MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 2, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0003

Establishment of a Monoclonal Antibody PMab-231
for Tiger Podoplanin

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Yoshikazu Furusawa,1–3 Mika K. Kaneko,1 Takuro Nakamura,1 Shunsuke Itai,1,4 Masato Fukui,3
Hiroyuki Harada,4 Shinji Yamada,1 and Yukinari Kato1,2

Podoplanin (PDPN), also known as T1alpha, has been used as a lung type I alveolar cell marker in thepathophysiological condition. Although we have established several monoclonal antibodies (mAbs) against
mammalian PDPNs, mAbs against tiger PDPN (tigPDPN), which are useful for immunohistochemical analysis,remain to be developed. In this study, we immunized mice with tigPDPN-overexpressing Chinese hamster
ovary (CHO)-K1 cells (CHO/tigPDPN) and screened hybridomas producing mAbs against tigPDPN using flow
cytometry. One of the mAbs, PMab-231 (IgG2a, kappa), specifically detected CHO/tigPDPN cells using flow
cytometry as well as recognized tigPDPN protein using western blotting. In addition, PMab-231 was found to
cross-react with cat PDPN (cPDPN). The dissociation constants (KD) of PMab-231 for CHO/tigPDPN andCHO/cPDPN cells were determined to be 1.2 · 10-8 and 1.9 · 10-8, respectively, indicating moderate affinity
for CHO/tigPDPN and CHO/cPDPN cells. PMab-231 stained type I alveolar cells of the feline lungs andpodocytes of the feline kidneys using immunohistochemistry. Our findings suggest the potential usefulness ofPMab-231 for the functional analyses of tigPDPN and cPDPN.
Keywords: tiger podoplanin, PDPN, PMab-231

rat,(16) rabbit,(17) bovine,(18) dog,(19) cat,(20) pig,(21) andhorse(22) PDPNs. However, anti-tiger PDPN mAbs have notbeen reported. In this study, we immunized mice with
CHO/tigPDPN cells and established hybridomas that could
produce mAbs against tigPDPN.

IntroductionP

odoplanin (PDPN), also known as a T1alpha, is a type
I transmembrane sialoglycoprotein that is expressed inseveral normal cells, including type I alveolar cells.(1)Therefore, PDPN is extremely useful for distinguishing type I
alveolar cells from type II alveolar cells in pathological diagnosis.(2) We previously reported that C-type lectin-likereceptor-2 (CLEC-2) is an endogenous receptor of PDPN.(3,4)
Importantly, the PDPN/CLEC-2 interaction facilitates embryonic blood/lymphatic vessel separation.(5) PDPN/CLEC-2
interaction also plays an essential role in murine lung development.(2)The expression of human PDPN (hPDPN) has been reported in several malignant tumors, including brain tumors,(6)oral squamous cell carcinomas,(7) lung cancers,(8) esophageal
cancers,(9) malignant mesotheliomas,(10) osteosarcomas,(11)chondrosarcomas,(12) and testicular tumors.(13) The hPDPN
expression is associated with cancer metastasis and malignant progression.(4,6,14) Till date, we have developed monoclonal antibodies (mAbs) against human,(15) mouse,(15)Materials and Methods
Cell lines and animals

P3X63Ag8U.1 (P3U1) and CHO-K1 cells were obtained
from the American Type Culture Collection (ATCC, Manassas, VA). The tigPDPN bearing an N-terminal LP tag (LPtigPDPN) was inserted into a pCAG-Neo vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan).(23) The
LP tag comprises 16 amino acids (NSVTGIRIEDLPTSES).
The CHO-K1 cells were transfected with pCAG-Neo/LPtigPDPN using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants
were selected by limiting dilution and then cultivated in a
medium containing 0.5 mg/mL of G418 (Nacalai Tesque,Inc., Kyoto, Japan). The P3U1, CHO-K1, CHO/tigPDPN,

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
3
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.
2

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CHO/cat PDPN (cPDPN),(20) CHO/hPDPN,(24) CHO/mouse
PDPN (mPDPN),(24) CHO/rat PDPN (rPDPN),(16) CHO/rabbit
PDPN (rabPDPN),(17) CHO/dog PDPN (dPDPN),(19) CHO/
bovine PDPN (bovPDPN),(18) CHO/pig PDPN (pPDPN),(21)CHO/horse PDPN (horPDPN),(25) CHO/alpaca PDPN
(aPDPN),(26) CHO/bear PDPN (bPDPN),(21) CHO/goat
PDPN (gPDPN),(27) CHO/sheep PDPN (sPDPN),(28) CHO/
Tasmanian devil (tasPDPN),(21) and CHO/whale PDPN
(wPDPN)(21) were cultured in 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 penicillin, 100 mg/mL streptomycin,
and 25 mg/mL amphotericin B (Nacalai Tesque, Inc.). The cells
were grown at 37C in a humidified environment with 5% CO2
and 95% air atmosphere.

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Hybridoma production

Female BALB/c mice (aged 6 weeks) were purchasedfrom CLEA Japan (Tokyo, Japan). The animals were housed
under specific pathogen-free conditions. The Animal Careand Use Committee of Tohoku University approved all
animal experiments. We employed a Cell-Based Immunization and Screening (CBIS) method(20,29–31) to develop sensitive and specific mAbs against tigPDPN. In brief,
two BALB/c mice were immunized with CHO/tigPDPN
cells (1 · 108), which were intraperitoneally (i.p.) administered with Imject Alum (Thermo Fisher Scientific, Inc.).
The procedure included three additional immunizations,followed by a final booster injection i.p. administered 2 days
before the harvesting of the spleen cells, resulting in total
five immunizations. These spleen cells were subsequently
fused with P3U1 cells using PEG1500 (Roche Diagnostics,
Indianapolis, IN), and the hybridomas were grown in RPMI
medium supplemented with hypoxanthine, aminopterin,
and thymidine for selection purpose (Thermo Fisher Scientific, Inc.). The culture supernatants were screened using
flow cytometry.
Flow cytometry

The cells were harvested after a brief exposure to 0.25%
trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). The cells were washed with 0.1% bovine serum
albumin/phosphate-buffered saline (PBS) and then treatedwith primary mAbs for 30 minutes at 4C. Next, the cells
were treated with Alexa Fluor 488-conjugated anti-mouse
IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA)
or Oregon Green anti-rat IgG (1:2000; Thermo Fisher Scientific, Inc.). Fluorescence data were collected using theSA3800 Cell Analyzers (Sony Corp., Tokyo, Japan).
Determination of binding affinity using flow cytometry

CHO/tigPDPN was suspended in 100 mL of serially diluted
PMab-231, followed by the addition of Alexa Fluor 488conjugated anti-mouse IgG (1:200; Cell Signaling Technology, Inc.). Fluorescence data were collected using the EC800
Cell Analyzer (Sony Corp.). The dissociation constant (KD)was obtained by fitting the binding isotherms to built-in onesite binding models in GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA).

FURUSAWA ET AL.
Western blotting

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.). The proteins were
subjected to electrophoresis on 5%–20% polyacrylamide gels
(Nacalai Tesque, Inc.) and subsequently transferred onto a
polyvinylidene difluoride membrane (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai
Tesque, Inc.), each membrane was incubated with primarymAbs, such as 1 mg/mL PMab-231, 10 mg/mL anti-LP tag(LpMab-17), 1 mg/mL anti-MAP tag (PMab-1), or 1 mg/mL
anti-b-actin (AC-15; Sigma-Aldrich Corp., St. Louis, MO),and subsequently with peroxidase-conjugated anti-mouse
IgG (1:1000; Agilent Technologies, Santa Clara, CA) or antirat IgG (1:10,000; Sigma-Aldrich Corp.). The bands werevisualized with the ImmunoStar LD (FUJIFILM Wako Pure
Chemical Corporation) using the Sayaca-Imager (DRC Co.
Ltd., Tokyo, Japan).
Immunohistochemical analyses

The normal feline tissues were purchased from Zyagen(San Diego, CA). Paraffin-embedded tissue sections (4 mm indiameter) were directly autoclaved in citrate buffer (pH 6.0;
Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes.
These tissue sections were then blocked with the SuperBlock
T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.),
incubated with PMab-231 (5 mg/mL) for 1 hour at room
temperature, and finally treated using the Envision+ Kit(Agilent Technologies, Inc.) for 30 minutes. Color was developed with 3,3¢-diaminobenzidine tetrahydrochloride(Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed with hematoxylin (FUJIFILM Wako
Pure Chemical Corporation).
Results and Discussion

In this study, we employed the CBIS method to develop
sensitive and specific mAbs against tigPDPN to facilitate
the immunohistochemical analysis of paraffin-embedded
tissue sections. Two mice were immunized with CHO/tigPDPN cells by following a screening procedure (Fig. 1).
The developed hybridomas were seeded into 96-well plates
and cultivated for 7 days. Wells positive for CHO/tigPDPN
and negative for CHO-K1 were selected using flow cytometry. Screening identified strong signals against
CHO/tigPDPN cells and weak or no signals against CHOK1 cells in 18 of 958 wells (1.9%). One of the six clones,
PMab-231 (IgG2a, kappa), was finally selected using immunohistochemistry against paraffin-embedded feline
normal tissue sections because tigPDPN and cPDPN shared
99% homology.(23)PMab-231 recognized CHO/tigPDPN, but showed no reaction with CHO-K1, as assessed using flow cytometry(Fig. 2). LpMab-17, an anti-LP tag mAb, detected CHO/
tigPDPN. PMab-231 cross-reacted with cPDPN and
horPDPN, but did not react with human, mouse, rat, rabbit,
dog, bovine, pig, alpaca, bear, goat, sheep, Tasmanian devil,
or whale PDPNs (Fig. 3).
A kinetic analysis performed using flow cytometry assessed the interaction of PMab-231 with CHO/tigPDPN andCHO/cPDPN cells. KD of PMab-231 for CHO/tigPDPN
and CHO/cPDPN cells were determined to be 1.2 · 10-8 andDownloaded by Yukinari Kato from www.liebertpub.com at 04/22/19. For personal use only.

PMab-231 AGAINST TIGER PDPN

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FIG. 1. Schematic illustration of CBIS method. Stabletransfectants expressing tigPDPN used as an immunogenwith no purification procedure. The selection of hybridomas
secreting specific mAbs performed by flow cytometry using
parental and transfectant cells. CBIS, Cell-Based Immunization and Screening; mAbs, monoclonal antibodies;
tigPDPN, tiger podoplanin.
1.9 · 10-8, respectively, indicating moderate affinity for
CHO/tigPDPN and CHO/cPDPN cells.
Western blotting performed using PMab-231 demonstrated that PMab-231 detected broad bands of tigPDPN andcPDPN between 35 and 48 kDa in CHO/tigPDPN andCHO/cPDPN cells, respectively (Fig. 4). LpMab-17, an antiLP tag mAb and PMab-1, an anti-MAP tag mAb, detectedeach band in CHO/tigPDPN and CHO/cPDPN cells, respectively.(24,32)
The immunohistochemical analyses revealed that PMab231 stained the feline lungs (Fig. 5) and kidneys (Fig. 6), but
PMab-231 did not react with lymphatic endothelial cells(data not shown). These results indicate that PMab-231 isuseful for the detection of cPDPN using immunohistochemistry. Because tigPDPN and cPDPN shared 99% homology,
PMab-231 may be useful for the detection of tigPDPN using
immunohistochemistry.
In conclusion, we established a mAb against tigPDPN,
PMab-231, which is suitable for use in flow cytometry,western blotting, and immunohistochemical analyses. The‰
FIG. 2. Detection of tigPDPN by flow cytometry using
PMab-231. CHO/tigPDPN and CHO-K1 cells were treatedwith PMab-231 (red line) or anti-LP tag (LpMab-17; blueline) at a concentration of 1 mg/mL or 0.1% BSA in PBS(gray) for 30 minutes, followed by incubation with secondary antibodies. BSA, bovine serum albumin; CHO,
Chinese hamster ovary; PBS, phosphate-buffered saline.

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FIG. 3. Cross-reaction of PMab-231 to PDPNs of the other species using flow cytometry. CHO-K1 cells transfected with
PDPNs of the other species were treated with PMab-231 (red line) or each positive control (blue line) at a concentration of
1 mg/mL or 0.1% BSA in PBS (gray) for 30 minutes, followed by incubation with secondary antibodies.

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FIG. 3.

(Continued).

FIG. 4. Western blotting. Cell lysates of CHO-K1, CHO/tigPDPN, and CHO/cPDPN (10 mg) electrophoresed andtransferred onto PVDF membranes. The membranes incubated with l mg/mL PMab-231, 10 mg/mL anti-LP tag (LpMab-17),
1 mg/mL anti-MAP tag (PMab-1), or 1 mg/mL anti-b-actin, and subsequently, with peroxidase-conjugated anti-mouse or -rat
IgG. cPDPN, cat PDPN; PVDF, polyvinylidene difluoride.
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FURUSAWA ET AL.

epitope of PMab-231 is required to be further investigated toclarify the sensitivity and specificity of PMab-231 against
tigPDPN. It is expected that PMab-231 would prove usefulfor the elucidation of the pathophysiological functions of
tigPDPN in future studies.
Acknowledgments

We thank Miyuki Yanaka, Masato Sano, Kayo Hisamatsu,
Saori Handa, 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.), and JP18ae0101028 (Y.K.), and by
JSPS KAKENHI grant number 17K07299 (M.K.K.) and
16K10748 (Y.K.).

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Author Disclosure Statement

Y.K. received research funding from Zenoaq ResourceCo., Ltd. The other authors have no conflict of interest.
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FIG. 5. Immunohistochemical analyses against the feline
lungs. Tissue sections of the feline lungs incubated with
5 mg/mL PMab-231 (A, B) or with a blocking buffer (C, D),followed by the Envision+ kit. (E, F) Hematoxylin andeosin staining. Scale bar = 100 mm.

FIG. 6. Immunohistochemical analyses against the feline
kidneys. Tissue sections of the feline kidneys incubated with
5 mg/mL PMab-231 (A, B) or with blocking buffer (C, D),followed by the Envision+ kit. (E, F) Hematoxylin andeosin staining. Scale bar = 100 mm.

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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp