MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 41, Number 3, 2022
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2022.0014
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Epitope Mapping of an Anti-Chinese/Golden Hamster
Podoplanin Monoclonal Antibody

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Nohara Goto,1,* Hiroyuki Suzuki,1,* Tomohiro Tanaka,2 Teizo Asano,2Mika K. Kaneko,2 and Yukinari Kato1,2

Chinese hamster (Cricetulus griseus) and golden hamster (Mesocricetus auratus) are important animal models
of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, which affect several organs,including respiratory tract, lung, and kidney. Podoplanin (PDPN) is a marker of lung type I alveolar cells,
kidney podocytes, and lymphatic endothelial cells. The development of anti-PDPN monoclonal antibodies(mAbs) for these animals is essential to evaluate the pathogenesis by SARS-CoV-2 infections. Using the CellBased Immunization and Screening method, we previously developed an anti-Chinese hamster PDPN
(ChamPDPN) mAb, PMab-281 (mouse IgG3, kappa), and further changed its subclass into IgG2a (281-mG2a-f),
both of which can recognize not only ChamPDPN but also golden hamster PDPN (GhamPDPN) by flow
cytometry and immunohistochemistry. In this study, we examined the critical epitope of 281-mG2a-f, using
enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA withpeptides derived from ChamPDPN and GhamPDPN extracellular domain, and found that 281-mG2a-f reactedwith the peptides, which commonly possess the KIPFEELxT sequence. Next, we analyzed the reaction with thealanine-substituted mutants, and revealed that 281-mG2a-f did not recognize the alanine-substituted peptides of
I75A, F77A, and E79A of ChamPDPN. Furthermore, these peptides could not inhibit the recognition of 281mG2a-f to ChamPDPN-expressing cells by flow cytometry. The results indicate that the binding epitope of 281mG2a-f includes Ile75, Phe77, and Glu79 of ChamPDPN, which are shared with GhamPDPN.
Keywords: hamster podoplanin, epitope mapping, monoclonal antibody, enzyme-linked immunosorbent assay
IntroductionP

odoplanin (PDPN) is a type I transmembrane mucinlike glycoprotein that plays critical roles in tumor progression(1) as well as normal development including lung,(2)kidney,(3) and lymphatic vessels.(4) The N-terminal extracellular domain has a repeat sequence named platelet
aggregation-stimulating (PLAG)1 to PLAG3 domains(5) that
promote platelet aggregation through interaction with aplatelet receptor, C-type lectin-like receptor 2 (CLEC-2).(6,7)
Furthermore, several PLAG-like domains (PLDs, one ofwhich is named PLAG4 domain) with similar sequences,were identified.(8)Furthermore, PDPN regulates the signal transduction
through its cytoplasmic tail, which is involved in cell proliferation, migration, invasion, epithelial-to-mesenchymal tran-

sition, and stemness.(9) PDPN expression is also elevatedin tumor stroma including cancer-associated fibroblasts(CAFs)(10) and lymphocytes.(11) CAFs remodel the extracellular matrix and the play a critical role in the formation of
immunosuppressive tumor microenvironment.(12,13)
PDPN is also important as a marker of lung type I alveolar
cells, kidney podocytes, and lymphatic endothelial cells.(1)We have developed anti-PDPN monoclonal antibodies (mAbs)
against 17 species,(14–30) which are useful for flow cytometry and immunohistochemistry. These mAbs are expected tocontribute not only to the research of each animal but also topathogenic diagnosis.
Using the Cell-Based Immunization and Screening (CBIS)
method,(14,15,31–45) we recently developed anti-PDPN mAbs
against Chinese hamster (Cham)/golden hamster (Gham)(46)
and ferret,(47) which are small animal models of severe acute

Departments of 1Molecular Pharmacology and 2Antibody Drug Development, Tohoku University Graduate School of Medicine,
Sendai, Japan.
*These authors contributed equally to this study.

163

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GOTO ET AL.

respiratory syndrome coronavirus 2 (SARS-CoV-2) infections.(48,49) These mAbs will contribute to the morphologicalalterations and evaluation of the pathogenesis of SARS-CoV2-infected lung type I alveolar cells and kidney podocytes.
In this study, we performed the epitope mapping using
enzyme-linked immunosorbent assay (ELISA) to clarify
further characteristics of an anti-Cham/GhamPDPN mAb,
281-mG2a-f.
Materials and Methods
PeptidesChamPDPN (accession no.: AB205160) and GhamPDPN(accession no.: XM_021233536) peptides (Table 1) and 20
alanine-substituted peptides (Table 2) were synthesized byutilizing PEPScreen (Sigma-Aldrich Corp., St. Louis, MO).

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ELISA

Synthesized ChamPDPN and GhamPDPN peptides(Tables 1 and 2) were immobilized on Nunc Maxisorp 96well immunoplates (Thermo Fisher Scientific, Inc., Waltham,MA) at a concentration of 10 lg/mL for 30 minutes at 37C.
After washing with phosphate-buffered saline (PBS) containing 0.05% Tween20 (PBST; Nacalai Tesque, Inc., Kyoto,
Japan), wells were blocked with 1% bovine serum albumin(BSA)-containing PBST for 30 minutes at 37C.
The plates were incubated with 1 lg/mL of 281-mG2a-f,
followed by peroxidase-conjugated anti-mouse immunoglobulins (1:2000 diluted; Agilent Technologies, Inc., Santa
Clara, CA). Enzymatic reactions were performed using theTable 2. Identification of the 281-mG2a-f Epitope
Using Alanine-Substituted Chinese Hamster
Podoplanin Peptides
PeptidesSequences281-mG2a-f

73–92
T73A
K74A
I75A
P76A
F77A
E78A
E79A
L80A
P81A
T82A
P83A
G84A
I85A
S86A
D87A
H88A
D89A
G90A
E91A
E92A

TKIPFEELPTPGISDHDGEEAKIPFEELPTPGISDHDGEETAIPFEELPTPGISDHDGEE
TKAPFEELPTPGISDHDGEE
TKIAFEELPTPGISDHDGEE
TKIPAEELPTPGISDHDGEE
TKIPFAELPTPGISDHDGEE
TKIPFEALPTPGISDHDGEE
TKIPFEEAPTPGISDHDGEE
TKIPFEELATPGISDHDGEE
TKIPFEELPAPGISDHDGEE
TKIPFEELPTAGISDHDGEE
TKIPFEELPTPAISDHDGEE
TKIPFEELPTPGASDHDGEE
TKIPFEELPTPGIADHDGEE
TKIPFEELPTPGISAHDGEE
TKIPFEELPTPGISDADGEE
TKIPFEELPTPGISDHAGEE
TKIPFEELPTPGISDHDAEETKIPFEELPTPGISDHDGAETKIPFEELPTPGISDHDGEA

+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

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

ELISA POD Substrate TMB Kit (Nacalai Tesque, Inc.).
Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA).
Flow cytometry

Table 1. Epitope Mapping of 281-mG2a-f Using
Deletion Mutants
PeptidesSequences

ChamPDPN
23–42
GAIGRLEDDIVTPGARDGMV
33–52
VTPGARDGMVTPGLEDRIGT
43–62
TPGLEDRIGTTGATEVLNES
53–72
TGATEVLNESTGKAPLVPTH
63–82
TGKAPLVPTHTKIPFEELPT
73–92
TKIPFEELPTPGISDHDGEE
83–102 PGISDHDGEEHTSTTTVRMV
93–112 HTSTTTVRMVTSHSADKETS
103–122 TSHSADKETSHPNRDNTADE
113–132 HPNRDNTADETQTTDKRDGL
123–135 TQTTDKRDGLAVVGhamPDPN
48–67
ALLKGLEDDIVTPGARDGMV
58–77
VTPGARDGMVTPGLEDRTTT
68–87
TPGLEDRTTTTGGLNEPTGK
78–97
TGGLNEPTGKAPLVPTHAKI
88–107 APLVPTHAKIPFEELSTPGV
98–117 PFEELSTPGVSDHDDKEHKS
108–127 SDHDDKEHKSTTTVRMVTSH
118–137 TTTVRMVTSHSSDKETSHPN
128–147 SSDKETSHPNIDNTADETQT
138–157 IDNTADETQTTDKRDGLAVV
148–157 TDKRDGLAVV

281-mG2a-f
+++
+++
+++
-

+++, OD655 S 0.3; -, OD655 < 0.1.
ChamPDPN, Chinese hamster PDPN; GhamPDPN, golden hamster PDPN; PDPN, podoplanin.

2 · RIEDL-ChamPDPN-overexpressed Chinese hamster
ovary-K1 (CHO/ChamPDPN)(46) was harvested after a brief
exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.) and washed with 0.1%
BSA (Nacalai Tesque, Inc.) in PBS (Nacalai Tesque, Inc.).
The 281-mG2a-f (0.01 lg/mL) was incubated with each
peptide (10 lg/mL) for 30 minutes at 4C. CHO/ChamPDPN
cells were treated with 281-mG2a-f + each peptide, and further treated with Alexa Fluor 488-conjugated anti-mouse IgG(1:1000). Fluorescence data were collected using the SA3800
Cell Analyzer (Sony Biotechnology Corp., Tokyo, Japan).
Results
Epitope mapping of 281-mG2a-f using deletion mutants

We previously established an anti-ChamPDPN mAb(PMab-281, mouse IgG3, kappa) by the CBIS method.(46) The
subclass of PMab-281 was converted from IgG3 to IgG2a
because the mouse IgG3 subclass is easy to aggregate. In
addition, a defucosylated anti-ChamPDPN mAb (281-mG2af) was produced using BINDS-09 cells (FUT8-deficient
ExpiCHO-S cells(50,51)). We found that 281-mG2a-f could
recognize both ChamPDPN and GhamPDPN by flow cytometry and immunohistochemistry.(46) To reveal the binding
epitope of 281-mG2a-f, we synthesized 22 peptides (Table 1),
which consist of 20 amino acids (aa) of extracellular domain
of ChamPDPN and GhamPDPN, and performed ELISA. As
shown in Figure 1A, 281-mG2a-f recognized both the 63–82
aa (TGKAPLVPTHTKIPFEELPT) and the 73–92 aa

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EPITOPE MAPPING OF ANTIHAMSTER PODOPLANIN MAB

FIG. 1. Determination of the 281-mG2a-f epitope for
ChamPDPN and GhamPDPN by ELISA using deletionmutants. (A) Synthesized peptides of ChamPDPN and
GhamPDPN were immobilized on immunoplates. The plates
were incubated with 281-mG2a-f (1 mg/mL), followed byincubation with peroxidase-conjugated anti-mouse immunoglobulins. (B) Sequence alignment of reacted peptides.
ChamPDPN, Chinese hamster PDPN; ELISA, enzymelinked immunosorbent assay; GhamPDPN, golden hamster
PDPN; PDPN, podoplanin.

(TKIPFEELPTPGISDHDGEE) sequences of ChamPDPN.
Furthermore, 281-mG 2a -f recognized the 88–107 aa(APLVPTHAKIPFEELSTPGV) sequence of GhamPDPN.
Compared with the sequences, the epitope of 281-mG2a-f
was suggested to exist in the 74–82 aa (KIPFEELPT) of
ChamPDPN (Fig. 1B).
Epitope mapping of 281-mG2a-f using
alanine-substituted PDPN peptides

Then, we synthesized 20 alanine-substituted peptides derived from the 73–92 aa peptide of ChamPDPN (Table 2).
The 281-mG2a-f exhibited reaction with T73A, K74A, P76A,
E78A, L80A, P81A, T82A, P83A, G84A, I85A, S86A,
D87A, H88A, D89A, G90A, E91A, E92A, and wild-type(WT, 73–92 aa) (Fig. 2A). In contrast, 281-mG2a-f did notreact with I75A, F77A, and E79A (Fig. 2A), indicating thatIle75, Phe77, and Glu79, which are shared with GhamPDPN,
are included in the critical epitope of 281-mG2a-f. The results
are summarized in Figure 2B.
Flow cytometry using 281-mG2a-f
with alanine-substituted PDPN peptide

We performed a blocking assay using flow cytometry. As
shown in Figure 3, 281-mG2a-f reacted with the CHO/
ChamPDPN cells. This reaction was almost completely
neutralized by WT and L80A, partially inhibited by K74A
and P76A, and slightly inhibited by E78A. In contrast, I75A,
F77A, and E79A did not block the reaction of 281-mG2a-f

165

FIG. 2. Determination of the 281-mG2a-f epitope of
ChamPDPN by ELISA using alanine-substituted PDPN peptides. (A) The alanine-substituted ChamPDPN peptides were
immobilized on immunoplates. The plates were incubated with
281-mG2a-f (1 mg/mL), followed by peroxidase-conjugatedanti-mouse immunoglobulins. (B) Schematic illustration of
ChamPDPN and the 281-mG2a-f epitope. The 281-mG2a-f
epitope involves Ile75, Phe77, and Glu79 of ChamPDPN.

with CHO/ChamPDPN. These results confirm that Ile75,
Phe77, and Glu79 of ChamPDPN are critical for 281-mG2a-f
detection.
Discussion

PDPN possesses three tandem repeats of the ‘‘EDxxVTPG’’sequences, which were defined as PLAG1, PLAG2, and
PLAG3 domains in the N-terminus.(5) Furthermore, there areseveral PLDs of the ‘‘E(D/E)xx(T/S)xx’’ sequences in thecentral part of PDPN.(8) In this study, we determined thecritical epitope of 281-mG2a-f as Ile75, Phe77, and Glu79 of
ChamPDPN (Fig. 2B). Glu79 is included in the first PLD(aa 78–82).
Since PLDs are reportedly important for the PDPN–
CLEC-2 interaction and induction of platelet aggregation,(8)further studies are needed to investigate the role of the first
PLD for platelet aggregation and the neutralizing activity of
281-mG2a-f. The O-glycosylation of Thr in the PLAG3 or
PLD has been reported to be essential for PDPN-induced
platelet aggregation.(8,52) However, Ser/Thr residues are notincluded in 281-mG2a-f epitope, indicating that 281-mG2a-f
was not categorized into GpMabs.(53)
We have not examined the crossreactivity of 281-mG2a-f
with other species excluding Gham. Therefore, we searched
the conservation of ‘‘IPFEE’’ sequence to other species
PDPN using standard protein BLAST (the Basic Local

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GOTO ET AL.

FIG. 3. Flow cytometry using 281-mG2a-f and peptides of ChamPDPN. The 281-mG2a-f (0.01 mg/mL), 281-mG2a-f(0.01 mg/mL) plus the alanine-substituted peptides (10 mg/mL), or control (blocking buffer) were reacted with CHO/
ChamPDPN cells for 30 minutes at 4C, followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG. CHO,
Chinese hamster ovary-K1.

Alignment Search Tool, NCBI). Only creeping vole (Microtus oregoni) possesses similar ‘‘IPFED’’ sequence, suggesting the crossreactivity by 281-mG2a-f.
In animal models of SARS-CoV-2, Ghams exhibit
similar pathogenesis and transmissibility found in humans
with SARS-CoV-2 infections.(54) The disease severity is
known to be typically lower in pediatric patients than in
adults, particularly the elderly patients.(55) Angiotensinconverting enzyme 2 (ACE2) serves as the entry receptor
for SARS-CoV-2.(56) Age-dependent upregulation of
ACE2 in PDPN-positive lung type I alveolar cells wasreported in mouse and human.(57) The 281-mG2a-f willcontribute to the analysis to detect PDPN-positive lungtype I alveolar cells in Gham and could provide an important information of the age-related correlation of disease severity in the animal model.

Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by Japan Agency for
Medical Research and Development (AMED) under Grant
Nos. JP22ama121008 (to Y.K.), JP22am0401013 (to Y.K.),
JP22bm1004001 (to Y.K.), JP22ck0106730 (to Y.K.), andJP21am0101078 (to Y.K.).
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Address correspondence to:
Yukinari Kato
Department of Molecular Pharmacology
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 12, 2022
Accepted: April 19, 2022