MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 2, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0002

Development of mPMab-1, a Mouse–Rat
Chimeric Antibody Against Mouse Podoplanin
Shinji Yamada,1,2 Mika K. Kaneko,1,2 Takuro Nakamura,1,2 Osamu Ichii,3 Satoru Konnai,3 and Yukinari Kato1,2,4

Podoplanin (PDPN), the ligand of C-type lectin-like receptor-2, is used as a lymphatic endothelial marker. We
previously established clone PMab-1 of rat IgG2a as a specific monoclonal antibody (mAb) against mouse
PDPN. PMab-1 is also very sensitive in immunohistochemical analysis; however, rat mAbs seem to be unfavorable for pathologists because anti-mouse IgG and anti-rabbit IgG are usually used as secondary antibodies incommercially available kits for immunohistochemical analysis. In this study, we develop a mouse–rat chimericantibody, mPMab-1 of mouse IgG2a, which was derived from rat PMab-1 mAb. Immunohistochemical analysis
shows that mPMab-1 detects podocytes of the kidney, lymphatic endothelial cells of the colon, and type I
alveolar cells of the lung. Importantly, mPMab-1 is more sensitive than PMab-1. This conversion strategy fromrat mAb to mouse mAb could be applicable to other mAbs.
Keywords: podoplanin, monoclonal antibody, chimeric antibody, PMab-1
mouse PDPN (mPMab-1), appropriate VH cDNA of rat PMab1 was subcloned into pFUSE-CHIGmG2a (InvivoGen, SanDiego, CA) and appropriate VL cDNA of rat PMab-1 and CL ofmouse kappa chain were subcloned into pCAG-Neo vector(Wako Pure Chemical Industries, Ltd., Osaka, Japan), respectively. Antibody expression vectors were transfected into CHOS cells (Thermo Fisher Scientific, Inc., Waltham, MA) using theLipofectamine LTX reagent (Thermo Fisher Scientific, Inc.).
Stable transfectants of CHO-S/mPMab-1 were selected bycultivating the transfectants in a medium containing 0.5 mg/mLof both geneticin and zeocin (InvivoGen). CHO-S/mPMab-1
cells were cultivated in CHO-S-SFM II medium (ThermoFisher Scientific, Inc.). mPMab-1 was purified using Protein GSepharose (GE healthcare Bio-Sciences, Pittsburgh, PA).

Introduction

Podoplanin (PDPN) is expressed in many normal tissues such as podocytes of the kidney, lymphatic endothelial cells of many tissues, and type I alveolar cells of thelung.(1) Several anti-mouse PDPN monoclonal antibodies(mAbs) such as clone 8.1.1 or PMab-1 are commerciallyavailable.(1,2) However, 8.1.1 is produced using hamsters andPMab-1 is produced using rats because it is difficult to develop
anti-mouse PDPN mAbs using mice. PMab-1 is produced
against the platelet aggregation-stimulating domain of mouse
PDPN(1); therefore, PMab-1 neutralizes the interaction between PDPN and C-type lectin-like receptor 2 (CLEC-2).
Administration of PMab-1 reduces lymphangiogenesis in thecorneal suture and ear wound healing models.(3) PMab-1 also
suppresses the infiltration of thioglycollate-induced macrophages at the site of wound healing. Furthermore, administration of PMab-1 leads to a significant suppression of therejection reaction in the corneal transplantation model, suggesting that PDPN is a novel therapeutic target for suppressing
lymphangiogenesis and inflammation.
In this study, we produce a mouse–rat chimeric antibody,mPMab-1, which is derived from PMab-1.

Flow cytometry

CHO/mPDPN cells were established and cultured as described previously.(4) Cell lines were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque, Inc.,Kyoto, Japan). After washing, the cells were treated with
PMab-1 or mPMab-1 (1 mg/mL) for 30 minutes at 4C, followed by treatment with Oregon Green 488 goat anti-rat or
anti-mouse IgG (Thermo Fisher Scientific, Inc.). Fluorescence data were collected using a Cell Analyzer EC800(Sony Corp., Tokyo, Japan).

Materials and Methods
Production of mPMab-1, a mouse–rat chimericanti-mouse PDPN antibody

Immunohistochemical analyses

PMab-1, a rat anti-mouse PDPN mAb, was developed aspreviously described.(1) To generate a mouse–rat chimeric anti-This study used normal mouse tissues, which were isolated
from one specific pathogen-free DBA/2Cr mouse (SLC

Departments of 1Antibody Drug Development and 2Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
3Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
4New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

77

78Japan, Shizuoka, Japan). Four-micrometer-thick histologic
sections were deparaffinized in xylene and rehydrated.
After antigen retrieval using citrate buffer (pH 6.0; Dako,Glostrup, Denmark), sections were incubated with 1 mg/mL
of PMab-1 or mPMab-1 for 1 hour at room temperature,
followed by treatment with Envision+ kit (Dako) for 30
minutes. Color was developed using 3,3-diaminobenzidine
tetrahydrochloride (DAB; Dako) for 30 seconds, and then thesections were counterstained with hematoxylin (Wako Pure
Chemical Industries Ltd.).
Results and Discussion

We previously established PMab-1 of rat IgG2a as a specific mAb against mouse PDPN. PMab-1 neutralizes the interaction between PDPN and CLEC-2. PMab-1 is also very
sensitive in the immunohistochemical analysis. In contrast,
rat mAbs seem to be unfavorable for pathologists because
anti-rat IgG is not included as secondary antibody in commercially available kits for immunohistochemical analysis
such as Envision+ (Dako). Nevertheless, mouse anti-mouse
PDPN antibodies have not been reported. Therefore, weherein produced a mouse–rat chimeric antibody, mPMab-1,
which was derived from PMab-1.

FIG. 1. Flow cytometric analysis of anti-mouse PDPN
mAbs. Cells were treated with PMab-1 and mPMab-1 (1 mg/mL) or control PBS (black) for 30 minutes at 4C, followed
by treatment with anti-rat or mouse IgG Oregon Green.
Fluorescence data were collected using a Cell Analyzer
EC800. mAb, monoclonal antibody; PBS, phosphatebuffered saline; PDPN, podoplanin.

YAMADA ET AL.

We first checked the reactivity of mPMab-1 in flow cytometry. The reactivity of mPMab-1 against CHO/mPDPN
cells is compatible to that of PMab-1 (Fig. 1). To investigate whether mPMab-1 is useful in immunohistochemical
analysis using commercially available kits, we next compared the reactivity between PMab-1 and mPMab-1 against
normal mouse tissues. mPMab-1 reacted with renal podocytes (Fig. 2A, B) in the same way as PMab-1 (Fig. 2C, D).
Importantly, the reactivity of mPMab-1 is higher than that ofPMab-1. Without primary antibodies, slight unspecificstaining was observed in red blood cells of the renal glomerulus (Supplementary Fig. S1A, B). Both mPMab-1(Fig. 3A, B) and PMab-1 (Fig. 3C, D) also stained lymphatic
endothelial cells and fibroblasts of the colon. The signal ofmPMab-1 is also higher compared with PMab-1. Furthermore, both mPMab-1 (Fig. 4A, B) and PMab-1 (Fig. 4C, D)
stained type I alveolar cells of the lung, indicating thatmPMab-1 is very useful for immunohistochemical analysis
using commercially available immunohistochemical kits.
In this study, PMab-1 was also shown to be useful in immunohistochemical analysis, although anti-mouse secondary
antibodies are included in immunohistochemical kits. Antimouse secondary antibodies usually cross-react with rat antibodies; cross-reaction seems to possess a lower reliability
for pathologists and scientists. mPMab-1 could be used as
mouse IgG and its reactivity is higher than that of PMab-1.
This conversion strategy from rat mAb to mouse antibody
could be applicable to other mAbs.

FIG. 2. An immunohistochemical analysis using antimouse PDPN mAbs to detect PDPN expression in the kidney. Sections were incubated with mPMab-1 (A, B) orPMab-1 (C, D) and made to react with the Envision+ kit.
Color was developed using DAB, and samples were thencounterstained with hematoxylin. Sections were also stainedwith hematoxylin and eosin (E, F). Scale bar = 100 mm.

MPMAB-1 AGAINST MOUSE PODOPLANIN

79
Acknowledgments

FIG. 3. An immunohistochemical analysis using antimouse PDPN mAbs to detect PDPN expression in the colon.
Sections were incubated with mPMab-1 (A, B) or PMab-1(C, D) and made to react with the Envision+ kit. Color wasdeveloped using DAB, and samples were then counterstained with hematoxylin. Sections were also stained withhematoxylin and eosin (E, F). Scale bar = 100 mm.

The authors thank Miyuki Yanaka, Noriko Saidoh, andKanae Yoshida for their excellent technical assistance. They
also thank Satoshi Ogasawara, Yuki Fujii, Hiroaki Uchida,and Hideaki Tahara for their specialized advice. This work
was supported, in part, by the Basic Science and Platform
Technology Program for Innovative Biological Medicine
from Japan Agency for Medical Research and development,
AMED (Y.K.), by the Translational Research Network
Program from AMED (Y.K.), by a project 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.), by JSPS KAKENHI Grant Number
26440019 (M.K.K.) and 16K10748 (Y.K.), and by theRegional Innovation Strategy Support Program from theMinistry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Y.K.). This work was performed,in part, under the Cooperative Research Program of Institutefor Protein Research, Osaka University, CR-16-05, and bythe Grant for Joint Research Project of the Institute ofMedical Science, the University of Tokyo. The authors
would like to thank Enago (www.enago.jp) for the Englishlanguage review.
Author Disclosure Statement

No competing financial interests exist.
References

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Immunohistochemical examination of novel rat monoclonal
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Yamamoto K, Matsumoto Y, Sawane M, Kerjaschki D,
Nakazawa T, Kinoshita S: The effect of podoplanin inhibition on lymphangiogenesis under pathological conditions.
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FIG. 4. An immunohistochemical analysis using antimouse PDPN mAbs to detect PDPN expression in the lung.
Sections were incubated with mPMab-1 (A, B) or PMab-1(C, D) and made to react with the Envision+ kit. Color wasdeveloped using DAB, and samples were then counterstained with hematoxylin. Sections were also stained withhematoxylin and eosin (E, F). Scale bar = 100 mm.

Address correspondence to:Dr. Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: January 17, 2017
Accepted: February 28, 2017