MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 4, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0018

Development and Characterization of Anti-Sheep Podoplanin
Monoclonal Antibodies PMab-253 and PMab-260

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

Mika K. Kaneko,1 Masato Sano,1 Junko Takei,1 Teizo Asano,1 Yusuke Sayama,1Hideki Hosono,1 Atsushi Kobayashi,2 Satoru Konnai,3,4 and Yukinari Kato1,5

Anti-podoplanin (PDPN) monoclonal antibodies (mAbs) are needed as markers for lymphatic endothelial cells ortype I alveolar cells in immunohistochemical analyses. We have developed anti-PDPN mAbs for many species,
including humans, mice, rats, rabbits, dogs, cats, bovines, pigs, Tasmanian devils, alpacas, tigers, whales, goats,horses, and bears. This study develops and characterizes anti-sheep PDPN (sPDPN) mAbs using Cell-Based
Immunization and Screening (CBIS) method. A RAP14 tag was added to the N-terminus of sPDPN, and an antiRAP14 tag mAb (PMab-2) was used to measure the expression level of sPDPN in flow cytometry and Western
blots. We immunized mice with sPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/sPDPN) cells
and screened mAbs against sPDPN using flow cytometry. Two of the mAbs, PMab-253 (immunoglobulin M[IgM], kappa) and PMab-260 (IgM, kappa), detected CHO/sPDPN cells specifically using flow cytometry andWestern blots. Both PMab-253 and PMab-260 stained the renal glomerulus and Bowman’s capsule, lymphatic
endothelial cells of the lung and colon, and type I alveolar cells of the lung, suggesting PMab-253 and PMab-260,
which were developed by CBIS method, can be applied to functional analyses of sPDPN. We also determined thebinding epitope of PMab-253 and PMab-260 using flow cytometry. Analysis of sPDPN deletion mutants revealed
that the N-terminus of the PMab-253 and PMab-260 epitope exists between amino acids 110 and 115 of sPDPN.
Analysis of sPDPN point mutations revealed that the critical epitope of PMab-253 and PMab-260 includes Thr112
and Ser113 of sPDPN, indicating that the PMab-253 and PMab-260 epitope are independent of the platelet
aggregation-stimulating (PLAG) domain or the PLAG-like domain of sPDPN.
Keywords: podoplanin, sheep, sPDPN, monoclonal antibody

Introduction

S

heep are used as experimental animals to study the
immune system,(1–3) development of the blood–brain
barrier,(4) and lymphopoiesis/lymphocyte recirculation,(5)among other subjects. However, detailed morphologicalstudies of sheep lymphatic vessels have not been performed
due to the lack of specific markers for lymphatic endothelial
cells. Podoplanin (PDPN) is a type I transmembrane
O-glycoprotein, which is expressed in renal podocytes, thelymphatic endothelial cells of every organ, and type I alveolar cells of the lung.(6,7) In morphological studies, PDPN is
an important specific marker of lymphatic vessels.(7) PDPN
possesses platelet aggregation-stimulating (PLAG) domains(8) and a PLAG-like domain,(9–13) which bind to C-type

lectin-like receptor-2 (CLEC-2).(14) The PDPN/CLEC-2 interaction induces platelet aggregation and separation of embryonic blood/lymphatic vessels.(15)
We previously developed monoclonal antibodies (mAbs)
against human,(16) mouse,(17) rat,(18) rabbit,(19) dog,(20)cat,(21) bovine,(22) pig,(23,24) Tasmanian devil,(25) alpaca,(26)tiger,(27) whale,(28) goat,(29,30) horse,(31,32) and bear(33,34)
PDPNs using Cell-Based Immunization and Screening(CBIS) method.(35–37) These mAbs have all proven useful inflow cytometry, Western blots, and immunohistochemical
analyses. In this study, we describe the development of antisheep PDPN (sPDPN) mAbs using CBIS to stain lymphatic
endothelial cells by immunohistochemistry. We also determined the binding epitope of anti-sPDPN mAbs using
flow cytometry.

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
3
Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University,
Sapporo, Japan.
4
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
5
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2

144

ANTI-SHEEP PDPN MAB

145

Materials and Methods

wild-type (WT) and mutation plasmids were transfected using Lipofectamine LTX with Plus Reagent (Thermo Fisher
Scientific, Inc., Waltham, MA). Stable transfectants wereselected by limiting dilution and cultivation in medium
containing 0.5 mg/mL of G418 (Nacalai Tesque, Inc., Kyoto,
Japan).
CHO-K1, P3U1, CHO/sPDPN,(39) CHO/human PDPN
(hPDPN),(8) CHO/mouse PDPN (mPDPN),(8) CHO/rat
PDPN (rPDPN),(18) CHO/rabbit PDPN (rabPDPN),(19) CHO/
dog PDPN (dPDPN),(20) CHO/bovine PDPN (bovPDPN),(22)CHO/cat PDPN (cPDPN),(21) CHO/pig PDPN (pPDPN),(24)CHO/horse PDPN (horPDPN),(31) CHO/tiger PDPN

Cell lines

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture
Collection (Manassas, VA). Synthesized DNA (Eurofins
Genomics KK, Tokyo, Japan) encoding sPDPN (Accession
No.: XM_004013802.4) and an N-terminal RAP14 tag was
subcloned into a pCAG-Neo vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan). The RAP14 tag
comprised 14 amino acids (aa), (DMVNPGLEDRIEDL) andis recognized by PMab-2 mAb(18) and LpMab-7.(38) sPDPN

FIG. 1. Detection of sPDPN by flow cytometry using anti-sPDPN mAbs. CHO/sPDPN and CHO-K1 cells were treatedwith PMab-253 (A), PMab-260 (B), or anti-RAP14 tag (C) at a concentration of 1 mg/mL or 0.1% BSA in PBS for 30 min,followed by incubation with secondary antibodies. PDPN, podoplanin; sPDPN, sheep PDPN; CHO, Chinese hamster ovary;
BSA, bovine serum albumin; PBS, phosphate-buffered saline; mAbs, monoclonal antibodies.

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

FIG. 2. Cross-reaction of anti-sPDPN mAbs with PDPNs of other species by flow cytometry. CHO-K1 cells transfectedwith PDPNs of other species were treated with PMab-253 (A) or PMab-260 (B) at a concentration of 5 mg/mL or 0.1% BSA
in PBS (gray) for 30 min, followed by incubation with secondary antibodies.
146

ANTI-SHEEP PDPN MAB(tigPDPN),(27) CHO/alpaca PDPN (aPDPN),(26) CHO/bear
PDPN (bPDPN),(24) CHO/Tasmanian devil PDPN
(tasPDPN),(24) CHO/goat PDPN (gPDPN),(27) and CHO/
whale PDPN (wPDPN)(28) were cultured in a Roswell Park
Memorial Institute (RPMI) 1640 medium (Nacalai Tesque,Inc.), supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mL of
penicillin, 100 lg/mL of streptomycin, and 0.25 lg/mL ofamphotericin B (Nacalai Tesque, Inc.). Cells were grownin a humidified incubator at 37C with an atmosphere of
5% CO2 and 95% air.

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

Animals

All animal experiments were performed according torelevant guidelines and regulations to minimize animal
suffering and distress in the laboratory. Animal experiments
were approved by the Animal Care and Use Committee of
Tohoku University (Permit number: 2016MdA-153). Mice
were monitored daily for health during the full 4-week duration of the experiment. A reduction of more than 25% of
total body weight was defined as a humane endpoint. Mice
were euthanized by cervical dislocation, and death was
verified by respiratory and cardiac arrest. Female BALB/cmice (6 weeks old) were purchased from CLEA Japan(Tokyo, Japan). The animals were housed under specificpathogen-free conditions.

147

ulin G (IgG, 1:2000; Cell Signaling Technology, Inc., Danvers, MA) or Oregon green anti-rat IgG (1:2000; Thermo
Fisher Scientific, Inc.). Fluorescence data were collectedusing the SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).
Determination of binding affinity by flow cytometry

CHO/sPDPN was suspended in 100 lL of serially diluted
PMab-253 and PMab-260, and Alexa Fluor 488-conjugatedanti-mouse IgG (1:200; Cell Signaling Technology, Inc.) was
added. Fluorescence data were collected using the EC800 cell
analyzer (Sony Corp.). The dissociation constant (KD) was
calculated by fitting binding isotherms to built-in, one-sitebinding models in GraphPad PRISM 6 (GraphPad Software,Inc., La Jolla, CA).
Western blot analysis

Cell lysates (10 lg) were boiled in a sodium dodecyl
sulfate sample buffer (Nacalai Tesque, Inc.). Proteins werethen electrophoresed on 5%–20% polyacrylamide gels
(FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride membranes (Merck

Production of sPDPN mutants

Deletion mutants of the sPDPN sequence were produced
using a HotStar HiFidelity polymerase kit (Qiagen, Inc.,
Hilden, Germany) with oligonucleotides. Substitutions ofamino acids with alanine in the sPDPN sequence were produced by QuikChange Lightning site-directed mutagenesiskits (Agilent Technologies, Inc., Santa Clara, CA). Polymerase chain reaction fragments bearing the desired mutations were inserted into the pCAG vector using the In-Fusion
HD cloning kit (Takara Bio, Inc., Shiga, Japan).
Hybridoma production

To develop mAbs against sPDPN, we employed
CBIS.(21,35–37) Briefly, three BALB/c mice were immunizedwith CHO/sPDPN cells (1 · 108) by the intraperitoneal route
together with the Imject Alum (Thermo Fisher Scientific,Inc.). The procedure included three additional immunizations
followed by a final booster intraperitoneal injection administered 2 days before the harvest of spleen cells. Harvested
spleen cells were subsequently fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN), and the
hybridomas were grown in an RPMI medium supplemented
with hypoxanthine, aminopterin, and thymidine for selection(Thermo Fisher Scientific, Inc.). Culture supernatants werescreened by flow cytometry.
Flow cytometry

Cells were harvested following a brief exposure to 0.25%
trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai
Tesque, Inc.). They were washed with 0.1% bovine serum
albumin in phosphate-buffered saline (PBS) and treated withprimary mAbs for 30 min at 4C. The cells were then treatedwith Alexa Fluor 488-conjugated anti-mouse immunoglob-

FIG. 3. Determination of binding affinity of anti-sPDPN
mAbs. CHO/sPDPN was suspended in 100 mL of seriallydiluted PMab-253 (A) or PMab-260 (B). Alexa Fluor 488conjugated anti-mouse immunoglobulin G was then added. Fluorescence data were collected using the EC800
cell analyzer.

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

148

KANEKO ET AL.

KGaA, Darmstadt, Germany). After blocking with 4% skim
milk (Nacalai Tesque, Inc.), the membranes were incubatedwith 5 lg/mL of PMab-253, 5 lg/mL of PMab-260, 1 lg/mLof anti-RAP14 tag (clone: PMab-2),(18) or 1 lg/mL of anti-bactin (clone AC-15; Sigma-Aldrich Corp., St. Louis, MO)
and incubated with peroxidase-conjugated anti-mouse immunoglobulins (Agilent Technologies, Inc.; diluted 1:2000).
The membranes were finally developed using the Pierce
Western Blotting Substrate Plus (Thermo Fisher Scientific,Inc.) or ImmunoStar LD (FUJIFILM Wako Pure Chemical
Corporation) with a Sayaca-Imager (DRC Co. Ltd., Tokyo,
Japan).

Results

Immunohistochemical analyses

Flow cytometric analyses

Normal sheep tissues from two sheep were collected after
autopsies at Hokkaido University, fixed in 10% neutral-bufferedformalin, and processed routinely to make formalin-fixedparaffin-embedded (FFPE) sections as in our previous study.(39)Histological sections 4-lm thick were directly autoclaved ina citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo,
Japan) for 20 min. After blocking with SuperBlock T20 (PBS)
blocking buffer (Thermo Fisher Scientific, Inc.), the sections
were incubated with PMab-253 and PMab-260 (5 lg/mL) for
1 h at room temperature and treated using an Envision+ kit(Agilent Technologies, Inc.) for 30 min. Color was developed
using 3,3¢-diaminobenzidine tetrahydrochloride (Agilent
Technologies, Inc.) for 2 min, and counterstaining was performed with hematoxylin (FUJIFILM Wako Pure Chemical
Corporation).

We performed flow cytometry using PMab-253 and PMab260 against CHO/sPDPN and CHO-K1. Both PMab-253 andPMab-260 recognized CHO/sPDPN, but not CHO-K1 (Fig. 1A,
B). As a positive control, an anti-RAP14 tag mAb (PMab-2)
detected CHO/sPDPN. The results indicate that PMab-253 andPMab-260 can detect sPDPN in flow cytometry analysis.
Next, the cross-reaction of PMab-253 and PMab-260 withthe other PDPNs, which are overexpressed in CHO-K1 cells,
was investigated. PMab-253 cross-reacted with hPDPN,
bovPDPN, cPDPN, gPDPN, tigPDPN, and wPDPN (Fig. 2A).
In contrast, PMab-260 cross-reacted with hPDPN, bovPDPN,
gPDPN, and wPDPN (Fig. 2B).
We then assessed the apparent binding affinity of PMab253 and PMab-260 with CHO/sPDPN using flow cytometry.
The KD of PMab-253 and PMab-260 for CHO/sPDPN was

Establishment of anti-sPDPN mAbs

To produce anti-sPDPN mAbs, we employed CBIS,
using stable transfectants for both immunization and flow
cytometric screening. Three mice were immunized with
CHO/sPDPN cells, which overexpress sPDPN. Hybridomas
were seeded into 96-well plates, and CHO/sPDPNpositive and CHO-K1-negative wells were selected.
PMab-253 (immunoglobulin M [IgM], kappa) and PMab260 (IgM, kappa) were selected using Western blotting
and immunohistochemistry.

FIG. 4. Western blot analysis. Cell lysates of CHO-K1 and CHO/sPDPN (10 mg) were electrophoresed and transferred
onto polyvinylidene fluoride membranes. The membranes were incubated with 5 mg/mL of PMab-253, 5 mg/mL of PMab260, 1 mg/mL of anti-RAP14 tag (PMab-2), and 1 mg/mL of anti-b-actin and subsequently with peroxidase-conjugated antimouse immunoglobulins.

ANTI-SHEEP PDPN MAB

2.6 · 10-8 M and 2.7 · 10-8 M, respectively (Fig. 3), indicating that PMab-253 and PMab-260 possess moderate affinity for CHO/sPDPN cells.
Western blot analyses

We next investigated whether PMab-253 and PMab-260
can be applied to Western blot analysis by using cell lysatesof CHO-K1 or CHO/sPDPN. As shown in Figure 4, PMab253 and PMab-260 detected sPDPN as a 55-kDa band(N-terminal RAP14 tag + sPDPN) in CHO/sPDPN cell lysates,
but these antibodies did not detect any band from CHO-K1
cells. Anti-RAP14 tag (PMab-2) as a positive control also
detected a band of the same molecular weight from
CHO/sPDPN. These results indicate that PMab-253 andPMab-260 can detect sPDPN in Western blot analysis.

149

lung, kidney, and colon tissues, all of which reportedly express PDPN in other species, including humans,(16) mice,(17)rats,(18) rabbits,(19) dogs,(20) cats,(21) bovines,(22) pigs,(23, 24)
Tasmanian devils,(25) alpacas,(26) tigers,(27) whales,(28)goats,(29,30) horses,(31,32) and bears.(33,34) As shown in
Figure 5A and B, PMab-253 stained the renal glomerulus
and Bowman’s capsule. Lymphatic endothelial cells (redarrow) of the colon were readily detected by PMab-253(Fig. 5C, D). Furthermore, PMab-253 strongly stained type I
alveolar cells and lymphatic endothelial cells (red arrow) of
the lung (Fig. 5E, F). Similarly, PMab-260 strongly stained
the renal glomerulus (Fig. 6A, B), lymphatic endothelial
cells (red arrow) of colon (Fig. 6C, D), and type I alveolar
cells and lymphatic endothelial cells (red arrow) of the lung(Fig. 6E, F).
Epitope mapping

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

Immunohistochemical analyses

We next investigated whether PMab-253 and PMab-260
can be applied to immunohistochemical analysis using FFPE
sheep sections. For this assessment, we selected normal sheep

Eleven deletion mutants of sPDPN in CHO-K1 cells were
produced as follows: dN30, aa 30–168; dN40, aa 40–168;
dN50, aa 50–168; dN60, aa 60–168; dN70, aa 70–168; dN80,
aa 80–168; dN100, aa 100–168; dN105, aa 105–168; dN110,

FIG. 5. Immunohistochemical analyses of sheep tissues using PMab-253. Formalin-fixed paraffin-embedded tissue sections of sheep kidneys (A, B), sheep colons (C, D), and sheep lungs (E, F) were incubated with 5 mg/mL of PMab-253,
followed by analysis with the Envision+ kit. Red arrows show lymphatic endothelial cells. Scale bar = 100 mm.

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

150

KANEKO ET AL.

FIG. 6. Immunohistochemical analyses of sheep tissues using PMab-260. Formalin-fixed paraffin-embedded tissue sections of sheep kidneys (A, B), sheep colons (C, D), and sheep lungs (E, F) were incubated with 5 mg/mL of PMab-260,
followed by analysis with the Envision+ Kit. Red arrows show lymphatic endothelial cells. Scale bar = 100 mm.
aa 110–168; dN115, aa 115–168; dN120, aa 120–168; or WT
sPDPN, aa 27–168. All deletion mutants and WTs containing
the N-terminal RAP14 tag were recognized by PMab-2(an anti-RAP14 tag mAb), indicating that the expression
level of each construct was high (Fig. 7A). PMab-253 recognized dN30, dN40, dN50, dN60, dN70, dN80, dN100,
dN105, and dN110, but did not react with dN115 and dN120(Fig. 7B), suggesting that the N-terminus of the PMab-253
epitope is between aa 110 and 115 of sPDPN. Similarly,
PMab-260 recognized dN30, dN40, dN50, dN60, dN70,
dN80, dN100, dN105, and dN110, but did not react with
dN115 and dN120 (Fig. 7C), suggesting that the N-terminusof the PMab-260 epitope is also between aa 110 and 115 ofsPDPN.
Next, we obtained a series of point mutants of sPDPN:
V105A, H106A, T107A, H108A, V109A, P110A, A111G,
T112A, S113A, H114A, S115A, and Q116A. PMab-2 reacted with all point mutants (Fig. 8A), but PMab-253 reacted
less with T112A and S113A (Fig. 8B), indicating that Thr112
and Ser113 of sPDPN are essential for PMab-253 binding.
PMab-260 reacted with T112A weakly, and did not react with
S113A (Fig. 8C), also indicating that Thr112 and Ser113 ofsPDPN are essential for PMab-260 binding. The epitope ofPMab-253 and PMab-260 is illustrated in Figure 9A and B,
respectively.
Discussion

We previously investigated possible cross-reactions between anti-PDPN mAbs and sPDPN.(39) Flow cytometry
analysis demonstrated that an anti-bovPDPN mAb (clone
PMab-44)(22) reacted with CHO/sPDPN. Other anti-PDPN
mAbs such as anti-mPDPN mAb (PMab-1),(17) anti-rPDPN
mAb (PMab-2),(18) anti-rabPDPN mAb (PMab-32),(19) antidPDPN mAb (PMab-38 and PMab-48), and anti-cPDPN
mAb (PMab-52)(21) did not react with CHO/sPDPN, indicating that only PMab-44 was useful for the detection ofsPDPN. Expression of sPDPN was then investigated by immunohistochemical analyses using PMab-44. Although typeI alveolar cells of sheep lungs were stained by PMab-44, therenal glomerulus and lymphatic endothelial cells were notstained by PMab-44.(39) As a result, novel anti-sPDPN mAbs
for detecting lymphatic endothelial cells by immunohistochemical analysis are needed.

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

FIG. 7. Epitope mapping of PMab-253 and PMab-260 using deletion mutations. (A) Deletion mutants of sPDPN wereanalyzed using flow cytometry. Mutants were incubated with PMab-2 (anti-RAP14 tag mAb; red line) or a buffer control(black line) for 30 min at 4C, and then stained with secondary antibodies. (B) Deletion mutants of sPDPN were analyzed
using flow cytometry. Mutants were incubated with PMab-253 (anti-sPDPN mAb; red line) or a buffer control (black line)
for 30 min at 4C, and stained with secondary antibodies. (C) Deletion mutants of sPDPN were analyzed using flow
cytometry. Mutants were incubated with PMab-260 (anti-sPDPN mAb; red line) or buffer control (black line) for 30 min at
4C and stained with secondary antibodies.

151

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

FIG. 8. Epitope mapping of PMab-253 and PMab-260 using point mutations. (A) Transient point mutants expressing
V105A, H106A, T107A, H108A, V109A, P110A, A111G, T112A, S113A, H114A, S115A, and Q116A of sPDPN wereincubated with PMab-2 (anti-RAP14 tag; red line), or a buffer control (black line) for 30 min at 4C, and stained withsecondary antibodies. (B) Transient point mutants expressing V105A, H106A, T107A, H108A, V109A, P110A, A111G,
T112A, S113A, H114A, S115A, and Q116A of sPDPN were incubated with PMab-253 (anti-sPDPN mAb; red line) or abuffer control (black line) for 30 min at 4C, followed by staining with secondary antibodies. (C) Transient point mutants
expressing V105A, H106A, T107A, H108A, V109A, P110A, A111G, T112A, S113A, H114A, S115A, and Q116A ofsPDPN were incubated with PMab-260 (anti-sPDPN mAb; red line) or a buffer control (black line) for 30 min at 4C andstained with secondary antibodies.
152

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

ANTI-SHEEP PDPN MAB

153

FIG. 9. Schematic illustration of the epitope recognized by PMab-253 and PMab-260. (A) Illustration of WT sPDPN and its 11
deletion mutants: dN30, dN40, dN50, dN60, dN70, dN80, dN100, dN105, dN110, dN115, and dN120. Black bars: positive
reactions of PMab-253. White bars: negative reactions of PMab-253. (B) Illustration of WT sPDPN and its 11 deletion mutants:
dN30, dN40, dN50, dN60, dN70, dN80, dN100, dN105, dN110, dN115, and dN120. Black bars: positive reactions of PMab-260.
White bars: negative reactions of PMab-260. PLAG, platelet aggregation-stimulating; PLD, PLAG-like domain; WT, wild type.
In this study, we immunized mice with CHO/sPDPN cells,
performed the first screening using only flow cytometry, andestablished two mAbs (PMab-253 and PMab-260). Although
our previous study showed that PMab-44 stained only type I
alveolar cells and not lymphatic endothelial cells from sheep
organs, PMab-253 and PMab-260 strongly stained lymphatic
endothelial cells of the lung or colon (Figs. 5 and 6). Our
recent study showed that one of the anti-bPDPN mAbs (clone
PMab-241) reacted with lymphatic endothelial cells, but notwith type I alveolar cells of bear lungs.(34) The other antibPDPN mAb (clone PMab-247) reacted not only with lymphatic endothelial cells but also type I alveolar cells and renal
podocytes,(33) suggesting that the binding epitope of eachmAb is critical to distinguishing PDPN-expressing lymphatic
endothelial cells from other PDPN-expressing tissues. We
therefore determined the binding epitope of PMab-253 andPMab-260. Using a series of point mutants of sPDPN, Thr112
and Ser113 of sPDPN were determined to be essential for
PMab-253 and PMab-260 binding. Until now, we have notfound the binding epitope of anti-PDPN mAbs between aa
100 and aa 120 near the transmembrane domain of PDPN,
indicating that the epitope of PMab-253/PMab-260 is novel.
PMab-253 and PMab-260 may be advantageous for detailed
morphological studies of sheep lymphatic vessels.
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

154

Numbers: JP20am0401013 (Y.K.), JP20am0101078 (Y.K.),
and JP20ae0101028 (Y.K.), and by Japan Society for the
Promotion of Science (JSPS) KAKENHI Grant Numbers:
17K07299 (M.K.K.), 19K07705 (Y.K.), and 20K16322 (M.S.).

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

References

1. Yen H-H, Murray CM, Washington EA, Kimpton WG, andDavies HMS: Characterisation of ovine lymphatic vessels
in fresh specimens. PLoS One 2019;14:e0209414.
2. Heath T, Brandon RB, and Fogarty SJ: The arterial supply
to lymph nodes in sheep. J Anat 1985;141:41–52.
3. Hein WR, and Griebel PJ: A road less travelled: Large
animal models in immunological research. Nat Rev Immunol 2003;3:79–84.
4. Dziegielewska KM, Evans CA, Malinowska DH, Mollgard
K, Reynolds JM, Reynolds ML, and Saunders NR: Studiesof the development of brain barrier systems to lipid insoluble
molecules in fetal sheep. J Physiol 1979;292:207–231.
5. Pearson LD, Simpson-Morgan MW, and Morris B: Lymphopoiesis and lymphocyte recirculation in the sheep fetus.
J Exp Med 1976;143:167–186.
6. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P,
Poczewski H, Kalt R, Schaffner G, and Kerjaschki D: Podoplanin, novel 43-kd membrane protein of glomerularepithelial cells, is down-regulated in puromycin nephrosis.
Am J Pathol 1997;151:1141–1152.
7. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R,
Amann G, Kriehuber E, Diem K, Weninger W, Tschachler
E, Alitalo K, and Kerjaschki D: Angiosarcomas express
mixed endothelial phenotypes of blood and lymphatic
capillaries: Podoplanin as a specific marker for lymphatic
endothelium. Am J Pathol 1999;154:385–394.
8. Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M,
and Tsuruo T: Molecular identification of Aggrus/T1alphaas a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem 2003;278:51599–51605.
9. Kato Y, Sayama Y, Sano M, and Kaneko MK: Epitope
analysis of an antihorse podoplanin monoclonal antibody
PMab-219. Monoclon Antib Immunodiagn Immunother2019;38:266–270.
10. Kato Y, Takei J, Furusawa Y, Sayama Y, Sano M, Konnai
S, Kobayashi A, Harada H, Takahashi M, Suzuki H, Yamada S, and Kaneko MK: Epitope mapping of anti-bear
podoplanin monoclonal antibody PMab-247. Monoclon
Antib Immunodiagn Immunother 2019;38:230–233.
11. Sano M, Kaneko MK, and Kato Y: Epitope mapping of
monoclonal antibody PMab-233 against tasmanian devil
podoplanin. Monoclon Antib Immunodiagn Immunother2019;38:261–265.
12. Sayama Y, Sano M, Furusawa Y, Kaneko MK, and Kato Y:
Epitope mapping of PMab-225 an anti-alpaca podoplanin
monoclonal antibody using flow cytometry. Monoclon
Antib Immunodiagn Immunother 2019;38:255–260.
13. Sayama Y, Sano M, Kaneko MK, and Kato Y: Epitope
analysis of an anti-whale podoplanin monoclonal antibody,PMab-237, using flow cytometry. Monoclon Antib Immunodiagn Immunother 2020;39:17–22.
14. Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A,
Ogasawara S, Matsuura N, Hasegawa Y, Suzuki-Inoue K,
Inoue O, Ozaki Y, and Narimatsu H: Molecular analysis of
the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.

KANEKO ET AL.

15. Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z,
Chen M, Chen CY, Xu B, Lu MM, Zhou D, Sebzda E,
Santore MT, Merianos DJ, Stadtfeld M, Flake AW, Graf T,
Skoda R, Maltzman JS, Koretzky GA, and Kahn ML:
Platelets regulate lymphatic vascular development through
CLEC-2-SLP-76 signaling. Blood 2010;116:661–670.
16. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K,
Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H, Mishima K, and Osawa M: Inhibition of tumor cell-induced
platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating
domain. Biochem Biophys Res Commun 2006;349:1301–
1307.
17. Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, and Sawa
Y: Immunohistochemical examination of novel rat monoclonal antibodies against mouse and human podoplanin.
Acta Histochem Cytochem 2012;45:227–237.
18. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Takagi M,
Kaneko MK, and Kato Y: Development of sensitive monoclonal antibody PMab-2 against rat podoplanin. Monoclon
Antib Immunodiagn Immunother 2015;34:396–403.
19. Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura
T, Kaneko MK, Takagi M, and Kato Y: Establishment of anovel monoclonal antibody PMab-32 against rabbit podoplanin. Monoclon Antib Immunodiagn Immunother 2016;35:41–47.
20. Honma R, Kaneko MK, Ogasawara S, Fujii Y, Konnai S,
Takagi M, and Kato Y: Specific detection of dog podoplanin
expressed in renal glomerulus by a novel monoclonal antibody PMab-38 in immunohistochemistry. Monoclon AntibImmunodiagn Immunother 2016;35:212–216.
21. Yamada S, Itai S, Nakamura T, Yanaka M, Saidoh N,
Chang YW, Handa S, Harada H, Kagawa Y, Ichii O,
Konnai S, Kaneko MK, and Kato Y: PMab-52: Specific andsensitive monoclonal antibody against cat podoplanin for
immunohistochemistry. Monoclon Antib Immunodiagn
Immunother 2017;36:224–230.
22. Honma R, Ogasawara S, Kaneko M, Fujii Y, Oki H, Nakamura T, Takagi M, Konnai S, and Kato Y: PMab-44 detects
bovine podoplanin in immunohistochemistry. Monoclon
Antib Immunodiagn Immunother 2016;35:186–190.
23. Kato Y, Yamada S, Furusawa Y, Itai S, Nakamura T, Yanaka M, Sano M, Harada H, Fukui M, and Kaneko MK:
PMab-213: A monoclonal antibody for immunohistochemical analysis against pig podoplanin. Monoclon AntibImmunodiagn Immunother 2019;38:18–24.
24. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,
Yanaka M, Fukui M, Harada H, Mizuno T, Sakai Y, Takasu
M, Kaneko MK, and Kato Y: PMab-210: A monoclonal
antibody against pig podoplanin. Monoclon Antib Immunodiagn Immunother 2019;38:30–36.
25. Furusawa Y, Yamada S, Itai S, Nakamura T, Takei J, Sano
M, Harada H, Fukui M, Kaneko MK, and Kato Y: Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil
podoplanin. Biochem Biophys Rep 2019;18:100631.
26. Kato Y, Furusawa Y, Yamada S, Itai S, Takei J, Sano M,
and Kaneko MK: Establishment of a monoclonal antibody
PMab-225 against alpaca podoplanin for immunohistochemical analyses. Biochem Biophys Rep 2019;18:
100633.
27. Furusawa Y, Kaneko MK, Nakamura T, Itai S, Fukui M,
Harada H, Yamada S, and Kato Y: Establishment of a
monoclonal antibody PMab-231 for tiger podoplanin.

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

ANTI-SHEEP PDPN MAB

Monoclon Antib Immunodiagn Immunother 2019;38:
89–95.
28. Kato Y, Furusawa Y, Itai S, Takei J, Nakamura T, Sano M,
Harada H, Yamada S, and Kaneko MK: Establishment ofan anticetacean podoplanin monoclonal antibody PMab237 for immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2019;38:108–113.
29. Furusawa Y, Yamada S, Nakamura T, Sano M, Sayama Y,
Itai S, Takei J, Harada H, Fukui M, Kaneko MK, and Kato
Y: PMab-235: A monoclonal antibody for immunohistochemical analysis against goat podoplanin. Heliyon 2019;5:e02063.
30. Takei J, Itai S, Harada H, Furusawa Y, Miwa T, Fukui M,
Nakamura T, Sano M, Sayama Y, Yanaka M, Handa S,
Hisamatsu K, Nakamura Y, Yamada S, Kaneko KM, andKato Y: Characterization of anti-goat podoplanin monoclonal antibody PMab-235 using immunohistochemistry
against goat tissues. Monoclon Antib Immunodiagn Immunother 2019;38:213–219.
31. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,
Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M, Harada H, Kaneko MK, and Kato Y: Establishment of monoclonal antibody PMab-202 against horse podoplanin.
Monoclon Antib Immunodiagn Immunother 2018;37:233–
237.
32. Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, andKaneko MK: Anti-horse podoplanin monoclonal antibody
PMab-219 is useful for detecting lymphatic endothelial
cells by immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2018;37:272–274.
33. Takei J, Furusawa Y, Yamada S, Nakamura T, Sayama Y,
Sano M, Konnai S, Kobayashi A, Harada H, Kaneko MK,
and Kato Y: PMab-247 detects bear podoplanin in immunohistochemical analysis. Monoclon Antib Immunodiagn
Immunother 2019;38:171–174.
34. Takei J, Yamada S, Konnai S, Ishinazaka T, Shimozuru M,
Kaneko MK, and Kato Y: PMab-241 specifically detects
bear podoplanin of lymphatic endothelial cells in the lung155of brown bear. Monoclon Antib Immunodiagn Immunother2019;38:282–284.
35. Itai S, Fujii Y, Nakamura T, Chang YW, Yanaka M, Saidoh
N, Handa S, Suzuki H, Harada H, Yamada S, Kaneko MK,
and Kato Y: Establishment of CMab-43, a sensitive andspecific anti-CD133 monoclonal antibody, for immunohistochemistry. Monoclon Antib Immunodiagn Immunother2017;36:231–235.
36. Yamada S, Itai S, Nakamura T, Yanaka M, Chang YW,
Suzuki H, Kaneko MK, and Kato Y: Monoclonal antibody
L1Mab-13 detected human PD-L1 in lung cancers. Monoclon Antib Immunodiagn Immunother 2018;37:110–115.
37. Yamada S, Itai S, Nakamura T, Yanaka M, Kaneko MK,
and Kato Y: Detection of high CD44 expression in oral
cancers using the novel monoclonal antibody, C44Mab-5.
Biochem Biophys Rep 2018;14:64–68.
38. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
39. Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, andKaneko MK: Immunohistochemical detection of sheep
podoplanin using an antibovine podoplanin monoclonal
antibody PMab-44. Monoclon Antib Immunodiagn Immunother 2018;37:265–268.

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
Received: April 14, 2020
Accepted: June 20, 2020