MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 3, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0006

Epitope Mapping of Monoclonal Antibody
PMab-48 Against Dog Podoplanin

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Shinji Yamada,1 Mika K. Kaneko,1 Shunsuke Itai,1,2 Yao-Wen Chang,1 Takuro Nakamura,1Miyuki Yanaka,1 Satoshi Ogasawara,3 Takeshi Murata,3 Hiroaki Uchida,4Hideaki Tahara,4 Hiroyuki Harada,2 and Yukinari Kato1,5Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on normal renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells. Increased expression of PDPN in cancers isassociated with poor prognosis and hematogenous metastasis through interactions with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously reported a novel PMab-48 antibody, which is an anti-dog PDPN(dPDPN) monoclonal antibody (mAb) recognizing PDPN expressed in lymphatic endothelial cells. However,the binding epitope of PMab-48 is yet to be clarified. In this study, an enzyme-linked immunosorbent assay andflow cytometry were used to investigate epitopes of PMab-48. The results revealed that the critical epitope ofPMab-48 comprises Asp29, Asp30, Ile31, Ile32, and Pro33 of dPDPN.
Keywords: dog podoplanin, dPDPN, monoclonal antibody, epitope mapping

Introduction

Materials and Methods

PCell lines

odoplanin (PDPN) is expressed in normal tissues suchas renal podocytes, pulmonary type I alveolar cells,
lymphatic endothelial cells, chondrocytes, myofibroblasts,
and mesothelial cells.(1) However, higher expression of PDPN
has been observed in different tumor types, including squamous cell carcinoma,(2) melanoma,(3) glioblastoma,(4) andmesothelioma.(5) Recent clinical studies have provided evidence of associations between increased PDPN expression, poor prognosis, and cancer metastasis,(6) indicating thatthe establishment and production of anti-PDPN monoclonal antibodies (mAbs) are necessary for developing novel
therapeutic strategies against cancer development and progression to metastasis.(7)Dog podoplanin (dPDPN) was first reported as gp40.(8)Recently, we developed two mAbs, PMab-38(2,3,9) andPMab-48,(10) both of which specifically recognize dPDPN.
Tyr67 and Glu68 were determined to be the critical features of the epitope of PMab-38.(11) Using point mutants ofdPDPN, the present study aimed to investigate the epitope ofPMab-48 by enzyme-linked immunosorbent assay (ELISA)
and flow cytometry.

CHO-K1 cells were transfected with the dPDPN-MAP tag
plasmid using Lipofectamine LTX (Thermo Fisher Scientific,Inc., Waltham, MA).(9) CHO/dPDPN cells were cultured in an
RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan),
supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc., Waltham, MA), 100 U/mL
penicillin, 100 mg/mL streptomycin, and 25 mg/mL amphotericin B (Nacalai Tesque, Inc.), and incubated at 37C in ahumidified atmosphere of 5% CO2 and 95% air.
Enzyme-linked immunosorbent assay

Synthesized dPDPN peptides (PEPScreen; Sigma-AldrichCorp., St. Louis, MO) were immobilized on Nunc Maxisorp 96well immunoplates (Thermo Fisher Scientific, Inc.) at 5 mg/mLfor 30 minutes. After blocking with SuperBlock T20 (phosphatebuffered saline [PBS]) Blocking Buffer (Thermo Fisher Scientific, Inc.), the plates were incubated with purified PMab-48(10 mg/mL), followed by a 1:2000 dilution of peroxidaseconjugated anti-mouse IgG (Agilent Technologies, Inc., Santa

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,Tokyo, Japan.
3
Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan.
4Division of Bioengineering, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo,Japan.
5
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
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163

Clara, CA). The enzymatic reaction was conducted using 1-Step
Ultra TMB-ELISA (Thermo Fisher Scientific, Inc.). Opticaldensity was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These
reactions were performed at 37C with a total sample volumeof 50–100 mL.
Flow cytometry

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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 PBS, thecells were treated with PMab-48 (10 mg/mL) or PMab-48(10 mg/mL) plus peptides (10 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). Fluorescence data were acquired using the cell analyzer
SA3800 (Sony Corp., Tokyo, Japan).
Results

We previously developed a novel PMab-48 mAb that exhibits high specificity and sensitivity against dPDPN and ishighly suitable for detection of lymphatic endothelial cells in
immunohistochemical analysis.(10) PMab-48 can be used todetect dPDPN expressed by squamous cell carcinomas.(10)These findings suggest that PMab-48 epitope mapping could
be a promising target for PDPN-based diagnosis and cancer
therapy.
In this study, we synthesized a series of point mutantsof dPDPN peptides from the 26th to the 40th amino acid
(Table 1). Using ELISA, PMab-48 detected V26A, R27A,
P28A, G34A, V35A, E36A, D37A, S38A, V39A, and V40A;
in contrast, PMab-48 did not react with D29A, D30A, I31A,
I32A, or P33A, indicating that the DDIIP sequence is a critical epitope of PMab-48.
Next, we performed a blocking assay using flow cytometry. PMab-48 reacted with the CHO/dPDPN cell line

Table 1. Determination of PMab-48 Epitopeby Enzyme-Linked Immunosorbent Assay
Mutation

Sequence

PMab-48

Wild type
V26A
R27A
P28A
D29A
D30AI31A
I32A
P33A
G34A
V35A
E36A
D37A
S38A
V39A
V40A

VRPDDIIPGVEDSVVARPDDIIPGVEDSVVVAPDDIIPGVEDSVVVRADDIIPGVEDSVVVRPADIIPGVEDSVVVRPDAIIPGVEDSVVVRPDDAIPGVEDSVVVRPDDIAPGVEDSVVVRPDDIIAGVEDSVVVRPDDIIPAVEDSVVVRPDDIIPGAEDSVVVRPDDIIPGVADSVVVRPDDIIPGVEASVVVRPDDIIPGVEDAVVVRPDDIIPGVEDSAVVRPDDIIPGVEDSVA+++++++++—————+++++++++++++++++++++, OD655 S 0.6; ++, 0.4 & OD655 < 0.6; +, 0.2 & OD655 <0.4; —, OD655 < 0.2.

FIG. 1. Flow cytometry using PMab-48 and point mutantsof dPDPN. PMab-48 or PMab-48 + peptides (WT, D29A,D30A, I31A, I32A, P33A, or V35A) were treated withCHO/dPDPN cells for 30 minutes at 4C, followed by addition of secondary antibodies. dPDPN, dog podoplanin;
WT, wild type; gray peak, negative control.
(Fig. 1). This reaction was completely neutralized by thewild-type peptide (VRPDDIIPGVEDSVV) and V35A; incontrast, D29A, D30A, I31A, I32A, and P33A did not block
the reaction of PMab-48 with CHO/dPDPN, indicatingthat the DDIIP sequence is a critical epitope of PMab-48(Fig. 2).

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YAMADA ET AL.

FIG. 2.

Schematic illustration of the epitope recognized by PMab-48. PLAG, platelet aggregation-stimulating.

Discussion

References

Previously, a critical epitope of another anti-dPDPN mAb,
PMab-38, was determined to comprise Tyr67 and Glu68,(11)indicating that epitopes of PMab-48 and PMab-38 are different(Fig. 2). PMab-48 reacted with lymphatic endothelial cells
in immunohistochemistry,(10) although PMab-38 did not,(9)demonstrating that the N-terminal region is a more adequate
epitope for detecting dPDPN of lymphatic endothelial cells
probably because N-terminus might not be O-glycosylated.(12)In contrast, PMab-38 showed cancer specificity in immunohistochemistry using canine tissues(2) in the same patternwith anti-human PDPN (hPDPN) cancer-specific mAbs, suchas LpMab-2(7,13) and LpMab-23.(6,14,15) We previously showedthat Thr55-Leu64 peptide of hPDPN, especially O-glycan attached in Thr55 and Ser56 of hPDPN, is a critical epitope ofLpMab-2.(7) We further showed that Gly54-Leu64 peptideof hPDPN is a critical epitope of LpMab-23.(15) The epitopeof PMab-38 for dPDPN is similar to those of LpMab-2 andLpMab-23 for hPDPN. Therefore, this kind of epitope mapping provides important evidence for clinical application ofanti-PDPN mAbs.

1. 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.
2. Kaneko MK, Honma R, Ogasawara S, Fujii Y, Nakamura
T, Saidoh N, Takagi M, Kagawa Y, Konnai S, and Kato Y:
PMab-38 recognizes canine podoplanin of squamous cell
carcinomas. Monoclon Antib Immunodiagn Immunother2016;35:263–266.
3. Ogasawara S, Honma R, Kaneko MK, Fujii Y, Kagawa Y,Konnai S, and Kato Y: Podoplanin expression in caninemelanoma. Monoclon Antib Immunodiagn Immunother2016;35:304–306.
4. 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.
5. 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-12
against malignant pleural mesothelioma in an orthotopic
xenograft model. Cancer Sci 2016;107:1198–1205.
6. Retzbach EP, Sheehan SA, Nevel E, Batra A, Phi T, Kato Y,
Baredes S, Fatahzadeh M, Shienbaum AJ, and Goldberg GS:
Podoplanin emerges as a functionally relevant oral cancer
biomarker and therapeutic target. Oral Oncol 2018;78:126–136.
7. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
8. Zimmer G, Klenk HD, and Herrler G: Identification of a 40kDa cell surface sialoglycoprotein with the characteristics of amajor influenza C virus receptor in a Madin-Darby canine
kidney cell line. J Biol Chem 1995;270:17815–17822.
9. Honma R, Kaneko MK, Ogasawara S, Fujii Y, Konnai S,Takagi M, and Kato Y: Specific detection of dog podoplanin
expressed in renal glomerulus by a novel monoclonal anti-Acknowledgments

The authors thank Noriko Saidoh, Saori Handa, andYoshimi Nakamura for their excellent technical assistance.
This research was supported, in part, by AMED under Grant
Numbers: JP17am0301010 (Y.K.), JP17am0101078 (Y.K.),
and JP17ae0101028 (Y.K.) and by JSPS KAKENHI Grant
Number 17K07299 (M.K.K.) and Grant Number 16K10748(Y.K.). This work was performed, in part, under the Cooperative Research Program of the Institute for Protein Research, Osaka University, CR-17-05, and by the Grant for
Joint Research Project of the Institute of Medical Science, theUniversity of Tokyo.
Author Disclosure Statement

No competing financial interests exist.

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EPITOPE OF PMAB-48

body PMab-38 in immunohistochemistry. Monoclon AntibImmunodiagn Immunother 2016;35:212–216.
10. Yamada S, Itai S, Kaneko MK, and Kato Y: PMab-48
recognizes dog podoplanin of lymphatic endothelial cells.
Monoclon Antib Immunodiagn Immunother 2018;37:63–66.
11. Chang YW, Yamada S, Kaneko MK, and Kato Y: Epitope
Mapping of Monoclonal antibody PMab-38 against dog
podoplanin. Monoclon Antib Immunodiagn Immunother 2017;36:291–295.
12. 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.
13. Kaneko MK, Yamada S, Nakamura T, Abe S, Nishioka Y,Kunita A, Fukayama M, Fujii Y, Ogasawara S, and Kato
Y: Antitumor activity of chLpMab-2, a human-mouse chimeric cancer-specific antihuman podoplanin antibody, via
antibody-dependent cellular cytotoxicity. Cancer Med 2017;
6:768–777.
14. Yamada S, Ogasawara S, Kaneko MK, and Kato Y:LpMab-23: A cancer-specific monoclonal antibody against
human podoplanin. Monoclon Antib Immunodiagn Immunother 2017;36:72–76.

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15. Kaneko MK, Nakamura T, Kunita A, Fukayama M, Abe S,
Nishioka Y, Yamada S, Yanaka M, Saidoh N, Yoshida K,Fujii Y, Ogasawara S, and Kato Y: ChLpMab-23: Cancerspecific human-mouse chimeric anti-podoplanin antibody
exhibits antitumor activity via antibody-dependent cellularcytotoxicity. Monoclon Antib Immunodiagn Immunother2017;36:104–112.

Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp;
yukinari-k@bea.hi-ho.ne.jp
Received: January 23, 2018
Accepted: March 7, 2018