MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 1, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0053

PMab-48 Recognizes Dog Podoplaninof Lymphatic Endothelial Cells

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Shinji Yamada,1 Shunsuke Itai,1 Mika K. Kaneko,1 and Yukinari Kato1,2Podoplanin, a type I transmembrane glycoprotein, is a specific marker of lymphatic endothelial cells (LECs).
Recently, we developed PMab-38, an anti-dog podoplanin monoclonal antibody that did not stain canine
LECs. In this study, we newly developed PMab-48 against dog podoplanin. Immunohistochemical analysis revealed that PMab-48 reacts not only with canine squamous cell carcinoma cells but also with LECsof the normal colon. Therefore, PMab-48 may be useful in investigating the function of dog podoplaninin LECs.
Keywords: dog podoplanin, monoclonal antibody, immunohistochemistry

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. Canine tissues wereobtained from North lab (Hokkaido, Japan).(12–14) Female
BALB/c mice (4-weeks old) were purchased from CLEA
Japan (Tokyo, Japan). Animals were housed under specificpathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved the animal experiments described herein.

Introduction

Dog podoplanin, a type I transmembrane sialoglycoprotein, was first reported as gp40.(1) In other species,
podoplanin is also known as Aggrus/T1a/PA2.26(2–4) and isexpressed in normal cells, including renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells(LECs).(3) Podoplanin binds to the C-type lectin-like receptor2 (CLEC-2) of platelets and induces platelet aggregation.(5)The interaction between podoplanin and CLEC-2 facilitates
lymphatic/blood vessel separation.(6) Human podoplanin has
been reported to be expressed in many malignant tumors, suchas lung cancers,(7) esophageal cancers,(8) and malignant mesotheliomas.(9) In addition, podoplanin expression is associated with malignant progression and cancer metastasis.(10,11)
Dog podoplanin has also been investigated in tumors using
PMab-38,(12–14) an immunohistochemically useful monoclonal antibody. In this study, we newly developed PMab-48
against dog podoplanin and investigated its reactivity in immunohistochemistry.

Hybridoma production

BALB/c mice were immunized by an intraperitoneal (i.p.)
injection of 100 mg of a synthetic peptide (dpp2640:
VRPDDIIPGVEDSVVC), conjugated with KLH together
with Imject Alum (Thermo Fisher Scientific, Inc.). Afterseveral additional immunizations of 100 mg, a booster injection of 100 mg was given i.p. 2 days before spleen cells wereharvested. The spleen cells were fused with P3U1 cells byusing PEG1500 (Roche Diagnostics, Indianapolis, IN). The
hybridomas were grown in RPMI medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.).

Materials and Methods
Cell lines, canine tissues, and animals

Enzyme-linked immunosorbent assay

P3U1 was purchased from the American Type Culture
Collection (ATCC, Manassas, VA), and it was cultured inRPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan)
supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc., Waltham, MA), 100 U/mL of1
2

The culture supernatants were screened by using
enzyme-linked immunosorbent assay (ELISA) for binding
to dpp2640, which is immobilized on Nunc Maxisorp
96-well immunoplates (Thermo Fisher Scientific, Inc.) at

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan.

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FIG. 1. Immunohistochemical analysis against canine tissues by PMab-48. (A) Sequence of dog podoplanin andimmunized peptide. (B) Histologic sections of canine tissues (normal kidney, normal colon, and oral squamous cellcarcinomas) were incubated with 50 mg/mL of PMab-48 for 1 hour at room temperature followed by treatment withEnvision+ kit for 30 minutes. Color was developed by using 3,3-diaminobenzidine tetrahydrochloride for 2 minutes, afterwhich the sections were counterstained with hematoxylin. Scale bar: 100 mm. Arrow heads, renal glomerulus; arrows:
lymphatic endothelial cells. SCC, squamous cell carcinomas.

PMAB-48 AGAINST DOG PODOPLANIN

1 mg/mL for 30 minutes. After blocking with 1% bovine
serum albumin in 0.05% Tween20/phosphate-buffered saline (Nacalai Tesque, Inc.), the plates were incubated withculture supernatant followed by 1:3000 diluted peroxidaseconjugated anti-mouse IgG (Agilent Technologies, Inc.,
Santa Clara, CA). The enzymatic reaction was conductedwith a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific,Inc.). The optical density was measured at 655 nm by using
an iMark microplate reader (Bio-Rad Laboratories, Inc.,Hercules, CA).

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Project for Supporting Drug Discovery and Life Science
Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED(Y.K.).
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd. All other authors have no conflicts of interest.
References

Downloaded by Yukinari Kato from online.liebertpub.com at 02/24/18. For personal use only.

Immunohistochemical analyses

Four-micrometer-thick histologic sections were deparaffinized in xylene and rehydrated, and they were autoclavedin citrate buffer (pH 6.0; Agilent Technologies, Inc.) for 20minutes. Sections were incubated with 50 mg/mL of primarymonoclonal antibodies for 1 hour at room temperature followed by treatment with Envision+ kit for 30 minutes(Agilent Technologies, Inc.). Color was developed by using
3,3-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, and then the sections were counterstained with hematoxylin (Wako Pure Chemical Industries,Ltd., Osaka, Japan).
Results and Discussion

We immunized mice with a synthetic peptide (dpp2640)
corresponding to amino acids 26–40 of dog podoplanin(Fig. 1A); screening was performed by using ELISA. The
ELISA-positive wells were screened a second time by flow
cytometry. Finally, the positive wells were selected in an
immunohistochemical analysis. After limiting dilution, oneof the clones, PMab-48 (IgG1, kappa) was established. PMab-48
reacted with Chinese hamster ovary (CHO)/dog podoplanin,
not with CHO/cat podoplanin, CHO/bovine podoplanin, CHO/rabbit podoplanin, CHO/mouse podoplanin, CHO/rat podoplanin, CHO/human podoplanin, and CHO/K1 (Supplementary Fig. S1), indicating that PMab-48 is specific against dog
podoplanin.
PMab-48 reacted with podocytes of renal glomerulus andLECs (Fig. 1B and Supplementary Fig. S2); podoplanin expression has been reported in such cells in other species.(3)Conversely, the previously established PMab-38 did not reactwith LECs.(12) In addition, PMab-48 strongly stained canine
squamous cell carcinomas (SCCs) (Fig. 1B and Supplementary Fig. S2). In this study, canine tissues were stained by
PMab-48 at a concentration of 10–50 mg/mL. PMab-48
stained SCCs strongly even at a concentration of 10 mg/mL(Supplementary Fig. S2); conversely, a higher concentration(50 mg/mL) was better for staining LECs and glomerulus cells(Fig. 1B).
These results indicate that PMab-48 is advantageous for
detecting LECs and may be useful in investigating thefunction of podoplanin in LECs.
Acknowledgments

This work was supported in part by the Basic Scienceand Platform Technology Program for Innovative Biological Medicine from Japan Agency for Medical Researchand Development, AMED (Y.K.) and by the Platform

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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: September 20, 2017
Accepted: November 16, 2017