,15) mesothelioma,(7) and breast cancers.(16,17) Recent clinical studies haveprovided evidence of association of increased PDPN expression with poor prognosis and cancer metastasis.(10,18,19)
Furthermore, PDPN elicits tumor cell-induced platelet aggregation response through the activation of platelet C-type
lectin-like receptor 2 to facilitate cancer progression and metastasis.(20–23) These evidences indicate that the establishment
and production of anti-PDPN monoclonal antibodies (mAbs) isimperative to develop novel therapeutic strategy against cancer
development and progression to metastasis.
Recently, we developed a novel PMab-38 mAb, which
specifically recognizes dPDPN in renal podocytes, in contrast
to LECs or type I alveolar cells, by immunohistochemical
analysis.(24) Moreover, PMab-38 also recognizes dPDPN ofSCCs, melanomas, and cancer-associated fibroblasts (CAFs)
12in cancer tissues.(12,25) Therefore, this study was initiated toinvestigate the epitope of PMab-38 using deletion or point
mutants of dPDPN in Chinese hamster ovary (CHO)-K1 cellsby flow cytometry.
Materials and Methods
Cell lines

CHO-K1 was procured from the American Type Culture
Collection (ATCC, Manassas, VA). CHO-K1 cells weretransfected with the dPDPN-MAP tag plasmid using Lipofectamine LTX (Thermo Fisher Scientific, Inc., Waltham,MA) according to manufacturer’s protocol. CHO-K1 andtransfectants were cultured in RPMI 1640 medium (NacalaiTesque, Inc., Kyoto, Japan) and supplemented with 10%
heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/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. Zeocin (0.5 mg/mL; InvivoGen, San Diego, CA) wasadded for producing and culturing each transfectant.
Production of dPDPN mutants

The amplified dPDPN complementary DNA was subcloned into a pCAG vector (Wako Pure Chemical Industries,Ltd.) and a MAP tag was added at the N-terminus. Deletion

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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mutants of dPDPN were generated using a HotStar HiFidelity
PCR (Qiagen, Inc., Hilden, Germany) with oligonucleotides
containing the desired mutations. PCR amplicons bearing thedesired mutations were subcloned into pCAG vector using anIn-Fusion PCR cloning kit (Clontech, Palo Alto, CA).
Substitutions of amino acids (aa) to alanine in the dPDPN
sequence were achieved using a QuikChange lightning sitedirected mutagenesis kit (Agilent Technologies, Inc., Santa
Clara, CA).
Flow cytometry

Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA) and Oregon Greenconjugated anti-rat IgG (1:1000; Thermo Fisher Scientific,Inc.), respectively. Fluorescence data were acquired using
Cell Analyzer EC800 (Sony, Corp., Tokyo, Japan).
Results and Discussion

Previously, we developed a novel PMab-38 mAb, which
exhibited high specificity and sensitivity toward dPDPN andis highly suitable for the detection of renal glomerulus podocytes in immunohistochemical analysis.(24) Interestingly,
PMab-38 can be used to detect dPDPN expressed by oral
and skin SCCs.(25) CAFs and canine melanoma cells werealso recognized by PMab-38.(12,25) Taken together, thesefindings suggest that the epitope mapping of PMab-38 could
provide a promising target for the PDPN-based diagnosis andcancer therapy.

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Cells were harvested after brief exposure to 0.25% trypsin/
1 mM ethylenediaminetetraacetic acid (Nacalai Tesque,Inc.). After washing with 0.1% bovine serum albumin in
phosphate-buffered saline, the cells were treated with 10 mg/
mL of PMab-38 and 5 mg/mL of anti-MAP tag (clone: PMab1) for 35 minutes at 4C followed by treatment with Alexa

CHANG ET AL.

FIG. 1. Epitope mapping of PMab-38 using deletion mutants of dPDPN. (A) Illustration of nine dPDPN deletion mutants:dN23, dN30, dN50, dN60, dN65, dN70, dN75, dN80, and dN110. (B) Epitope mapping of deletion mutants of dPDPN byflow cytometric analysis. Deletion mutants were incubated with anti-MAP tag PMab-1 (left panel, red), PMab-38 (rightpanel, blue), or buffer control (black) for 35 minutes at 4C, followed by addition of corresponding secondary antibodies,respectively. dPDPN, dog podoplanin.

EPITOPE OF PMAB-38

sponding to 60–169 aa), dN65 (corresponding to 65–169 aa),dN70 (corresponding to 70–169 aa), dN75 (corresponding to75–169 aa), dN80 (corresponding to 80–169 aa), and dN110(corresponding to 110–169 aa). All dPDPN deletion mutants
were tagged with N-terminal MAP and therefore wereDownloaded by Yukinari Kato from online.liebertpub.com at 12/21/17. For personal use only.

For this study, nine deletion mutants of dPDPN weregenerated (Fig. 1A). Stable transfections of dPDPN-mutant
clones were established in CHO-K1 cells, including dN23
(corresponding to 23–169 aa), dN30 (corresponding to 30–169 aa), dN50 (corresponding to 50–169 aa), dN60 (corre-293

FIG. 2. Epitope mapping of PMab-38 using point mutants of dPDPN. (A, B) Flow cytometry. Stable point mutants
expressing D60A, G61A, A62G, T63A, E64A, E65A, P66A, Y67A, E68A, S69A, and G70A of dPDPN were treated with
PMab-1 (A) or PMab-38 (B) for 35 minutes at 4C, followed by addition of corresponding secondary antibodies, respectively. (C) Schematic illustration of the epitope recognized by PMab-38.

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analyzed using flow cytometry for epitope mapping of PMab38. Flow cytometric analysis revealed that PMab-1 (anti-MAPtag mAb) detected all the dPDPN deletion mutants, including
dN23, dN30, dN50, dN60, dN65, dN70, dN75, dN80, anddN110 (Fig. 1B, left). However, a significant loss in the reaction of PMab-38 was observed with dN70, dN75, dN80, anddN110 (Fig. 1B, right). These results indicate that N-terminusof PMab-38 epitope is located between 65th and 70th aa.
Next, to further investigate the crucial epitope of PMab-38, weproduced a series of point mutants of dPDPN in CHO-K1 cells,
including D60A, G61A, A62G, T63A, E64A, E65A, P66A,
Y67A, E68A, S69A, and G70A. All deletion mutants wererecognized by PMab-1 (Fig. 2A). Notably, PMab-38 reactedwith all these mutants, except with Y67A and E68A (Fig. 2B).
These results provide strong direct evidence that Tyr67 andGlu68 are the crucial epitopes of PMab-38 (Fig. 2C). This YE
motif of dPDPN is not conserved among the other species.
Although detection of PMab-38 for renal glomerulus,SCCs, melanoma, and CAFs using immunohistochemistry
and flow cytometry was successful,(12,24,25) our results revealed that PMab-38 has limitation for dPDPN recognition byWestern blot analysis (data not shown). One reason might bethat PMab-38 has high affinity binding to undenatured dPDPN.
Taken together, these results demonstrated that PMab-38
is a potential diagnostic target for the detection of dPDPN
using flow cytometry. Furthermore, the study also revealed
Tyr67 and Glu68 as the crucial epitopes of dPDPN for specific binding with PMab-38. This study also provides evidence on the potential application of PMab-38 in unveiling
pathophysiological function of dPDPN.
Acknowledgments

This work was supported, in part, by the Basic Science andPlatform Technology Program for Innovative Biological
Medicine from Japan Agency for Medical Research and development, AMED (Y.K.), by Project for utilizing glycans in
the development of innovative drug discovery technologies
from AMED (Y.K.), and by Platform Project for Supporting
Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED.
Author Disclosure Statement

Y.K. received research funding from Ono Pharmaceutical,Co., Ltd.
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-kuSendaiMiyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp,
yukinarikato@med.tohoku.ac.jp
Received: September 8, 2017
Accepted: October 19, 2017