MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 3, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0008

Detection of Lion Podoplanin Using the Antitiger
Podoplanin Monoclonal Antibody PMab-231

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Yukinari Kato,1,2 Junko Takei,1 Masato Sano,1 Teizo Asano,1 Yusuke Sayama,1Kazuyuki Uchida,3 Takayuki Nakagawa,4 and Mika K. Kaneko1Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells,
are needed for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including
human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, andsheep PDPNs. However, no antilion PDPN (lioPDPN) antibody has been developed. In this study, possible
cross-reaction between available anti-PDPN mAbs and lioPDPN was examined. Immunohistochemical analysis
showed that antitiger PDPN mAb PMab-231 (IgG2a, kappa) reacted with type I alveolar cells from lion lung,indicating that PMab-231 is useful for the detection of lioPDPN.
Keywords: lion podoplanin, PDPN, lioPDPN, PMab-231

LpMab-3, LpMab-9, LpMab-12, LpMab-19, and LpMab-21
are antiglycopeptide mAbs (GpMabs), epitopes of which
include both peptides and glycans. Cancer-specific mAbs(CasMabs), such as LpMab-2(23,32) and LpMab-23,(33,34) may
be used for PDPN-targeted therapy(23,32–35) since PDPN isexpressed in many healthy tissues.
To date, antilion PDPN (lioPDPN) mAbs have not beenreported. In the present study, cross-reaction between antiPDPN mAbs for various species and lioPDPN were examinedusing immunohistochemical analyses.

Introduction

Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein that induces platelet aggregation throughplatelet C-type lectin-like receptor-2 (CLEC-2).(1) It is
composed of three platelet aggregation-stimulating (PLAG)
domains, termed PLAG1–3 (EDxxVTPG sequence).(2) Previously, PLAG3 is the most important domain for platelet
aggregation by human PDPN.(2–4) Recently, a PLAG-likedomain (PLD; EDxxT sequence) was reported to be anothercritical sequence for PDPN–CLEC-2 interaction.(5)Anti-PDPN monoclonal antibodies (mAbs) are useful indistinguishing lymphatic from vascular endothelial cells ortype I from type II alveolar cells of the lung.(2,6) PDPNs frommany animals have been characterized using specific antiPDPN mAbs, including antimouse PDPN (PMab-1),(7)antirat PDPN (PMab-2),(8) antirabbit PDPN (PMab-32),(9)antidog PDPN (PMab-38(10) and PMab-48,(11)) antibovine
PDPN (PMab-44),(12) anticat PDPN (PMab-52),(13) antipig
PDPN (PMab-213),(14) anti-Tasmanian devil PDPN (PMab233),(15) antialpaca PDPN (PMab-225),(16) antitiger PDPN(PMab-231),(17) antiwhale PDPN (PMab-237),(18) antigoat
PDPN (PMab-235),(19) antihorse PDPN (PMab-219),(20) antibear PDPN (PMab-247),(21) and antisheep PDPN (PMab256).(22) Furthermore, many antihuman PDPN mAbs havebeen developed, including NZ-1.2,(7) LpMab-3,(23) LpMab7,(23,24) LpMab-9,(23) LpMab-10,(25) LpMab-12,(26) LpMab13,(27) LpMab-17,(28) LpMab-19,(29) and LpMab-21.(30,31)

Materials and Methods
Immunohistochemical analyses

Lion lungs were collected at necropsy at the Universityof Tokyo, fixed in 10% neutral-buffered formalin, andprocessed routinely to produce formalin-fixed paraffinembedded tissue (FFPE) sections. Four micrometers histological sections were autoclaved in citrate buffer (pH 6.0;
Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes.
After blocking with SuperBlock T20 (PBS) Blocking
Buffer (Thermo Fisher Scientific, Inc., Waltham, MA) for
15 minutes, sections were incubated with anti-PDPN mAbs
(10 lg/mL) for 1 hour at room temperature and treated using
Envision+ Kit (Agilent Technologies, Inc., Santa Clara, CA)
for 30 minutes. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies,Inc.) for 2 minutes, and finally, sections were counterstained

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
4
Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
2391

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92

FIG. 1. Immunohistochemical analyses of lion lung tissues. Histological sections of the lion lung were directly
autoclaved in citrate buffer for 20 minutes. After blocking,sections were incubated with 10 mg/mL of PMab-231 (A, B)
or PMab-237 (C, D), followed by detection using Envision+Kit. (E, F) HE staining. Scale bar = 100 mm. HE, hematoxylin and eosin.
with hematoxylin/eosin (FUJIFILM Wako Pure ChemicalCorporation, Osaka, Japan).
Results

In this study, cross-reaction between anti-PDPN mAbs
against several mammalian species and lioPDPN was examined because a specific anti-lioPDPN mAb has not been
available. Immunohistochemical analysis revealed that antitiger PDPN mAb PMab-231 (IgG2a, kappa)(17) reacted withtype I alveolar cells in lion lung (Fig. 1A, B). In contrast,
PMab-237 (an antiwhale PDPN mAb) did not react with thelion lung using the next consecutive tissue section (Fig. 1C,D). Similarly, other anti-PDPN mAbs, including all mAbs
already listed did not react with the lion lung using additional
consecutive tissue sections (data not shown). Hematoxylin
and eosin staining using the final consecutive tissue section
indicated that tissue was well preserved (Fig. 1C).
Using the same methods, PMab-231 staining of other tissues was investigated, since PDPN is expressed in tissues
other than lung.(6,36) PMab-231 did not stain renal podocytes,
Bowman’s capsule, or lymphatic endothelial cells (data notshown).
Discussion

To date, antilioPDPN mAbs have not been established dueto lack of the relevant amino acid or DNA sequence oflioPDPN using fresh lion lung tissues. Instead, anti-PDPN
mAbs developed for several mammalian tissues were tested
for cross-reaction with lion lung, collected at necropsy, using
immunohistochemical analyses. Only antitiger PDPN mAb

KATO ET AL.

(PMab-231) stained type I alveolar cells of lion lung (Fig. 1).
Previously, PMab-231 specifically detected tiger PDPN using flow cytometry, Western blot, and immunohistochemicalanalyses.(17) PMab-231 cross-reacted with cat PDPN, andstained type I alveolar cells using immunohistochemistry.
Dissociation constants (KD) of PMab-231 for PDPNexpressing CHO-K1 cells (CHO/tigPDPN and cPDPN) weredetermined to be 1.2 · 10-8 and 1.9 · 10-8, for tiger and cat,respectively, indicating similar affinity across species.
Although the staining intensity of PMab-231 against lion
lung seems weak (Fig. 1), intensity of PMab-231 in cat lungwas previously moderate to strong.(17) This difference islikely due to variation in binding epitopes between cat andlion, or PDPN protein in FFPE sections of lion tissues might
have been damaged. Lack of reaction of PMab-231 withkidney and colon tissues remains unexplained. Similar data
were observed previously using an antibovine PDPN mAb(PMab-44) against sheep,(37) goat,(38) and alpaca tissues.(39)Thus, expression levels of PDPN in the lung may be higher
than levels in kidney or colon, or, perhaps binding epitopes ofmAbs are different among tissues due to post-translational
modification, such as glycosylation. Additional lion tissues
could be investigated to further characterize immunoreactivity of PMab-231.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by AMED underGrant Nos. JP20am0401013 (Y.K.), JP20am0101078 (Y.K.),
and JP20ae0101028 (Y.K.), and by JSPS KAKENHI Grant
No. 17K07299 (M.K.K.) and Grant 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 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 9, 2020
Accepted: April 15, 2020