MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 1, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0038

PMab-210:
A Monoclonal Antibody Against Pig Podoplanin

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Yoshikazu Furusawa,1–3 Shinji Yamada,1 Shunsuke Itai,1,4 Masato Sano,1 Takuro Nakamura,1Miyuki Yanaka,1 Masato Fukui,3 Hiroyuki Harada,4 Takuya Mizuno,5 Yusuke Sakai,6Masaki Takasu,7 Mika K. Kaneko,1 and Yukinari Kato1,2

Podoplanin (PDPN) is a type I transmembrane glycoprotein that is expressed in normal tissues, including renal
corpuscles and type I lung alveolar cells. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog,
cat, and bovine PDPNs have already been established; however, antipig PDPN (pPDPN) mAbs have not. We
therefore immunized mice with pPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/pPDPN),
and screened hybridomas, which are producing anti-pPDPN mAbs. One of mAbs, PMab-210 (an IgG1, kappa),
was able to specifically detect CHO/pPDPN cells by flow cytometry and detect pPDPN by Western blot
analysis. Furthermore, PMab-210 strongly stained type I lung alveolar cells and weakly stained renal corpuscles
by immunohistochemistry. PMab-210 is expected to be useful in investigating the function of pPDPN.
Keywords: pig podoplanin, PDPN, PMab-210

specific mAbs against pPDPN are needed to investigate its
expression and function. In this study, we therefore immunized mice with CHO/pPDPN cells and established cell lines
producing anti-pPDPN mAbs.

IntroductionP

odoplanin (PDPN)/T1a/Aggrus is a type I transmembrane glycoprotein expressed in normal tissues, including
renal corpuscles, type I lung alveolar cells, and lymphatic endothelial cells.(1,2) PDPN induces platelet aggregation bybinding to C-type lectin-like receptor-2 (CLEC-2),(1,3–9) and theinteraction between PDPN on lymphatic endothelial cells and
CLEC-2 on platelets facilitates embryonic blood/lymphatic
vessel separation.(10) PDPN is also expressed in human fetal ribs
and in chondrocytes of the proliferative and hypertrophic regions of the growth plate.(11) The expression of human PDPN
has been reported in several malignant tumors, such as esophageal cancers,(12) malignant mesotheliomas,(13,14) lungcancers,(15) testicular tumors,(16) osteosarcomas,(17–19) chondrosarcomas,(18) oral squamous cell carcinomas,(20) and malignant brain tumors.(21–24) PDPN expression is also associatedwith malignant progression and cancer metastasis.(6,21,25)
We have previously developed monoclonal antibodies(mAbs) against human,(26) mouse,(26) rat,(27) rabbit,(28) dog,(29)cat,(30) and bovine(31) PDPNs. However, antipig PDPN
(pPDPN) mAbs have not yet been produced. Sensitive andMaterials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 and P3U1 were obtained
from the American Type Culture Collection (ATCC, Manassas, VA). The pig kidney cell line, PK-15, was obtained fromthe Japanese Collection of Research Bioresources Cell Bank
(Osaka, Japan). pPDPN bearing an N-terminal PA16 tag(PA16-pPDPN) was inserted into a pCAG-Ble vector (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The
PA16 tag consists of 16 amino acids (GLEGGVAMPGAEDDVV).(32) CHO-K1 cells were transfected with pCAGBle/PA16-pPDPN using Lipofectamine LTX with Plus
Reagent (Thermo Fisher Scientific, Inc., Waltham, MA).
Stable transfectants were selected by limiting dilution andcultivated in a medium containing 0.5 mg/mL zeocin (InvivoGen, San Diego, CA). CHO-K1, CHO/pPDPN, and P3U1

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
ZENOAQ RESOURCE CO., LTD., Koriyama, Japan.
4
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Bunkyo-ku, Japan.
5
Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
6
Laboratory of Veterinary Pathology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
7
Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
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PMAB-210 AGAINST PIG PDPN

cells were cultured in RPMI 1640 medium (Nacalai Tesque,Inc., Kyoto, Japan), and PK-15 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Nacalai Tesque,Inc.). All media were supplemented with 10% heat-inactivatedfetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/mLof penicillin, 100 mg/mL of streptomycin, and 25 mg/mL ofamphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified
atmosphere of 5% CO2 and 95% air.

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Hybridoma production

Female BALB/c mice (6-week old) were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed under
specific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved all the animal
experiments described here. BALB/c mice were immunizedagainst CHO/pPDPN cells (1 · 108), which were administered intraperitoneally (i.p.) together with Imject Alum
(Thermo Fisher Scientific, Inc.). After an additional threeimmunizations, a final booster injection was administered i.p.
2 days before spleen cells were harvested. The spleen cells
were then fused with P3U1 cells using PEG1500 (Roche
Diagnostics, Indianapolis, IN), and the hybridomas weregrown in RPMI medium supplemented with hypoxanthine,aminopterin, and thymidine for selection (Thermo Fisher
Scientific, Inc.). The culture supernatants were screened byflow cytometry.
Flow cytometry

Cells were harvested after brief exposure to 0.25% trypsin/1 mM EDTA (Nacalai Tesque, Inc.). After washing with
0.1% bovine serum albumin/phosphate-buffered saline (PBS),

31

the cells were treated with primary mAbs for 30 minutes at
4C, followed by treatment with Alexa Fluor 488-conjugatedantimouse IgG (1:2000; Cell Signaling Technology, Inc.,
Danvers, MA) or Oregon green antirat IgG (1:2000; Thermo
Fisher Scientific Inc.). Fluorescence data were collected using SA3800 Cell Analyzers (Sony Corp., Tokyo, Japan).
Western blot analysis

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.). The proteins were
electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation), and then transferred onto a polyvinylidene difluoride membrane (Merck
KGaA, Darmstadt, Germany). After blocking with 4% skim
milk (Nacalai Tesque, Inc.), the membrane was incubatedwith primary mAbs, and subsequently with peroxidaseconjugated antimouse IgG (1:1000; Agilent Technologies,
Santa Clara, CA) or antirat IgG (1:10,000; Sigma-AldrichCorp., St. Louis, MO). Bands were visualized with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation)using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Determination of binding affinity using flow cytometry

CHO/pPDPN or PK-15 (2 · 105 cells) was suspended in
100 mL of serially diluted PMab-210, followed by addition of
Alexa Fluor 488-conjugated antimouse IgG (1:200; Cell
Signaling Technology, Inc.). Fluorescence data were collected using EC800 Cell Analyzer (Sony Corp.). The dissociation constants (KD) were obtained by fitting the binding
isotherms to built-in one-site binding models in GraphPad
PRISM 6 (GraphPad Software, Inc., La Jolla, CA).

FIG. 1. Schematic illustration of CBIS. Stable transfectants expressing the protein of interest are used as an immunogenwith no purification procedure. The selection of hybridomas secreting specific mAbs is performed by flow cytometry using
the parental and transfectant cells. CBIS, Cell-Based Immunization and Screening; mAbs, monoclonal antibodies.

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FURUSAWA ET AL.

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Immunohistochemical analyses

Pig tissues were collected at autopsy at Yamaguchi University and fixed in 10% neutral-buffered formalin. Micromini pig (Fuji Micra, Inc., Shizuoka, Japan) tissues werecollected at autopsy at Gifu University and fixed in 4%
paraformaldehyde. Tissues were processed routinely to produce 4 mm paraffin-embedded tissue sections, which weredirectly autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) or EnVision FLEX Target Retrieval Solution, High pH (Agilent Technologies, Inc.) for
20 minutes. After blocking with SuperBlock T20 (PBS)
Blocking Buffer (Thermo Fisher Scientific, Inc.), sections
were incubated with PMab-210 (10 or 20 mg/mL) for 1 hour atroom temperature and treated using an Envision+ Kit (Agilent Technologies, Inc.) for 30 minutes. Color was developed
using 3,3¢-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2 minutes, and counterstaining
was performed with hematoxylin (FUJIFILM Wako Pure
Chemical Corporation).
Results and Discussion

To produce sensitive and specific anti-pPDPN mAbs for
immunohistochemical analysis of paraffin-embedded tissue
sections, we first immunized mice with two different synthetic
pPDPN-derived peptides; however, we were unsuccessful(data not shown). We therefore instead employed a CellBased Immunization and Screening (CBIS) method for producing anti-pPDPN mAbs. We have previously successfully
utilized a CBIS method to establish mAbs against various
types of membrane proteins such as CD44,(32) CD133,(33) PDL1,(34) and cat PDPN (cPDPN),(30) so here we immunized one
mouse with pPDPN-overexpressing CHO-K1 (CHO/pPDPN)
cells using the immunization and screening procedure depicted in Figure 1. The hybridomas were seeded into andcultured in 96-well plates, and supernatants were used to label
and screen cells by flow cytometry. CHO/pPDPN cells werescreened to identify potential anti-pPDPN antibodies,
whereas CHO-K1 cells were screened to exclude antibodies
against CHO-K1 cells antigens. PK-15 cells were also used to
identify antibodies that reacted with the endogenous pig
protein. Screening identified strong signals against
CHO/pPDPN cells and weak or no signals against CHO-K1
cells in 44 of 480 wells (9.2%). Among these 44 wells, sevenproduced strong signals against PK-15 cells and were negative
against CHO-K1 cells. PMab-210 (IgG1, kappa) was finallyestablished by limiting dilution from one of these wells.
PMab-210 recognized CHO/pPDPN but did not reactwith CHO-K1, as assessed by flow cytometry (Fig. 2). It
also reacted with the PK-15 pig kidney cell line, indicatingthat it could recognize endogenous pPDPN. PMab-210 did
not react with human, mouse, rat, rabbit, dog, cat, or bovine
PDPNs (Fig. 3). Furthermore, it did not react with horse,
Tasmanian devil, tiger, alpaca, bear, goat, sheep, or whale
PDPNs (Supplementary Fig. S1), indicating that PMab-210
is specific to pPDPN. Flow cytometry was next used toperform a kinetic analysis of the interaction of PMab-210
with CHO/pPDPN and PK-15 cells. The dissociation constant (KD) of PMab-210 for CHO/pPDPN and PK-15 cells
was determined to be 4.0 · 10-8 M and 4.4 · 10-8 M, respectively, indicating a moderate affinity for either cell
type.

FIG. 2. Detection of pPDPN by flow cytometry using
PMab-210. CHO/pPDPN, CHO-K1, and PK-15 cells weretreated with PMab-210 (red line) at a concentration of
10 mg/mL or control (gray) for 30 minutes, followed by the
incubation with secondary antibodies. CHO, Chinese hamster ovary; pPDPN, pig podoplanin.
PMab-210 was able to identify pPDPN as a 40 kDa band in
the Western blot of CHO/pPDPN cells (Fig. 4), but did notdetect it in PK-15 cells, possibly due to low expression of
pPDPN in PK-15 cells or a low affinity of PMab-210 against
PK-15 cells. An anti-PA16 tag mAb (NZ-1) detected twostrong bands in CHO/pPDPN cells: the upper is reported
to be highly glycosylated form and the lower one is unglycosylated.(3,24) In contrast, PMab-210 recognized only theupper glycosylated pPDPN, suggesting that the mAb may
recognize glycosylated pPDPN more selectively. Our previous studies have similarly found that antihuman PDPN
(hPDPN) mAbs such as LpMab-3,(35) LpMab-9,(36) LpMab19,(37) and LpMab-21(38) also detect only glycosylated
hPDPN. It would be interesting to identify the specific epitope of PMab-210 in the future.
When used in immunohistochemistry, PMab-210 strongly
stained type I pulmonary alveolar cells of pigs (Fig. 5) and33

FIG. 4. Western blot analysis. Cell lysates of CHO-K1,
CHO/pPDPN, and PK-15 (10 mg) were electrophoresed andtransferred onto a PVDF membrane. The membrane wasincubated with 10 mg/mL of PMab-210, 1 mg/mL of anti-PAtag (NZ-1), or 1 mg/mL of anti-b-actin, and subsequently
with peroxidase-conjugated antimouse or -rat IgG. PVDF,
polyvinylidene difluoride.

micromini pigs (Fig. 6). Furthermore, PMab-210 weakly
stained renal corpuscles of pigs (Fig. 7) and micromini pigs
(Fig. 8), indicating that the mAb is useful for the detection of
PDPN by immunohistochemistry. PDPN is known to be a
specific lymphatic endothelial cell marker; however, PMab210 could not detect any lymphatic endothelial cells in lungs,
kidneys, or colons of pigs or micromini pigs (data notshown). We could not obtain primary cells or established celllines of pig lymphatic endothelial cells in this study, and so
we could not determine whether PMab-210 could not reactwith lymphatic endothelial cells in paraffin sections orwhether pig lymphatic endothelial cells exhibit tissuespecific glycosylation and so do not carry the PMab-210
epitope.
Taken together, these data demonstrate that we have established an anti-pPDPN mAb, PMab-210, which is applicable for use in flow cytometry, Western blotting, andimmunohistochemical analyses. The epitope of PMab-210
should be determined in a future study to further clarify thesensitivity and specificity of PMab-210 against pPDPN. ThismAb should prove useful for elucidating the pathophysiological functions of pPDPN in the future studies.

‰

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PMAB-210 AGAINST PIG PDPN

FIG. 3. Specific detection of pPDPN by flow cytometry
using PMab-210. CHO/pPDPN, CHO/hPDPN, CHO/
mPDPN, CHO/rPDPN, CHO/rabPDPN, CHO/dPDPN, CHO/
bovPDPN, and CHO/cPDPN cells were treated with PMab210 (red line) at a concentration of 10 mg/mL or control (gray)for 30 minutes, followed by the addition of secondary antibodies. The same cells were also treated with positive control(blue line) at a concentration of 1 mg/mL or control (gray) for
30 minutes, followed by the addition of secondary antibodies.

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FIG. 5. Immunohistochemical analyses using pig lungtissue. Histological sections were incubated with 10 mg/mL
of PMab-210 (A, B) or with blocking buffer (C, D), followed by an Envision+ Kit. (E, F) Hematoxylin and eosin
staining. Scale bar = 100 mm.

FIG. 7. Immunohistochemical analyses using pig kidneys.
Histological sections of pig kidney tissue were incubatedwith 10 mg/mL of PMab-210 (A, B) or with blocking buffer(C, D), followed by an Envision+ Kit. (E, F) Hematoxylin
and eosin staining. Scale bar = 100 mm.

FIG. 6. Immunohistochemical analyses using micromini
pig lungs. Histological sections of micromini pig lung tissue
were incubated with 10 mg/mL of PMab-210 (A, B) or withblocking buffer (C, D), followed by an Envision+ Kit. (E, F)
Hematoxylin and eosin staining. Scale bar = 100 mm.

FIG. 8. Immunohistochemical analyses using micromini
pig kidneys. Histological sections of the micromini pig
kidney tissue were incubated with 20 mg/mL of PMab-210(A, B) or with blocking buffer (C, D), followed by an Envision+ Kit. (E, F) Hematoxylin and eosin staining. Scalebar = 100 mm.
34

PMAB-210 AGAINST PIG PDPN
Acknowledgments

We thank Saori Handa, Kayo Hisamatsu, and Yoshimi
Nakamura for their excellent technical assistance. This research was supported in part by AMED under grant numbers
JP18am0101078 (Y.K.), JP18am0301010 (Y.K.), andJP18ae0101028 (Y.K.), and by JSPS KAKENHI grant number
17K07299 (M.K.K.) and grant number 16K10748 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.
Supplementary Material

Supplementary Figure S1

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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: September 21, 2018
Accepted: December 20, 2018