MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 5, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0027

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PMab-52: Specific and Sensitive Monoclonal Antibody
Against Cat Podoplanin for Immunohistochemistry
Shinji Yamada,1 Shunsuke Itai,1,2 Takuro Nakamura,1 Miyuki Yanaka,1 Noriko Saidoh,1
Yao-Wen Chang,1 Saori Handa,1 Hiroyuki Harada,2 Yumiko Kagawa,3 Osamu Ichii,4Satoru Konnai,5 Mika K. Kaneko,1 and Yukinari Kato1,6

Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes ofrenal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type
lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN,
mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN
(cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary(CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs. One of the clones, PMab-52 (IgM,
kappa), detected cPDPN specifically in flow cytometry and Western blot analysis. PMab-52 is also useful for
detecting feline squamous cell carcinoma cells in immunohistochemical analysis. PMab-52 is expected to be
useful for investigating the function of cPDPN in feline carcinomas.
Keywords: cat podoplanin, PDPN, monoclonal antibody, immunohistochemistry

Introduction

P

Materials and Methods(1–4)

odoplanin (PDPN), also known as T1a/Aggrus,isa type I transmembrane glycoprotein. PDPN activates
platelet aggregation by binding to C-type lectin-like receptor2 (CLEC-2) on platelet.(5–8) The interaction between PDPN
and CLEC-2 facilitates blood/lymphatic vessel separation.(9)PDPN is also expressed in human fetal rib and chondrocytes ofthe proliferative and hypertrophic regions of the growthplate.(10) The expression of human PDPN has been reported inmany malignant tumors such as oral squamous cell carcinomas,(11) malignant brain tumors,(12–15) lung cancers,(16) esophageal cancers,(17) malignant mesotheliomas,(18,19) testiculartumors,(20) osteosarcomas,(21–23) and chondrosarcomas.(22)
PDPN expression is also associated with malignant progression
and cancer metastasis.(5,12,24)
Although we previously established monoclonal antibodies (mAbs) against human,(25) mouse,(26) rat,(27) rabbit,(28) dog,(29) and bovine PDPN,(30) anticat PDPN (cPDPN)
mAbs have not been developed yet. To investigate the expression and function of cPDPN, specific and sensitive mAbs
against cPDPN are required. In this study, we immunized
mice with cPDPN and developed anti-cPDPN mAbs.

Cell lines, feline tissues, and animals

Chinese hamster ovary (CHO)-K1 and P3U1 were obtained from the American Type Culture Collection (ATCC,
Manassas, VA). Feline kidney cell line, CrFK, was obtained
from the Japanese Collection of Research Bioresources( JCRB) Cell Bank (Osaka, Japan). CHO-K1, stable CHO
transfectants, and P3U1 were cultured in RPMI 1640 medium(Nacalai Tesque, Inc., Kyoto, Japan), and CrFK was cultured
in Dulbecco’s modified Eagle’s medium (Nacalai Tesque,Inc.), supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Thermo Fisher Scientific, Inc., Waltham, MA),
100 U/mL of penicillin, 100 mg/mL of streptomycin, and25 mg/mL of amphotericin B (Nacalai Tesque, Inc.) at 37C
in a humidified atmosphere of 5% CO2 and 95% air. Female
BALB/c mice (4 weeks old) were purchased from CLEA
Japan (Tokyo, Japan). Feline tissues were obtained from
North Lab (Hokkaido, Japan). Animals were housed under
specific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved the animal experiments described herein.

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
3
North Lab, Sapporo, Japan.
Departments of 4Basic Veterinary Sciences and 5Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
6
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2

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Hybridoma production

The cPDPN with N-terminal MAP tag (MAP-cPDPN) was
inserted into pCAG-zeo vector (Wako Pure Chemical Industries Ltd., Osaka, Japan). MAP tag consists of 12 aminoacids (GDGMVPPGIEDK).(31) CHO-K1 was transfected with
pCAG-zeo/MAP-cPDPN using Lipofectamine LTX reagent(Thermo Fisher Scientific, Inc.). Stable transfectant of CHO/
cPDPN was established by limiting dilution. BALB/c mice
were immunized by intraperitoneal (i.p.) injection of CHO/
cPDPN cells together with Imject Alum (Thermo Fisher
Scientific, Inc.). After several additional immunizations of
CHO/cPDPN cells, a booster injection of CHO/cPDPN cells
was given i.p. 2 days before spleen cells were harvested. The
spleen cells were fused with P3U1 cells using PEG1500(Roche Diagnostics, Indianapolis, IN). The hybridomas weregrown in RPMI medium with hypoxanthine, aminopterin, andthymidine selection medium supplement (Thermo Fisher
Scientific, Inc.). The culture supernatants were screened using
flow cytometry for the binding to CHO/cPDPN and CHO-K1.
Flow cytometry

Stable transfectants [CHO/PA-dog PDPN-RAP-MAP(CHO/dPDPN), CHO/PA-bovine PDPN-RAP-MAP (CHO/
bovPDPN), CHO/human PDPN-FLAG (CHO/hPDPN),
CHO/mouse PDPN-FLAG (CHO/mPDPN), CHO/rat PDPNHis (CHO/rPDPN), and CHO/PA-rabbit PDPN (CHO/
rabPDPN)] were previously established.(1,27–30) Cells were
harvested by brief exposure to 0.25% trypsin/1 mM EDTA
(Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albumin/phosphate-buffered saline, the cells were treated with a primary mAb (1 mg/mL) for 30 minutes at 4Cfollowed by treatment with Alexa Fluor 488-conjugated antimouse IgG (1:1000; Cell Signaling Technology, Inc.,
Danvers, MA). Fluorescence data were collected using
EC800 or SA3800 Cell Analyzers (Sony Corp.).
Western blot analysis

Cell lysates (10 mg) of CHO/cPDPN, CHO/dPDPN, CHO/
bovPDPN, CHO/hPDPN, CHO/mPDPN, CHO/rPDPN, CHO/
rabPDPN, and CHO-K1 were boiled in SDS sample buffer(Nacalai Tesque, Inc.). The proteins were electrophoresed on
5–20% polyacrylamide gels (Wako Pure Chemical Industries
Ltd.) and were transferred onto a polyvinylidene difluoride(PVDF) membrane (Merck KGaA, Darmstadt, Germany).
After blocking with 4% skim milk (Nacalai Tesque, Inc.), themembrane was incubated with 1 mg/mL of PMab-52, anti-bactin (clone AC-15; Sigma-Aldrich Corp., St. Louis, MO), antiIDH1 (clone RMab-3), and then with peroxidase-conjugatedantimouse IgG (1:1000 diluted; Agilent Technologies, Inc.,
Santa Clara, CA), and developed with ImmunoStar LD (Wako
Pure Chemical Industries Ltd.) using a Sayaca-Imager (DRC
Co. Ltd., Tokyo, Japan).
Determination of the binding affinity
using flow cytometry5

CHO/cPDPN or CrFK (2 · 10 cells) was suspended in
100 mL of serially diluted PMab-52, followed by addition of
Alexa Fluor 488-conjugated antimouse IgG (1:200; Cell
Signaling Technology, Inc.). Fluorescence data were collected using EC800 Cell Analyzer. The dissociation con-225

stants (KD) were obtained by fitting the binding isotherms
using the built-in one-site binding models in GraphPad
PRISM 6 (GraphPad software, Inc., La Jolla, CA).
Immunohistochemical analyses

Four micrometer thick histological sections were deparaffinized in xylene and rehydrated, and were autoclaved incitrate buffer (pH 6.0; Agilent Technologies, Inc.) for 20
minutes. Sections were incubated with 1 mg/mL of PMab-52
for 1 hour at room temperature followed by treatment withEnvision+ kit for 30 minutes (Agilent Technologies, Inc.).
Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2
minutes, and then the sections were counterstained with hematoxylin (Wako Pure Chemical Industries Ltd.).
Results
Production of mAbs against cPDPN

We immunized mice with CHO/cPDPN cells, and performed flow cytometric screening. Culture supernatants of1100 wells were mixed with CHO/cPDPN cells and CHO-K1
cells, and 84 wells (84/1100; 7.6%) that were positive against
CHO/cPDPN cells and negative against CHO-K1 cells wereselected. Of these 84 wells, 43 (51%) were positive against afeline kidney cell line, CrFK cells, in flow cytometry, indicating that about half of the wells reacted with endogenous
cPDPN. We further performed Western blot analysis using
CHO/cPDPN and CrFK cells. After limiting dilution ofseveral wells that displayed specific and sensitive signals in
Western blot analysis, one of the clones, PMab-52 (IgM,
kappa), was established.
Specificity of PMab-52 against cPDPN in Western blot
and flow cytometry

In flow cytometry, PMab-52 recognized only cPDPN from
CHO/cPDPN, and did not react with PDPNs of CHO/dPDPN,
CHO/bovPDPN, CHO/hPDPN, CHO/mPDPN, CHO/
rPDPN, or CHO/rabPDPN cells (Fig. 1A). Furthermore,
PMab-52 displayed the *40-kDa band of CHO/cPDPN, but
did not give bands with CHO/dPDPN, CHO/bovPDPN,
CHO/hPDPN, CHO/mPDPN, CHO/rPDPN, or CHO/
rabPDPN cells in Western blot analysis (Fig. 1B). These results indicate that PMab-52 specifically detects cPDPN.
Determination of the binding affinity of PMab-52

We then performed a kinetic analysis of the interaction ofPMab-52 with CHO/cPDPN or CrFK using flow cytometry.
The dissociation constant KD of PMab-52 was determined to
be 4.6 · 10-8 M against CHO/cPDPN (Fig. 1C) and 3.6 · 10-8
M against CrFK (Fig. 1D), indicating that PMab-52 possesses
moderate affinity for CHO/cPDPN and CrFK.
Immunohistochemical analysis

Finally, we investigated the immunohistochemical utilityof PMab-52 in feline squamous cell carcinomas. As shown in
Figure 2A and B, PMab-52 stained membranes of cancer
cells in the squamous cell carcinomas. PMab-52 also stained
lymphatic endothelial cells (Fig. 2A, C), indicating that

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FIG. 1. Characterization of PMab-52. (A) Flow cytometry with PMab-52. Cells were treated with 1 mg/mL of PMab-52
followed by treatment with Oregon green-conjugated antimouse IgG. Black line: negative control. (B) Western blot with
PMab-52. Cell lysates (10 mg) were electrophoresed and transferred onto a PVDF membrane. After blocking, the membrane
was incubated with 1 mg/mL of PMab-52, anti-b-actin (AC-15), and anti-IDH1 (clone RMab-3), and then incubated withperoxidase-conjugated antimouse IgG. (C, D) Determination of the binding affinity of PMab-52 using flow cytometry.
CHO/cPDPN (C) and CrFK (D) were suspended in 100 mL of serially diluted PMab-52 (6 mg/mL–100 mg/mL), followed byaddition of secondary antimouse IgG. Fluorescence data were collected using a cell analyzer. CHO, Chinese hamster ovary;cPDPN, cat PDPN; PDPN, podoplanin; PVDF, polyvinylidene difluoride.
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MABS AGAINST CAT PODOPLANIN

227

FIG. 2. Immunohistochemical analysis by PMab-52. (A–C) Sections of feline squamous cell carcinomas were autoclaved incitrate buffer (pH 6.0). After blocking, they were incubated with 1 mg/mL of PMab-52, followed by EnVision+ kit; color wasdeveloped using DAB and counterstained with hematoxylin. (D, E) Hematoxylin and eosin staining was performed against
serial sections. Arrows: lymphatic endothelial cells; scale bar: 100 mm. DAB, 3,3-diaminobenzidine tetrahydrochloride.
PMab-52 is useful for immunohistochemistry using paraffinembedded tissues.
Discussion

In recent studies, we successfully developed specific andsensitive mAbs against dPDPN(29) and bovPDPN,(30) andinvestigated the expression of PDPN in canine and bovine
normal tissues, respectively. Furthermore, we investigatedthe expression of dPDPN in canine cancers, such as canine
squamous cell carcinomas(32) and melanomas.(33) Until now,
anti-cPDPN mAbs have not been developed; therefore, theexpression of cPDPN has not been clarified in immunohistochemical analysis.
Our previous reports revealed that PMab-38 (anti-dPDPN
mAb, mouse IgG1),(29) PMab-44 (anti-bovPDPN mAb,

mouse IgG1),(30) LpMab-12 (anti-hPDPN mAb, mouse
IgG1),(34) PMab-1 (anti-mPDPN mAb, rat IgG2a),(35) PMab-2(anti-rPDPN mAb, mouse IgG1),(27) and PMab-32 (antirabPDPN mAb, mouse IgG1)(28,36) specifically react with
CHO/dPDPN, CHO/bovPDPN, CHO/hPDPN, CHO/
mPDPN, CHO/rPDPN, and CHO/rabPDPN, respectively.
Many other anti-hPDPN mAbs also specifically detect
hPDPN.(14,15,19,21,37–48) In this study, we immunized micewith CHO/cPDPN cells, and developed several anti-cPDPN
mAbs including PMab-52. By using CHO/cPDPN cells for
immunization and first screening, we did not require purifying recombinant cPDPN proteins. This method may be applicable for developing mAbs, especially against membraneproteins that cannot be easily purified. PMab-52 was determined to be IgM class although previously established antiPDPN mAbs were determined to be IgG class, indicating that

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immunization with CHO/cPDPN might not be enough for aclass switch and affinity maturation. Indeed, the binding affinity of PMab-52, which was determined by flow cytometry,
was not high (Fig. 1C, D).
PMab-52 could be useful for uncovering the pathophysiological function of cPDPN in feline tissues. PMab-52 mightalso be applicable for antibody-based therapy against feline
tumors after producing cat–mouse chimeric antibodies.

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Acknowledgments

This work was mainly supported by the Platform Projectfor Supporting Drug Discovery and Life Science Researchfrom Japan Agency for Medical Research and Development, AMED (Y.K.). This work was also supported, in part,by the Basic Science and Platform Technology Program for
Innovative Biological Medicine from AMED (Y.K.), byproject for utilizing glycans in the development of innovative drug discovery technologies from AMED (Y.K.), by
the Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) from AMED (Y.K.), and by JSPS
KAKENHI Grant Numbers 17K07299 and 26440019(M.K.K.) and Grant Number 16K10748 (Y.K.). This work
was performed, in part, under the Cooperative Research
Program of Institute for Protein Research, Osaka University, CR-17-05, and by the Grant for Joint Research
Project of the Institute of Medical Science, the University ofTokyo. The authors would like to thank Enago (www.enago.jp) for the English language review.
Author Disclosure Statement

Y. K. received research funding from Ono PharmaceuticalCo., Ltd.
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YAMADA ET AL.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
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: May 14, 2017
Accepted: June 25, 2017