MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 5, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0023

Epitope Mapping of Antipig Podoplanin Monoclonal
Antibody PMab-213

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Shinji Yamada,1 Shunsuke Itai,1 Yoshikazu Furusawa,1,2 Mika K. Kaneko,1 and Yukinari Kato1,2

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on lymphatic endothelial cells,
podocytes of the kidneys, and type I alveolar cells of the lungs. PDPN, a platelet aggregation-inducing factor,
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 a mouse
antipig PDPN (pPDPN) monoclonal antibody (mAb) clone, PMab-213, which was developed using the CellBased Immunization and Screening (CBIS) method. PMab-213 is very useful in flow cytometry, Western
blotting, and immunohistochemical analyses; however, the binding epitope of PMab-213, which was developedby CBIS method, remains to be elucidated. Therefore, this study aimed to investigate the epitope of PMab-213
using enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemical analyses. The results
revealed that the critical epitopes of PMab-213 are Pro53, Arg54, Arg56, and Tyr60 of pPDPN.
Keywords: podoplanin, PDPN, PMab-213, epitope mapping

IntroductionP

odoplanin (PDPN) is a type I transmembrane sialoglycoprotein that is expressed in normal tissues, including type I lung alveolar cells, renal corpuscles, and lymphatic
endothelial cells.(1,2) PDPN binds to C-type lectin-like receptor2 (CLEC-2) and thus induces platelet aggregation.(1,3–9) The
interaction between PDPN on lymphatic endothelial cells and
CLEC-2 on platelets facilitates the separation of embryonicblood/lymphatic vessels.(10) Studies have reported the expression of human PDPN in several malignant tumors, such as oral
squamous cell carcinomas,(11) malignant brain tumors,(12–15)
esophageal cancers,(16) malignant mesotheliomas,(17,18) lungcancers,(19) testicular tumors,(20) and osteosarcomas.(21–23)
PDPN expression has also been associated with malignant
progression and cancer metastasis.(6,12,24) Although pathophysiological studies of alveolar cells of the lungs, podocytesof the kidneys, or lymphatic endothelial cells of the colon andskin using antipig PDPN monoclonal antibodies (mAbs) are
important in many disorders, mAbs against pPDPN remain
to be developed.
In our previous study, we used the Cell-Based Immunization and Screening (CBIS) method to develop specific andsensitive mAbs against the pig PDPN (pPDPN) of 159 aminoacids (aas) to facilitate the immunohistochemical analysis ofparaffin-embedded tissue sections.(25) Flow cytometry revealed that PMab-213 (mouse IgG2b, kappa), an establishedclone, recognized Chinese hamster ovary (CHO)/pPDPN but
1
2

did not react with CHO-K1. PMab-213 also reacted with the
PK-15 pig kidney cell line, indicating that PMab-213 recognizes endogenous pPDPN. Western blotting performed
demonstrated the ability of PMab-213 to detect pPDPN as 40and 25-kDa bands in CHO/pPDPN cells. Moreover, the KD of
PMab-213 for CHO/pPDPN was determined to be 2.1 · 10-9 M,
indicating its high affinity for CHO/pPDPN. Immunohistochemical analyses revealed that PMab-213 strongly stained
pulmonary type I alveolar cells, podocytes, and Bowman’s
capsules in the kidney, and lymphatic endothelial cells obtained from microminipigs. This study aimed to determine
the binding epitope of PMab-213 to pPDPN using enzymelinked immunosorbent assay (ELISA), flow cytometry, andimmunohistochemical analyses.
Materials and Methods
Cell line

CHO-K1 was obtained from the American Type Culture
Collection (ATCC; Manassas, VA). The pig kidney cellline PK-15 was obtained from the Japanese Collection of
Research Bioresources Cell Bank (JCRB; Osaka, Japan).
pPDPN (accession no.: XM_005665017) bearing an Nterminal PA16 tag (PA16-pPDPN) was previously inserted
into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan). The PA16 tag comprised 16 aas(GLEGGVAMPGAEDDVV).(25–27) pCAG-Ble/PA16-pPDPN

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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EPITOPE OF PMAB-213

225

was transfected into CHO-K1 cells using Lipofectamine LTX
with Plus Reagent (Thermo Fisher Scientific, Inc., Waltham,MA). Stable transfectants were selected by limiting the dilution
and cultivating them in a medium containing 0.5 mg/mL zeocin(InvivoGen, San Diego, CA). CHO/pPDPN cells were cultured
in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan),whereas PK-15 cells were cultured in DMEM medium (Nacalai
Tesque, Inc.). All media were supplemented with 10% heatinactivated fetal bovine serum (Thermo Fisher Scientific, Inc.),
100 U/mL penicillin, 100 mg/mL streptomycin, and 25 mg/mL
amphotericin B (Nacalai Tesque, Inc.) and stored at 37C in ahumidified chamber under 5% CO2 and 95% air atmosphere.

fixed in 4% paraformaldehyde. The tissues were processedto produce 4-mm paraffin-embedded tissue sections, whichwere directly autoclaved in citrate buffer (pH 6.0; Nichirei
Biosciences, Inc., Tokyo, Japan) for 20 min. These tissue
sections were then blocked with the SuperBlock T20 (PBS)
Blocking Buffer (Thermo Fisher Scientific, Inc.), incubatedwith PMab-213 (1 mg/mL) or PMab-213 (1 mg/mL) + peptides(1 mg/mL) for 1 hour at room temperature, and treated using
an Envision + Kit (Agilent Technologies, Inc.) for 30 min.
Color was developed with 3,3¢-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2 min, andcounterstaining was performed using hematoxylin (FUJIFILM
Wako Pure Chemical Corporation).

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Enzyme-linked immunosorbent assay

Synthesized pPDPN peptides using PEPScreen (SigmaAldrich Corp., St. Louis, MO) were immobilized on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientific,Inc.) at 1 mg/mL for 30 min at 37C. After blocking with
SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher
Scientific, Inc.), the plates were incubated with purified
PMab-213 (1 mg/mL), followed by a peroxidase-conjugatedantimouse 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.
Flow cytometry

Cells were harvested after a brief exposure to 0.25%
trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). The cells were then washed with 0.1% bovine
serum albumin/phosphate-buffered saline (PBS) and treatedwith PMab-213 (1 mg/mL) or PMab-213 (1 mg/mL) + peptides (10 mg/mL) for 30 min at 4C. The cells were thentreated with Alexa Fluor 488-conjugated antimouse IgG(1:2000; Cell Signaling Technology, Inc., Danvers, MA).
Fluorescence data were collected using the SA3800 Cell
Analyzers (Sony Corp., Tokyo, Japan).
Immunohistochemical analyses

Microminipig (Fuji Micra, Inc., Shizuoka, Japan) tissues
were collected on autopsy at the Gifu University and thenTable 1. Determination of PMab-213 Epitopeby Enzyme-Linked Immunosorbent Assay
PeptideSequence

pp23-42
pp33-52
pp43-62
pp53-72
pp63-82
pp73-92
pp83-102
pp93-112
pp103-122
pp113-125

ASTVLPEDGVTPGVEGSSKA
TPGVEGSSKASPGVEDYTVTSPGVEDYTVTPRTREEPYATPRTREEPYATPLVPTRTKGT
PLVPTRTKGTTGSPTEDVFTTGSPTEDVFTVGSTTHSHKG
VGSTTHSHKGSQSTTTQNVVSQSTTTQNVVTSQSHDKGDETSQSHDKGDEEKSKTVTKDGEKSKTVTKDGLGT+++, OD655 ‡ 0.6; -, OD655 < 0.1.

PMab-213++++++-

Results

We first synthesized a series of pPDPN peptides, which aresummarized in Table 1. Using ELISA, PMab-213 detected
43–62 and 53–72 peptides corresponding to the 43rd–62nd
and 53rd–72nd aas of pPDPN, respectively. We then synthesized point mutants of 43–62 peptides (Table 2). Using ELISA,
PMab-213 detected S43A, P44A, G45A, V46A, E47A, D48A,
Y49A, T50A, V51A, T52A, T55A, E57A, E58A, P59A,
A61G, and T62A. Conversely, it did not detect P53A, R54A,
R56A, and Y60A, indicating that Pro53, Arg54, Arg56, andTyr60 are critical epitopes of PMab-213.
We then performed a blocking assay using flow cytometry.
PMab-213 reacted with the PK-15 cell line (Fig. 1A). This
reaction was completely neutralized by S43A. Conversely,
P53A, R54A, R56A, and Y60A did not block the reaction ofPMab-213 with PK-15. Similarly, PMab-213 reacted with the
CHO/pPDPN cell line, and this reaction was partially neutralized by S43A (Fig. 1B). Conversely, P53A, R54A, R56A,
and Y60A did not block the reaction of PMab-213 with
CHO/pPDPN, confirming that Pro53, Arg54, Arg56, andTyr60 of pPDPN are critical for PMab-213 detection.

Table 2. Determination of PMab-213 Epitopeby Enzyme-Linked Immunosorbent Assay
Mutation

Sequence

S43A
P44A
G45A
V46A
E47A
D48A
Y49A
T50A
V51A
T52A
P53A
R54A
T55A
R56A
E57A
E58A
P59A
Y60A
A61G
T62A

APGVEDYTVTPRTREEPYATSAGVEDYTVTPRTREEPYATSPAVEDYTVTPRTREEPYATSPGAEDYTVTPRTREEPYATSPGVADYTVTPRTREEPYATSPGVEAYTVTPRTREEPYATSPGVEDATVTPRTREEPYATSPGVEDYAVTPRTREEPYATSPGVEDYTATPRTREEPYATSPGVEDYTVAPRTREEPYATSPGVEDYTVTARTREEPYATSPGVEDYTVTPATREEPYATSPGVEDYTVTPRAREEPYATSPGVEDYTVTPRTAEEPYATSPGVEDYTVTPRTRAEPYATSPGVEDYTVTPRTREAPYATSPGVEDYTVTPRTREEAYATSPGVEDYTVTPRTREEPAATSPGVEDYTVTPRTREEPYGTSPGVEDYTVTPRTREEPYAA

Bold indicates the replaced amino acids.
+++, OD655 ‡ 0.6; -, OD655 < 0.1.

PMab-213
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

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YAMADA ET AL.

We further performed a blocking assay using immunohistochemistry. PMab-213 reacted with the podocytes of thekidneys (Fig. 2). These reactions were completely neutralized
by S43A. Conversely, Y60A (Fig. 2) and P53A, R54A, and
R56A (data not shown) did not block these reactions ofPMab-213. These data confirmed that Pro53, Arg54, Arg56,
and Tyr60 of pPDPN are critical for PMab-213 detection.
Discussion

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We have previously produced several mAbs against CD44,
PD-L1, and CD133 using the CBIS method.(26,28–31) However,the binding epitopes of these mAbs (C44Mab-5 against CD44,
L1Mab-13 against PD-L1, and CMab-43 against CD133)

FIG. 1. Flow cytometry using PMab-213 and point mutantsof pPDPN. PMab-213 (1 mg/mL), PMab-213 (1 mg/mL) plus
peptides (10 mg/mL), or control (without primary mAbs;gray) was reacted with (A) PK-15 or (B) CHO/pPDPN cells
for 30 min at 4C, followed by the addition of secondary
antibodies. CHO, Chinese hamster ovary; mAbs, monoclonal
antibodies; pPDPN, pig podoplanin.

FIG. 2. Immunohistochemical analyses using PMab-213
and point mutants of pPDPN. Histological sections of thekidneys were directly autoclaved in citrate buffer for 20 min.
After blocking with SuperBlock T20 (PBS) blocking buffer,the created sections were incubated with PMab-213 (1 mg/mL),
PMab-213 (1 mg/mL) + peptides (1 mg/mL), or control (blocking buffer), followed by treatment with the Envision + Kit. Scalebar: 100 mm. HE, hematoxylin and eosin; PBS, phosphatebuffered saline.

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EPITOPE OF PMAB-213

remain to be determined because these mAbs did not reactwith synthesized peptides. 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.(32,33) Although PMab-213
was produced using the CBIS method in this study, the epitope of PMab-213 was readily determined by ELISA(Tables 1 and 2).
To date, we have developed mAbs against human,(34)mouse,(34) rat,(35) rabbit,(36) dog,(37) cat,(30) bovine,(38)horse,(27,39,40) and pig(25) PDPNs. PDPN comprises threetandem repeats of the ‘‘EDxxVTPG’’ sequences, which were
defined to be platelet aggregation-stimulating (PLAG) domains(PLAG1, 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.(41) Almost all mAbs against PDPNs reportedly react with
PLAG domains or PLDs.(41,42) Flow cytometry (Fig. 1) and immunohistochemical analyses (Fig. 2) further confirmed Pro53,

227

Arg54, Arg56, and Tyr60 of pPDPN (Table 2) as the critical
epitope of PMab-213 in this study. As summarized in Figure 3,
Pro53 and Arg54 are included in the PLAG3 domain, whereas
Arg56 and Tyr60 are included in the first PLD. Therefore, PLAG3
and the adjoining PLD of pPDPN were also clarified to be advantageous epitopes for several applications, such as flow cytometry, Western blotting, and immunohistochemical analyses.
PMab-213 showed no reaction with human, mouse, rat,
rabbit, dog, cat, bovine, horse, Tasmanian devil, tiger, alpaca, bear, goat, sheep, or whale PDPNs, thereby indicatingthat PMab-213 is specific to pPDPN.(25) Indeed, the corresponding sequence of PMab-213 ‘‘PRTREEPY’’ of human
PDPN is ‘‘PGTSEDRY,’’ which indicates that only two of
the four aas are the same (share 50% homology). Similarly,‘‘TGATGGLN’’ of mouse PDPN (0/4 aas: 0% homology),‘‘TDTTGELD’’ of rat PDPN (0/4 aas: 0% homology),‘‘DGATEEPY’’ of dog PDPN (1/4 aas: 25% homology), and‘‘PAASKESL’’ of bovine PDPN (1/4 aas: 25% homology)
are different from the corresponding sequence of the PMab213 epitope.(4)FIG. 3. Schematic illustration of the epitope recognized by PMab-213. PLAG, platelet aggregation-stimulating; PLD,
PLAG-like domain.

228

In summary, the critical epitopes of PMab-213 are Pro53,
Arg54, Arg56, and Tyr60 of pPDPN. Among these epitopes,
Pro53 and Arg54 are included in the PLAG3 domain. Ser/Thr
residues, which are involved in O-glycosylation, are not included in PMab-213 epitope, indicating that PMab-213 was
not categorized into GpMabs.(42) Conversely, PLAG3 and the
adjoining PLD of pPDPN were clarified to be advantageous
epitopes for several applications. These findings can be applied
to the production of more functional anti-pPDPN mAbs.
Author Disclosure Statement

No competing financial interests exist.

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Funding Information

This research was supported, in part, by AMED underGrant Nos. JP19am0101078 (Y.K.), JP18am0301010 (Y.K.),
and JP19ae0101028 (Y.K.), and by JSPS KAKENHI Grant
No. 17K07299 (M.K.K.), Grant No. 16K10748 (Y.K.), andGrant No. 19K07705 (Y.K.).
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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: May 15, 2019
Accepted: July 30, 2019