MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 3, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0013

Establishment of an Anticetacean Podoplanin Monoclonal
Antibody PMab-237 for Immunohistochemical Analysis

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Yukinari Kato,1,2,* Yoshikazu Furusawa,1,2,* Shunsuke Itai,1,3,* Junko Takei,1,3 Takuro Nakamura,1Masato Sano,1 Hiroyuki Harada,3 Shinji Yamada,1 and Mika K. Kaneko1

Podoplanin (PDPN) has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses. Therefore, sensitive and specific monoclonal antibodies (mAbs) targeting PDPN are needed for
immunohistochemical analyses using formalin-fixed paraffin-embedded tissues. Recently, anti-PDPN mAbs
against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse were established inour studies. However, anticetacean (whale) PDPN (wPDPN) has not been established yet. In this study, we
immunized mice with wPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/wPDPN) cells, andscreened mAbs against wPDPN using flow cytometry. One of the mAbs, PMab-237 (IgG1, kappa), specificallydetected CHO/wPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential
usefulness of PMab-237 for the functional analyses of wPDPN.
Keywords: whale podoplanin, PDPN, lymphatic endothelial cells, PMab-237

IntroductionP

odoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed in many cell types, including
pulmonary type I alveolar cells, renal epithelial cells such as
podocytes, and lymphatic endothelial cells of every organ.(1)PDPN has been used to distinguish lymphatic endothelial
cells from vascular endothelial cells in pathophysiologicalstudies.(2) As an endogenous receptor of PDPN, C-type
lectin-like receptor-2 (CLEC-2) was previously reported inour studies.(3,4) The PDPN-CLEC-2 interaction facilitates
the separation of embryonic blood and lymphatic vessels.(5)The expression of human PDPN (hPDPN) has been reported in many malignant tumors, including oral squamouscell carcinomas,(6) lung squamous cell carcinomas,(7)esophageal squamous cell carcinomas,(8) malignant brain
tumors,(9) and malignant mesotheliomas.(10) The expression of hPDPN is associated with malignant progression
and cancer metastasis.(11)We recently developed monoclonal antibodies (mAbs)
against human,(9) mouse,(12) rat,(13) rabbit,(14) bovine,(15) dog,(16)cat,(17) pig,(18) and horse(19) PDPNs. An anti-cat PDPN (cPDPN)
mAb (PMab-52) was shown to cross-react with tiger PDPN
(tigPDPN).(20) An anti-bovine PDPN (bovPDPN) mAb (PMab44) recognized not only bovPDPN but also goat,(21) sheep,(22)and alpaca(23) PDPNs. In this study, we immunized mice with
CHO/whale PDPN (wPDPN) cells and established mAbs
against wPDPN.
Materials and Methods
Cell lines

P3X63Ag8U.1 (P3U1) and CHO-K1 cells were obtained
from the American Type Culture Collection (ATCC, Manassas, VA). Synthesized DNA (Eurofins Genomics KK, Tokyo,
Japan) encoding wPDPN (accession no.: XM_007104824.2)
plus an N-terminal RIEDL tag, which are recognized by ananti-RIEDL tag mAb (LpMab-7), was subcloned into a pCAGNeo vector (FUJIFILM Wako Pure Chemical Corporation,
Osaka, Japan). Plasmids were transfected using Lipofectamine
LTX with Plus Reagent (Thermo Fisher Scientific, Inc., Waltham, MA). Stable transfectants were selected by limiting dilution and cultivation in a medium containing 0.5 mg/mL of
G418 (Nacalai Tesque, Inc., Kyoto, Japan).
The P3U1, CHO-K1, CHO/wPDPN, CHO/hPDPN,(24)CHO/mouse PDPN (mPDPN),(24) CHO/rat PDPN (rPDPN),(13)CHO/rabbit PDPN (rabPDPN),(14) CHO/dog PDPN
(dPDPN),(16) CHO/bovPDPN,(15) CHO/cPDPN,(17) CHO/pig PDPN (pPDPN),(25) CHO/horse PDPN (horPDPN),(26)CHO/tigPDPN,(20) CHO/alpaca PDPN (aPDPN),(23)1Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
3
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
*Equally contributed.
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CHO/bear PDPN (bPDPN),(25) CHO/Tasmanian devil PDPN
(tasPDPN),(25) CHO/goat PDPN (gPDPN),(21) and CHO/sheep
PDPN (sPDPN)(22) were cultured in a Roswell Park Memorial
Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.), supplemented with 10% of heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc.), 100 units/mL of penicillin,
100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B(Nacalai Tesque, Inc.). The cells were grown in an incubator
at 37C with humidity and 5% CO2 and 95% air atmosphere.
Animals

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Female BALB/c mice (6 weeks of age) 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.
Hybridoma production

We employed a Cell-Based Immunization and Screening(CBIS) method(17,27–29) to develop mAbs against wPDPN. In
brief, two BALB/c mice were immunized with CHO/wPDPN
cells (1 · 108) intraperitoneally (i.p.) together with the Imject

FIG. 1. Schematic illustration of the CBIS method. Stabletransfectants expressing the protein of interest were used as
an immunogen with no purification procedure. The selectionof hybridomas secreting specific mAbs was performed byflow cytometry using parental and transfectant cells. CBIS,
Cell-Based Immunization and Screening; mAbs, monoclonal antibodies.

FIG. 2. Detection of wPDPN by flow cytometry using
PMab-237. CHO/wPDPN and CHO-K1 were treated with
PMab-237 (red line) or anti-RIEDL tag (LpMab-7; blueline) at a concentration of 1 mg/mL or 0.1% BSA in PBS(gray) for 30 minutes, followed by incubation with secondary antibodies. CHO, Chinese hamster ovary; BSA,
bovine serum albumin; PBS, phosphate-buffered saline;
PDPN, podoplanin; wPDPN, whale PDPN.

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FIG. 3. Cross-reaction of PMab-237 to PDPNs of the other species by flow cytometry. CHO-K1 cells transfected with
PDPNs of the other species were treated with PMab-237 (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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PMAB-237 AGAINST WHALE PDPN

Alum (Thermo Fisher Scientific, Inc.). The procedure included three additional immunizations, followed by a final
booster injection administered i.p. 2 days before the harvestof spleen cells. Subsequently, these spleen cells were fusedwith P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN), and the hybridomas were grown in an RPMI
medium supplemented with hypoxanthine, aminopterin, andthymidine for selection (Thermo Fisher Scientific, Inc.). The
culture supernatants were screened by flow cytometry.

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Flow cytometry

The cells were harvested after a brief exposure to 0.25%
trypsin and 1 mM ethylenediaminetetraacetic acid (Nacalai
Tesque, Inc.). The cells were washed with 0.1% bovine serum
albumin in phosphate-buffered saline (PBS) and treated withprimary mAbs for 30 minutes at 4C. Thereafter, the cells weretreated 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.). Then, fluorescence data were collected using the SA3800
Cell Analyzer (Sony Corporation, Tokyo, Japan).
Determination of binding affinity by flow cytometry

CHO/wPDPN was suspended in 100 mL of serially diluted
PMab-237. Then, Alexa Fluor 488-conjugated anti-mouse
IgG (1:200; Cell Signaling Technology, Inc.) was added.
Fluorescence data were collected using the EC800 Cell
Analyzer (Sony Corporation). The dissociation constant (KD)was calculated by fitting the binding isotherms to built-in
one-site binding models in the GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA).

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tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed using hematoxylin(FUJIFILM Wako Pure Chemical Corporation).
Results and Discussion

Two mice were immunized with CHO/wPDPN cells
using CBIS method (Fig. 1). The developed hybridomas
were seeded into 96-well plates and cultivated for 10 days.
Wells positive for CHO/wPDPN and negative for CHO-K1
were selected by flow cytometry. The screening approach
identified strong signals from CHO/wPDPN cells and weakor no signals from CHO-K1 cells in 71 of the 960 wells(7.4%). PMab-237 (IgG1, kappa) was finally selected.
PMab-237 recognized CHO/wPDPN cells, but showed noreaction with CHO-K1 cells, as assessed by flow cytometry(Fig. 2). PMab-237 cross-reacted with human, bovine, goat,
sheep, alpaca, and tiger PDPNs (Fig. 3). Those expression
levels were confirmed by each positive control mAb. In addition, kinetic analysis conducted by flow cytometry was
employed to assess the interaction of PMab-237 with
CHO/wPDPN cells. KD of PMab-237 for CHO/wPDPN cells
was determined to be 4.6 · 10-9 M, indicating a high affinityof PMab-237 for CHO/wPDPN cells.
Western blot analysis performed using PMab-237 demonstrated that PMab-237 detects wPDPN as a 40-kDa bandin CHO/wPDPN cells (Fig. 4). An anti-RIEDL tag mAb(LpMab-7) also detected a 40-kDa band in CHO/wPDPN
cells. The immunohistochemical analyses revealed that
PMab-237 strongly stained CHO/wPDPN cells (Fig. 5B),
whereas it did not react with CHO-K1 cells (Fig. 5A).

Western blot analysis

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.). Proteins were thenelectrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred
onto polyvinylidene difluoride membranes (Merck KGaA,
Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.), membranes were incubated with
1 mg/mL of PMab-237, 10 mg/mL of LpMab-7, and 1 mg/mLof anti-b-actin (clone AC-15; Sigma-Aldrich Corporation,
St. Louis, MO), followed by incubation with peroxidaseconjugated anti-mouse IgG (diluted 1:1000; Agilent Technologies, Inc., Santa Clara, CA), and were finally developed
using ImmunoStar LD (FUJIFILM Wako Pure Chemical
Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo,
Japan).
Immunohistochemical analyses

Cell blocks were produced using iPGell (Genostaff Co.,
Ltd., Tokyo, Japan). Histological sections of 4-mm thickness
were deparaffinized in xylene, then rehydrated, and autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc.,Tokyo, Japan) for 20 minutes. Then, sections were blocked
using the SuperBlock T20 (PBS) Blocking Buffer (Thermo
Fisher Scientific, Inc.), incubated with PMab-237 (1 mg/mL)
for 1 hour at room temperature, and treated with the Envision+ Kit for mouse (Agilent Technologies, Inc.) for 30
minutes. Color was developed using 3,3¢-diaminobenzidine

FIG. 4. Western blot analysis. Cell lysates of CHO-K1
and CHO/wPDPN (10 mg) were electrophoresed and transferred onto PVDF membranes. The membranes were incubated with l mg/mL of PMab-237, 10 mg/mL of anti-RIEDL
tag (LpMab-7), or 1 mg/mL of anti-b-actin and subsequently
with peroxidase-conjugated anti-mouse IgG. Red arrow,
wPDPN. PVDF, polyvinylidene difluoride.

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FIG. 5. Immunohistochemical analyses for cell lines.
CHO-K1 cells (A, C) and CHO/wPDPN cells (B, D) wereincubated with 1 mg/mL of PMab-237 (A, B) or blocking
buffer (C, D), followed by that with the Envision+ Kit. (E,F) Hematoxylin and eosin staining. Scale bar = 100 mm.
In conclusion, we established PMab-237 against wPDPN,
which is suitable for use in flow cytometry, Western blot, andimmunohistochemical analyses using CBIS method. The epitope of PMab-237 needs further investigation to clarify thesensitivity and specificity of PMab-237 against wPDPN. We
believe that PMab-237 should prove to be useful in elucidating
the pathophysiological functions of wPDPN in future studies.
Acknowledgments

We thank Miyuki Yanaka, Kayo Hisamatsu, Saori Handa,
and Yoshimi Nakamura for their excellent technical assistance. This research was supported in part by AMED under
grant nos. JP18am0101078 (Y.K.), JP18am0301010 (Y.K.),
and JP18ae0101028 (Y.K.), and by JSPS KAKENHI grantno. 17K07299 (M.K.K.) and grant no. 16K10748 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.
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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: yukinarikato@med.tohoku.ac.jp