MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 3, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0012

Epitope Mapping of Anti-Tiger Podoplanin
Monoclonal Antibody PMab-231

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

Podoplanin (PDPN) is expressed on podocytes of the kidneys, type I alveolar cells of the lungs, and lymphatic
endothelial cells. PDPN comprises three platelet aggregation-stimulating (PLAG) domains (PLAG1, PLAG2,
and PLAG3) in the N-terminus and PLAG-like domains in the middle of the PDPN protein. We have previously
reported on an anti-tiger PDPN (tigPDPN) monoclonal antibody (mAb), PMab-231, which was developed using
the Cell-Based Immunization and Screening (CBIS) method. PMab-231 is very useful in flow cytometry,
Western blotting, and immunohistochemical analyses; however, the binding epitope of PMab-231 remains to beelucidated. This study aimed to investigate the epitopes of PMab-231, which was developed by CBIS method,using enzyme-linked immunosorbent assay. The results revealed that the critical epitopes of PMab-231 are
Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35, Gly36, and Glu38 of tigPDPN, which is corresponding to
PLAG1/2. The findings of our study can be applied to the production of more functional anti-tigPDPN mAbs.
Keywords: podoplanin, PDPN, PMab-231, epitope mapping

IntroductionP

odoplanin (PDPN) is a type I transmembrane sialoglycoprotein that is expressed in normal tissues, including renal corpuscles, type I lung alveolar cells, andlymphatic endothelial cells.(1,2) PDPN induces platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on
platelets.(1,3–9) The interaction between PDPN on lymphatic
endothelial cells and CLEC-2 on platelets facilitates the separation of embryonic blood/lymphatic vessels.(10) Studies havereported the expression of human PDPN in several malignant
tumors, such as oral squamous cell carcinomas,(11) malignant
mesotheliomas,(12,13) lung cancers,(14) and malignant brain
tumors.(15–18) PDPN expression has also been associated withmalignant progression and cancer metastasis.(6,15,19) Althoughpathophysiological studies of alveolar cells of the lungs, podocytes of the kidneys, or lymphatic endothelial cells of thecolon using anti-tigPDPN monoclonal antibodies (mAbs) are
important in many disorders, mAbs against tigPDPN remain to
be developed.
In our previous study, we used the Cell-Based Immunization and Screening (CBIS) method to develop specificand sensitive mAbs against the tigPDPN of 163 amino acids
to facilitate the immunohistochemical analysis of paraffin-

embedded tissue sections.(20) Screening identified strong
signals against Chinese hamster ovary (CHO)/tigPDPN cells
and weak or no signals against CHO-K1 cells in 18 of 958
wells (1.9%). One of the six clones, PMab-231 (IgG2a,kappa), was finally selected using immunohistochemistry
against paraffin-embedded feline normal tissue sections
because tigPDPN and cat PDPN (cPDPN) shared 99% homology.(21) The dissociation constants (KD) of PMab-231
for CHO/tigPDPN and CHO/cPDPN cells were determined
to be 1.2 · 10-8 M and 1.9 · 10-8 M, respectively, indicating
moderate affinity for CHO/tigPDPN and CHO/cPDPN cells.
PMab-231 stained type I alveolar cells of the feline lungs
and podocytes of the feline kidneys using immunohistochemistry. This study aimed to determine the binding epitope ofPMab-231 to tigPDPN using enzyme-linked immunosorbent
assay (ELISA).
Materials and Methods
Enzyme-linked immunosorbent assay

Synthesized tigPDPN peptides using PEPScreen (SigmaAldrich Corp., St. Louis, MO) were immobilized on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientific,
Inc., Waltham, MA) at 10 mg/mL for 30 minutes at 37C.

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
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
4
ZENOAQ RESOURCE CO., LTD., Koriyama, Japan.
2

129

130

TAKEI ET AL.

Table 1. Determination of PMab-231 Epitopeby Enzyme-Linked Immunosorbent Assay
PeptideSequence

PMab-231

24–43
34–53
44–63
54–73
64–83
74–93
84–103
94–113
104–123
114–129

STVRPEDDIMTPGVEDGMVTTPGVEDGMVTLGAEDNVVTTLGAEDNVVTTGAGADPEESTGAGADPEESTGPTGLVPTNTGPTGLVPTNTESITDFHIEDESITDFHIEDGPTQESTGHAGPTQESTGHANEESQSTTTL
NEESQSTTTLNVVTSHSIEKNVVTSHSIEKIGEDTETTVEIGEDTETTVEKDGLAT++-++, 0.3 & OD655 < 0.6; -, OD655 < 0.1.

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Table 2. Determination of PMab-231 Epitopeby Enzyme-Linked Immunosorbent Assay
Mutation

Sequence

PMab-231

S24A
T25A
V26A
R27A
P28A
E29A
D30A
D31A
I32A
M33A
T34A
P35A
G36A
V37A
E38A
D39A
G40A
M41A
V42A
T43A

ATVRPEDDIMTPGVEDGMVTSAVRPEDDIMTPGVEDGMVTSTARPEDDIMTPGVEDGMVTSTVAPEDDIMTPGVEDGMVTSTVRAEDDIMTPGVEDGMVTSTVRPADDIMTPGVEDGMVTSTVRPEADIMTPGVEDGMVTSTVRPEDAIMTPGVEDGMVTSTVRPEDDAMTPGVEDGMVTSTVRPEDDIATPGVEDGMVTSTVRPEDDIMAPGVEDGMVTSTVRPEDDIMTAGVEDGMVTSTVRPEDDIMTPAVEDGMVTSTVRPEDDIMTPGAEDGMVTSTVRPEDDIMTPGVADGMVTSTVRPEDDIMTPGVEAGMVTSTVRPEDDIMTPGVEDAMVTSTVRPEDDIMTPGVEDGAVTSTVRPEDDIMTPGVEDGMATSTVRPEDDIMTPGVEDGMVA

+
+
+
+
+
+
+
+
+
+
+

+, 0.1 & OD655 < 0.3; -, OD655 < 0.1.

After blocking with SuperBlock T20 (PBS) Blocking Buffer(Thermo Fisher Scientific, Inc.), the plates were incubated withpurified PMab-231 (1 mg/mL), followed by a peroxidaseconjugated anti-mouse IgG (Agilent Technologies, Inc., Santa
Clara, CA) dilution of 1:2000. Enzymatic reactions were
performed using 1-Step Ultra TMB-ELISA (Thermo Fisher
Scientific, Inc.). Optical density was measured at 655 nm using
an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). These reactions were performed at 37C using a
total sample volume of 50–100 mL.
Results and Discussion

We have previously produced mAbs against HER2(22) or
EGFR(23) using CasMab technology and mAbs against CD44,
PD-L1, or CD133 using the CBIS method.(24–28) However,the binding epitopes of these mAbs (H2Mab-77 against HER2,
EMab-51 against EGFR, C44Mab-5 against CD44, L1Mab-13
against PD-L1, and CMab-43 against CD133) remain to be
determined because these mAbs did not react with synthesizedpeptides. Conversely, the binding epitopes of mAbs produced
by immunizing with synthesized peptides have been easily
determined using deletion mutants and point mutants of synthesized peptides.(29,30)
We first synthesized a series of tigPDPN peptides, which
are summarized in Table 1. Using ELISA, PMab-231 detected
24–43 peptides corresponding to the 24th–43rd amino acids(aas) of tigPDPN. We then synthesized point mutants of 24–43
peptides (Table 2). Using ELISA, PMab-231 detected S24A,
T25A, V26A, R27A, P28A, V37A, D39A, G40A, M41A,
V42A, and T43A. Conversely, it did not detect E29A, D30A,
D31A, I32A, M33A, T34A, P35A, G36A, and E38A, indicating that Glu29, Asp30, Asp31, Ile32, Met33, Thr34, Pro35,
Gly36, and Glu38 are included in the critical epitope of PMab231. Although PMab-231 was produced using the CBIS method,the epitope of PMab-231 was fortunately determined by ELISA.
We previously developed mAbs against human,(31)mouse,(31) rat,(32) rabbit,(33) dog,(34) cat,(27) bovine,(35)horse,(36–38) and pig(39) PDPNs. PDPN comprises three tandemrepeat of the ‘‘EDxxVTPG’’ sequences, which were defined to
be platelet aggregation-stimulating (PLAG) domains (PLAG1,

FIG. 1. Schematic illustration of the epitope recognized by PMab-231. The critical epitope of PMab-231 are shown ingray letters. PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain.

EPITOPE OF PMAB-231

PLAG2, and PLAG3) in the N-terminus of the PDPN protein.(4) There are several PLAG-like domains (PLDs) of the‘‘E(D/E)xx(T/S)xx’’ sequence in the middle of the PDPN
protein. PLDs are reportedly important for PDPN–CLEC-2
interactions.(40) Almost all mAbs against PDPNs reportedly
react with PLAG domains or PLDs.(40,41) As summarized in
Figure 1, PMab-231 also detected PLAG1 (Glu29, Asp30,
Asp31, Ile32, Met33, Thr34, Pro35, and Gly36) and a part ofPLAG2 (Glu38) of tigPDPN. Therefore, PLAG1 of tigPDPN
was clarified to be the advantageous epitope for several applications, such as flow cytometry, Western blotting, and immunohistochemical analyses. These findings can be applied
to the production of more functional anti-tigPDPN mAbs.

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Acknowledgments

This research was supported in part by AMED underGrant Numbers: JP18am0101078 (Y.K.), JP18am0301010(Y.K.), and JP18ae0101028 (Y.K.), and by JSPS KAKENHI
Grant Number 17K07299 (M.K.K.) and Grant Number
16K10748 (Y.K.).
Author Disclosure Statement

Y.K. received research funding from ZENOAQ RESOURCE CO., LTD. The other authors have no conflict ofinterest.
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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: February 27, 2019
Accepted: April 24, 2019