MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 5, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0025

Epitope Mapping of Anti-Bear Podoplanin Monoclonal
Antibody PMab-247

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Yukinari Kato,1,2 Junko Takei,1,3 Yoshikazu Furusawa,1,2 Yusuke Sayama,1 Masato Sano,1 Satoru Konnai,4,5Atsushi Kobayashi,6 Hiroyuki Harada,3 Maki Takahashi,7 Hiroyoshi Suzuki,7Shinji Yamada,1 and Mika K. Kaneko1Podoplanin (PDPN)/T1alpha is a type I transmembrane sialoglycoprotein, which is expressed on podocytes ofthe kidneys and type I alveolar cells of the lungs. PDPN is also known as Aggrus, a platelet aggregationinducing factor, which comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2,and PLAG3) in the N-terminus and PLAG-like domains (PLDs) in the middle of the PDPN protein. We havepreviously established a mouse anti-bear PDPN (bPDPN) monoclonal antibody (mAb) clone, PMab-247 using
the Cell-Based Immunization and Screening (CBIS) method. PMab-247 is very useful in flow cytometry,
Western blotting, and immunohistochemical (IHC) analyses; however, the binding epitope of PMab-247 has notbeen elucidated. In this study, we aimed to investigate the epitope of PMab-247 using enzyme-linked immunosorbent assay and IHC analyses. The results revealed that the critical epitopes of PMab-247 are Asp76,Arg78, Glu80, and Arg82 of bPDPN. The Glu80 and Arg82 are included in PLD of bPDPN. The findings of ourstudy can be applied to the production of more functional anti-bPDPN mAbs.
Keywords: podoplanin, PDPN, PMab-247, epitope mapping

Introduction

Podoplanin (PDPN)/T1alpha/Aggrus is a type I
transmembrane sialoglycoprotein that is expressed innormal tissues, such as renal corpuscles, type I alveolar cellsof lung, and lymphatic endothelial cells of every organ.(1)PDPN induces platelet aggregation by binding to C-type
lectin-like receptor-2 (CLEC-2).(2) The interaction between
PDPN on lymphatic endothelial cells and CLEC-2 on platelets facilitates the separation of embryonic blood/lymphaticvessels.(3) Human PDPN is overexpressed in several malignanttumors, such as malignant mesotheliomas,(4,5) oral squamouscell carcinomas,(6) malignant brain tumors,(7) and lung cancers.(8) PDPN expression has also been associated with malignant progression and cancer metastasis.(9)In our previous study, we used the Cell-Based Immunization and Screening (CBIS) method to develop sensitive and specific monoclonal antibodies (mAbs) against bear

PDPN (bPDPN) to facilitate the immunohistochemical (IHC)
analysis of paraffin-embedded tissue sections.(10) Flow cytometry revealed that PMab-247 (mouse IgG1, kappa), oneof the established clones, recognized CHO/bPDPN but did
not react with CHO-K1. Western blotting demonstrated
that PMab-247 could detect bPDPN as a 48-kDa band in
CHO/bPDPN cells. IHC analyses revealed that PMab-247
strongly stained pulmonary type I alveolar cells, lymphatic
endothelial cells, and podocytes of the kidney.(11) This study
aimed to determine the binding epitope of PMab-247 tobPDPN using enzyme-linked immunosorbent assay (ELISA)and IHC analyses.
Materials and Methods
Enzyme-linked immunosorbent assay

Synthesized bPDPN peptides using PEPScreen (SigmaAldrich Corp., St. Louis, MO) were immobilized on Nunc

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Miyagi, Japan.
New Industry Creation Hatchery Center, Tohoku University, Miyagi, Japan.
3
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,Tokyo, Japan.
4
Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University,Hokkaido, Japan.
5
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
6
Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
7
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Miyagi, Japan.
2

230

EPITOPE OF PMAB-247

231

Table 1. Determination of PMab-247 Epitopeby Enzyme-Linked Immunosorbent Assay
PeptideSequence

PMab-247

18–37
28–47
38–57
48–67
58–77
68–87
78–97
88–107
98–117
108–127

DPAEGASTVRPEDDITPGVEPEDDITPGVEDNMVTLGVEDDNMVTLGVEDNMVTAGTSEENMVTAGTSEEPYESGLTAAVPYESGLTAAVPINREGVTDLPINREGVTDLRLEDRTTESTRLEDRTTESTVRAEEESQSTVRAEEESQSTTTLNVVTSHPTTLNVVTSHPTEKVGEDTETTEKVGEDTETTVEKDGLATV+++-

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+++, OD655 ‡ 0.6; -, OD655 < 0.1.

Maxisorp 96-well immunoplates (Thermo Fisher Scientific,Inc., Waltham, MA) at 10 mg/mL for 30 minutes at 37C.
After blocking with SuperBlock T20 (PBS) Blocking
Buffer (Thermo Fisher Scientific, Inc.), the plates were incubated with purified PMab-247 (1 mg/mL), followed by aperoxidase-conjugated anti-mouse IgG (dilution of 1:2000;
Agilent Technologies Inc., Santa Clara, CA). Enzymatic reactions were performed using 1-Step Ultra TMB-ELISA(Thermo Fisher Scientific, Inc.). Optical density wasmeasured at 655 nm using an iMark microplate reader(Bio-Rad Laboratories, Inc., Berkeley, CA). These reactions were performed at 37C using a total sample volumeof 50–100 mL.
Immunohistochemical analyses

Malayan Sun Bear (Helarctos malayanus) tissues werecollected after autopsy at Hokkaido University, fixed in 10%

Table 2. Determination of PMab-247 Epitopeby Enzyme-Linked Immunosorbent Assay
Using Point Mutants
Mutation

Sequence

PMab-247

P68A
I69A
N70A
R71A
E72A
G73A
V74A
T75A
D76A
L77A
R78A
L79A
E80A
D81A
R82A
T83A
T84A
E85A
S86A
T87A

AINREGVTDLRLEDRTTEST
PANREGVTDLRLEDRTTEST
PIAREGVTDLRLEDRTTESTPINAEGVTDLRLEDRTTESTPINRAGVTDLRLEDRTTEST
PINREAVTDLRLEDRTTEST
PINREGATDLRLEDRTTEST
PINREGVADLRLEDRTTESTPINREGVTALRLEDRTTEST
PINREGVTDARLEDRTTEST
PINREGVTDLALEDRTTEST
PINREGVTDLRAEDRTTEST
PINREGVTDLRLADRTTEST
PINREGVTDLRLEARTTEST
PINREGVTDLRLEDATTEST
PINREGVTDLRLEDRATEST
PINREGVTDLRLEDRTAESTPINREGVTDLRLEDRTTAST
PINREGVTDLRLEDRTTEATPINREGVTDLRLEDRTTESA++++++++++++++++++++++++++++++++++++++++++++++++++++, OD655 ‡ 0.6; +, 0.1 £ OD655 < 0.4; -, OD655 < 0.1.

FIG. 1. IHC analyses of bear lungs using PMab-247 and point
mutants of bPDPN. Histological sections of the bear lungs weredirectly autoclaved in citrate buffer for 20 minutes. After
blocking with SuperBlock T20 (PBS) Blocking Buffer, thesections were incubated with (A, B) PMab-247 (5 mg/mL), (C,D) PMab-247 (5 mg/mL) + P68A (50 mg/mL), (E, F) PMab-247(5 mg/mL) + R78A (50 mg/mL), or (G, H) control (blockingbuffer), followed by treatment with the Envision + Kit. (I, J) HEstaining. Scale bar: 100 mm. bPDPN, bear podoplanin; HE, hematoxylin and eosin; IHCs, immunohistochemical.

neutral-buffered formalin, and routinely processed to makeparaffin-embedded tissue sections. Histological sections (4mm thick) were directly autoclaved in citrate buffer (pH 6.0;
Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes.
These tissue sections were then blocked with the SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific,Inc.), incubated with PMab-247 (5 mg/mL) or PMab-247(5 mg/mL) + peptides (50 mg/mL) for 1 hour at room

232

KATO ET AL.

temperature, and treated using an Envision + Kit (AgilentTechnologies, Inc.) for 30 minutes. Color was developed with 3,3¢-diaminobenzidine tetrahydrochloride (AgilentTechnologies, Inc.) for 2 minutes, and counterstaining wasperformed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

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Results and Discussion

FIG. 2. IHC analyses of bear kidneys using PMab-247 andpoint mutants of bPDPN. Histological sections of the bearkidneys were directly autoclaved in citrate buffer for 20minutes. After blocking with SuperBlock T20 (PBS) Blocking Buffer, the sections were incubated with (A, B) PMab247 (5 mg/mL), (C, D) PMab-247 (5 mg/mL) + P68A
(50 mg/mL), (E, F) PMab-247 (5 mg/mL) + R78A (50 mg/mL),or (G, H) control (blocking buffer), followed by treatment
with the Envision + Kit. (I, J) HE staining. Scale bar: 100 mm.

We first synthesized a series of bPDPN peptides, which
are summarized in Table 1. Using ELISA, PMab-247 detected the 68–87 peptide corresponding to the 68th–87th
amino acids (AAs) of bPDPN. We then synthesized pointmutants of 68–87 peptides (Table 2). Using ELISA, PMab247 strongly detected P68A, I69A, N70A, R71A, E72A,G73A, V74A, T75A, L77A, L79A, D81A, T83A, T84A,E85A, S86A, and T87A, and weakly detected R82A. Conversely, it did not detect D76A, R78A, and E80A, indicatingthat Asp76, Arg78, Glu80, and Arg82 are the critical epitopesof PMab-247.
We then performed a blocking assay using IHC to confirm the PMab-247 epitope. PMab-247 reacted with lungtype I alveolar cells (Fig. 1A, B). These reactions werecompletely neutralized by P68A (Fig. 1C, D). Conversely,
R78A did not block these reactions of PMab-247 (Fig. 1E,F). Likewise, D76A, E80A, and R82A did not inhibit thePMab-247 reaction (Supplementary Fig. S1). Negativecontrol (blocking buffer) did not show any staining(Fig. 1G, H). As shown in Figure 2A and B, PMab-247
reacted with the podocytes and Bowman’s capsules of thekidneys. The reactions of PMab-247 in the kidneys werecompletely neutralized by P68A (Fig. 2C, D) although
R78A did not block these reactions of PMab-247 (Fig. 2E,F). Negative control (blocking buffer) did not show anystaining (Fig. 2G, H). These IHC results indicate that
Asp76, Arg78, Glu80, and Arg82 of bPDPN are essential
for PMab-247 binding to bPDPN.
In summary, the critical epitopes of PMab-247 are Asp76,Arg78, Glu80, and Arg82 of bPDPN. Among these AAs,Glu80 and Arg82 are included in PLAG-like domains ofbPDPN, which is important for PDPN/CLEC-2 interaction(Fig. 3). These findings can be applied to the production ofmore functional anti-bPDPN mAbs.

FIG. 3. Schematic illustration of the epitope recognized by PMab-247. PLAG and PLD. Red AAs indicate the critical
epitope of PMab-247. AAs, amino acids; PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain.

EPITOPE OF PMAB-247
Acknowledgment

We thank Yoshikazu Satoh for excellent technical assistance.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by AMED undergrant numbers JP19am0101078 (Y.K.), JP18am0301010(Y.K.), and JP19ae0101028 (Y.K.), and by JSPS KAKENHI grant numbers 17K07299 (M.K.K.) and 19K07705(Y.K.).
Supplementary Material

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Supplementary Figure S1
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: June 5, 2019
Accepted: August 20, 2019