MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 6, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0033

Epitope Mapping of PMab-225 an Anti-Alpaca Podoplanin
Monoclonal Antibody Using Flow Cytometry

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Yusuke Sayama,1 Masato Sano,1 Yoshikazu Furusawa,1,2 Mika K. Kaneko,1 and Yukinari Kato1,2

Podoplanin (PDPN) is a mucin-type membrane glycoprotein, and possesses three platelet aggregationstimulating (PLAG) domains: PLAG1, PLAG2, and PLAG3 at the N-terminus of PDPN, and one or two PLAGlike domains (PLDs) in the middle of PDPN. PDPN is expressed on normal tissues, such as podocytes of thekidney and type I alveolar cells of the lung, and is also overexpressed in numerous malignant cancers.
Previously, we reported a novel anti-alpaca podoplanin (aPDPN) monoclonal antibody (mAb), PMab-225,using Cell-Based Immunization and Screening (CBIS) method. PMab-225 specifically detected aPDPNoverexpressed Chinese hamster ovary (CHO)-K1 (CHO/aPDPN) cells using flow cytometry and western
blotting, and strongly stained alpaca tissues such as lung type I alveolar cells by immunohistochemistry.
However, the specific binding epitope of aPDPN for PMab-225 remains unclear. Thus, in this study, a series ofdeletion or point mutations of aPDPN were utilized for investigating the binding epitope of PMab-225 using
flow cytometry. The analysis of deletion mutants showed that N-terminus of PMab-225 epitope might exist
between 80 amino acid (aa) and 85 aa of aPDPN. Furthermore, the analysis of point mutants demonstrated thatThr84 of aPDPN, which exists in PLD, could be included in the critical epitope of PMab-225.
Keywords: alpaca podoplanin, monoclonal antibody, epitopeIntroduction

Podoplanin (PDPN/T1alpha/Aggrus), a type I transmembrane glycoprotein, is expressed in normal tissues,
such as lymphatic endothelial cells, type I lung alveolar cells,
and renal podocytes.(1–3) PDPN has been also known as aplatelet aggregation-inducing factor in highly metastatic tumor cells.(1,4) PDPN is frequently overexpressed in various
malignant tumors, including several squamous cell carcinomas, mesothelioma, glioblastoma, and osteosarcoma.(5–7)
PDPN possesses three platelet aggregation-stimulating(PLAG) domains: PLAG1, PLAG2, and PLAG3; these domains exist at the N-terminus of PDPN.(8) Furthermore, one
or two PLAG-like domains (PLDs) have been reported to bepresent in the middle of PDPN.(9) PLAG1–3 and/or PLD
elicit tumor cell-induced platelet aggregation response
through the activation of C-type lectin-like receptor 2(CLEC-2) to facilitate cancer progression and metastasis.(1,3)
These results imply the importance of the developing antiPDPN monoclonal antibodies (mAbs) for cancer therapeutictreatment.
Alpaca (lama pacos) is a livestock that is used in various
applications in Latin America. This animal has also beenused for the production of antigen-specific single domain
12

antibodies (nanobodies).(10,11) However, membrane proteinsof alpaca have not been investigated due to the lack of specific antibodies. Because PDPN is important for blood/
lymphatic vessel separation,(12,13) high endothelial venule
integrity,(14) and embryonic cardiac development,(15) sensitive and specific mAbs against alpaca PDPN (aPDPN) arenecessary to analyze the expression and function of aPDPN.
Recently, we developed a novel anti-aPDPN mAb, PMab225, using Cell-Based Immunization and Screening (CBIS)
method. PMab-225 specifically detected aPDPN by flow
cytometry, western blotting, and immunohistochemical
analysis.(16) However, the binding epitope of PMab-225 for
aPDPN remains to be clarified. Thus, this study aimed toidentify the epitope of PMab-225 using deletion mutants orpoint mutants of aPDPN.
Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 was obtained fromthe American Type Culture Collection (ATCC, Manassas,VA). The aPDPN mutation plasmids containing N-terminal
RAP16 tag were transfected into CHO-K1 cells using

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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FIG. 1. Epitope mapping of PMab-225 using deletion mutants of aPDPN. (A) Illustration of WT and 10 deletion mutants
aPDPN: dN30, dN40, dN50, dN60, dN70, dN80, dN85, dN90, dN95, and dN100. Black bars: the positive reaction of PMab225. White bars: the negative reaction of PMab-225. (B) Deletion mutants of aPDPN were analyzed using flow cytometry.
Deletion mutants were incubated with PMab-2 (anti-RAP16 tag; left panel, red line), PMab-225 (anti-aPDPN mAb; rightpanel, blue line), or buffer control (black line) for 30 minutes at 4C, followed by secondary antibodies. aPDPN, alpaca
podoplanin; mAb, monoclonal antibody; PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain; WT, wild type.

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FIG. 2. Epitope mapping of PMab-225 using point mutants of aPDPN. (A, B) Transient point mutants expressing E80A,
D81A, L82A, P83A, T84A, A85G, E86A, S87A, T88A, V89A, H90A, and V91A of aPDPN were treated with PMab-2(A) (red line), PMab-225 (B) (blue line), or buffer control (A, B) (black line) for 30 minutes at 4C, followed by secondaryantibodies. (C) Schematic illustration of the epitope recognized by PMab-225. Underlined amino acid is a critical epitope ofPMab-225.

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Lipofectamine LTX (Thermo Fisher Scientific, Inc., Waltham, MA). The RAP16 tag comprises 16 amino acids (aa;GPGDDMVNPGLEDRIE).(17) Transiently transfected cellswith deletion/point-mutants were cultured in Roswell Park
Memorial Institute (RPMI) 1640 medium (Nacalai Tesque,Inc., Kyoto, Japan), supplemented with 10% heat-inactivatedfetal bovine serum (Thermo Fisher Scientific, Inc.), 100
units/mL of penicillin, 100 mg/mL of streptomycin, and25 mg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37Cin a humidified atmosphere of 5% CO2 and 95% air.

Next, to identify the binding epitope of PMab-225, we
produced a series of point mutations of aPDPN in CHO-K1
cells, including E80A, D81A, L82A, P83A, T84A, A85G,E86A, S87A, T88A, V89A, H90A, and V91A. PMab-2 reacted with all point mutants (Fig. 2A). In contrast, PMab-225
reacted with all these mutants, except for T84A (Fig. 2B),
indicating that Thr84 of aPDPN could be included in thecritical epitope of PMab-225.

Production of aPDPN mutants

We have demonstrated that critical epitope of PMab-225
could be Thr84 of aPDPN using deletion mutants and point
mutants of aPDPN. This result does not conclude that only
Thr84 is a PMab-225-binding amino acid. In our experiences, the binding epitope of mAbs has been determined tobe 5–13 aa(18,19); therefore, PMab-225 might bind to thepeptide or the glycopeptide around Thr84 of aPDPN. We
should perform X-ray crystallography to exactly determine
the binding epitope of PMab-225 to aPDPN in the future
study.
We have developed mAbs against many mammalianPDPNs, such as human,(20) mouse,(20) rat,(21) rabbit,(22)dog,(23) cat,(24) pig,(25,26) bovine,(27) horse,(28) Tasmaniandevil,(29) tiger,(30) and bear(31) PDPNs. Almost all mAbs
against PDPNs have been reported to react with the PLAG1–3 domains or PLD.(9,24,32) Actually, Thr84 of aPDPN exists in
PLD (Fig. 2C). In the future study, we should investigate
whether PMab-225 could neutralize the aPDPN-induced
platelet aggregation.
In conclusion, Thr84 of aPDPN, which is located in PLD,
is critical for PMab-225-specific binding to aPDPN. PMab225 could be a useful tool for elucidating the pathophysiological function of aPDPN.

The coding sequence of aPDPN was subcloned into apCAG vector (FUJIFILM Wako Pure Chemical Corporation,Osaka, Japan), and a RAP16 tag was added at the N-terminus.
Deletion mutants of aPDPN sequence were performed usinga HotStar HiFidelity PCR (polymerase chain reaction; Qiagen, Inc., Hilden, Germany) with oligonucleotides containing
the desired mutations. Substitutions of amino acids to alanine
in aPDPN sequence were conducted by QuikChange lightning site-directed mutagenesis kit (Agilent Technologies,Inc., Santa Clara, CA). PCR fragments bearing the desired
mutations were inserted into pCAG vector using In-Fusion
PCR cloning kit (Takara Bio, Inc., Shiga, Japan).
Flow cytometry

Transiently transfected CHO-K1 cells were detached
by 0.25% trypsin/1 mM ethylenediaminetetraacetic acid
(EDTA; Nacalai Tesque, Inc.) and collected in RPMI 1640
medium. After washing with 0.1% bovine serum albumin/phosphate buffered saline, the cells were incubated with
anti-aPDPN antibody (PMab-225; 1 mg/mL) or control antiRAP16 tag antibody (PMab-2; 1 mg/mL) for 30 minutes at4C. Alexa Fluor 488-conjugated anti-mouse IgG (1:1000;Cell Signaling Technology, Inc., Danvers, MA) were added
to each cell, and incubated for 30 minutes at 4C. Fluorescence data were collected using a Cell Analyzer EC800(Sony Corp., Tokyo, Japan), and were analyzed using FlowJo(BD Biosciences, Franklin Lakes, NJ).
Results

Ten deletion mutants and wild type (WT) of aPDPN weregenerated (Fig. 1A). Transient transfectants were produced
using CHO-K1 cells, including WT (corresponding to 27–162 aa), dN30 (corresponding to 30–162 aa), dN40 (corresponding to 40–162 aa), dN50 (corresponding to 50–162 aa),dN60 (corresponding to 60–162 aa), dN70 (corresponding to70–162 aa), dN80 (corresponding to 80–162 aa), dN85
(corresponding to 85–162 aa), dN90 (corresponding to 90–162 aa), dN95 (corresponding to 95–162 aa), and dN100(corresponding to 100–162 aa). All deletion mutants andWT of aPDPN containing N-terminal RAP16 tag were analyzed using flow cytometry for the epitope mapping of
PMab-225. PMab-2 (an anti-RAP16 tag mAb) detected all
deletion mutants of aPDPN (Fig. 1B, left), indicating that theexpression level of each deletion mutant is very high. In
contrast, PMab-225 did not react with dN85, dN90, dN95,
and dN100 (Fig. 1B, right), suggesting that N-terminus of
PMab-225 epitope might exist between 80 aa and 85 aa ofaPDPN.

Discussion

Author Disclosure Statement

The authors have no conflict of interest.
Funding Information

This research was supported in part by AMED under grant
numbers JP19am0401013 (Y.K.), JP19am0101078 (Y.K.),
and JP19ae0101028 (Y.K.), and by JSPS KAKENHI grant
numbers 17K07299 (M.K.K.) and 19K07705 (Y.K.).
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SAYAMA ET AL.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: July 13, 2019
Accepted: September 17, 2019