MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 43, Number 2, 2024
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2024.0003
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PMab-314:
An Anti-Giant Panda Podoplanin
Monoclonal Antibody

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Tsunenori Ouchida,* Guanjie Li,* Hiroyuki Suzuki, Miyuki Yanaka, Takuro Nakamura, Saori Handa,
Tomohiro Tanaka, Mika K. Kaneko, and Yukinari Kato

The giant panda (Ailuropoda melanoleuca) is one of the important species in worldwide animal conservation.
Because it is essential to understand the disease of giant panda for conservation, histopathological analyses oftissues are important to understand the pathogenesis. However, monoclonal antibodies (mAbs) against giant
panda-derived proteins are limited. Podoplanin (PDPN) is an essential marker of lung type I alveolar epithelial
cells, kidney podocytes, and lymphatic endothelial cells. PDPN is also overexpressed in various human tumors,which are associated with poor prognosis. Here, an anti-giant panda PDPN (gpPDPN) mAb, PMab-314 (mouse
IgG1, kappa) was established using the Cell-Based Immunization and Screening method. PMab-314 recognizedN-terminal PA16-tagged gpPDPN-overexpressed Chinese hamster ovary-K1 cells (CHO/PA16-gpPDPN) inflow cytometry. The KD value of PMab-314 for CHO/PA16-gpPDPN was determined as 1.3 · 10-8 M. Furthermore, PMab-314 is useful for detecting gpPDPN in western blot analysis. These findings indicate that
PMab-314 is a useful tool for the analyses of gpPDPN-expressed cells.
Keywords: giant panda, podoplanin, monoclonal antibody, flow cytometry, western blotting

invasion and metastasis.13 Therefore, anti-PDPN monoclonal
antibodies (mAbs) are useful to distinguish the specific cell
types and investigate the PDPN-expressing tumor cells.
We have developed mAbs against PDPN of various species including human,14 mouse,15 rat,16 rabbit,17 dog,18 bovine,19 cat,20 tiger,21 horse,22 pig,23 goat,24 sheep,25 alpaca,26
Tasmanian devil,27 bear,28 whale,29 California sea lion,30
hamster,31 ferret,32 elephant,33 and giraffe.34 Here, we established an anti-giant panda PDPN (gpPDPN) mAb, PMab314, using the Cell-Based Immunization and Screening(CBIS) method and evaluated the applications.

IntroductionT

he giant panda (Ailuropoda melanoleuca) is an endangered animal in the world. The Fourth National
Survey Report on the Giant Panda reported that there are 1864
wild giant pandas in 33 segregated local populations. However, 25 of these populations are too small to be self-sustaining
and are at high risk of extinction.1 The main causes of death ofgiant pandas were gastrointestinal diseases, parasites, and viral
infections.2,3 With an increased population of captive-bred
giant pandas, chronic kidney disease (CKD)4 and tumor5,6 in
the giant pandas were also reported. These reports showed thehistological analysis of affected kidney,4 pancreatic ductal
adenocarcinoma (PDAC),5 and testicular seminoma.6
Podoplanin (PDPN) is a type I transmembrane mucin-likeprotein7 conserved in various species8 and is an important
marker of lung type I alveolar cells,9,10 kidney podocytes,7and lymphatic endothelial cells.11,12 In humans, PDPN isoverexpressed in tumors and plays crucial roles in tumor

Materials and Methods
Preparation of cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture
Collection (Manassas, VA). The full-length open reading

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Miyagi, Japan.
*Contributed equally to this work.

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frame of gpPDPN (Accession No.: XM_011232779) was
synthesized by Eurofins Genomics KK. The gpPDPN sequence, in which the original signal sequence of the
N-terminus (1-MWKVPVLLLVLGSVWLWDLAEG-22) was
deleted, was obtained by polymerase chain reaction. Interleukin 2-signal sequence and PA16 tag or MAP16 tag of the
N-terminus of gpPDPN were inserted into pCAGzeo vectors.
The amino acid sequences of the PA16 tag and the MAP16 tagwere GLEGGVAMPGAEDDVV and PGTGDGMVPPGIEDKI, respectively. The PA16 tag and the MAP16 tag can bedetected by NZ-114,35 and PMab-1,15,36 respectively.
The gpPDPN plasmids were transfected into CHO-K1
cells, using a Neon transfection system (Thermo Fisher Scientific, Inc., Waltham, MA). Clones stably expressing
gpPDPN were established through cell sorting using a cell
sorter (SH800; Sony Corp., Tokyo, Japan), after which cultivation in a medium, containing 0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA) was conducted.
CHO-K1, gpPDPN-overexpressed CHO-K1 (CHO/
MAP16-gpPDPN and CHO/PA16-gpPDPN), and P3U1 cells
were cultured in a Roswell Park Memorial Institute (RPMI)1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), with
10% heat-inactivated fetal bovine serum (FBS; Thermo
Fisher Scientific, Inc.), 100 units/mL of penicillin,
100 lg/mL of streptomycin, and 0.25 lg/mL of amphotericin
B (Nacalai Tesque, Inc.). All cells were grown in a humidified incubator at 37C, in an atmosphere of 5% CO2 and 95%
air.
Production of hybridomas

Two five-week-old BALB/c female mice were purchasedfrom CLEA Japan (Tokyo, Japan). The animals were housed
under specific pathogen-free conditions. All animal experiments were performed according to the relevant guidelines
and regulations to minimize animal suffering and distress in
the laboratory. The Animal Care and Use Committee of Tohoku University (Permit number: 2022MdA-001) approved
animal experiments.
To establish mAbs against gpPDPN, mice (aged six weeks)
were intraperitoneally immunized with CHO/MAP16gpPDPN cells (1 · 108 cells/mouse) plus Alhydrogel adjuvant
2% (InvivoGen). The procedure included three additional injections every week (1 · 108 cells/mouse), which was followed
by a final booster intraperitoneal injection (1 · 108 cells/mouse), two days before harvesting splenocytes. The splenocytes were fused with P3U1 cells, using PEG1500 (Roche
Diagnostics, Indianapolis, IN). Hybridomas were selected andcultivated in the RPMI-1640 medium with 10% FBS, 100
units/mL of penicillin, 100 lg/mL of streptomycin,
0.25 lg/mL of amphotericin B, 5 lg/mL of Plasmocin, 5%
Briclone (NICB, Dublin, Ireland), and hypoxanthine, aminopterin, and thymidine (Thermo Fisher Scientific, Inc.). The
supernatants were subsequently screened by flow cytometry
using CHO/PA16-gpPDPN and CHO-K1.
Purification of PMab-314

The cultured supernatants of PMab-314-producing hybridomas were filtrated with Steritop (0.22 lm; Merck
KGaA, Darmstadt, Germany) and subsequently applied to
1 mL of Ab-Capcher ExTra (ProteNova, Inc., Kagawa, Japan). After washing with phosphate-buffered saline (PBS),

OUCHIDA ET AL.

bound antibodies were eluted with an IgG elution buffer(Thermo Fisher Scientific, Inc.), followed by immediate
neutralization of eluates, using 1M Tris-HCl (pH 8.0). Finally, the eluates were concentrated, after which the buffer
was replaced with PBS using Amicon Ultra (Merck KGaA).
Flow cytometric analyses

CHO-K1 and CHO/PA16-gpPDPN cells were harvested
by exposure to 1 mM ethylenediaminetetraacetic acid
(EDTA; Nacalai Tesque, Inc.). The cells were washed with
0.1% bovine serum albumin in PBS and treated with PMab314 (0.01–10 lg/mL) and NZ-1 (1 lg/mL) for 30 min at 4C.
Then, the cells were treated with Alexa Fluor 488-conjugatedanti-mouse IgG (1:1000; Cell Signaling Technology, Inc.,
Danvers, MA) for PMab-314 or Alexa Fluor 488-conjugatedanti-rat IgG (1:1000; Cell Signaling Technology, Inc.) for
NZ-1. The fluorescence data were collected using theSA3800 Cell Analyzer (Sony Corp.).
To determine the dissociation constant (KD), PMab-314
was serially 1:1 diluted from 20 mg/mL to 1.2 ng/mL. Then,the cells were treated with Alexa Fluor 488-conjugated antimouse IgG (1:200). The geometric mean of fluorescenceintensity of CHO/PA16-gpPDPN at each concentration was
calculated by FlowJo (BD Biosciences, Franklin Lakes, NJ).
The KD was calculated by fitting saturation binding curves tothe built-in; one-site binding models in GraphPad PRISM 6(GraphPad Software, Inc., La Jolla, CA).
Western blotting

CHO-K1 and CHO/PA16-gpPDPN cells were harvested
by exposure to 0.25% trypsin and 1 mM EDTA (Nacalai
Tesque, Inc.). Cell lysates were boiled in sodium dodecyl
sulfate sample buffer (Nacalai Tesque, Inc.), after which
proteins were separated on 5%–20% polyacrylamide gels
(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and transferred to polyvinylidene difluoride membranes(Merck KGaA). After blocking with 4% skim milk (Nacalai
Tesque, Inc.) in 0.05% Tween 20-containing PBS, membranes were incubated with 1 lg/mL of PMab-314, an antiPA16 tag mAb (NZ-1), or an anti-isocitrate dehydrogenase 1(IDH1) mAb (RcMab-1).37 Next, they were incubated again
with horseradish peroxidase-conjugated anti-mouse immunoglobulins (1:1000; Agilent Technologies, Inc., Santa
Clara, CA) for PMab-314 or horseradish peroxidaseconjugated anti-rat immunoglobulins (diluted 1:10,000;
Sigma-Aldrich Corp., St. Louis, MO) for NZ-1 and RcMab-1.
Finally, protein bands were detected using ECL Plus Western
Blotting Substrate (Thermo Fisher Scientific, Inc.) and a
Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).
Results
Establishment of a novel anti-gpPDPN mAb

For the establishment of anti-gpPDPN mAb, we used the
CBIS method. The CBIS method is composed of 2 steps:
immunization of antigen-overexpressed cells and screeningof hybridoma supernatants using flow cytometry. The CBIS
method is a useful approach for the establishment of mAbs
against glycoproteins38,39 and multiple transmembrane proteins.40–42 In this study, we immunized mice with CHO/
MAP16-gpPDPN cells (Fig. 1A). The splenocytes from theseDownloaded by Yukinari Kato from www.liebertpub.com at 04/09/24. For personal use only.

FIG. 1. The scheme of establishment of PMab-314 by the CBIS method. (A) Immunization and production of hybridomas. CHO/MAP16-gpPDPN cells were injected intraperitoneally into two BALB/c mice. The splenocytes were fused with
P3U1 cells using polyethylene glycol. (B) Screening of hybridomas. The culture supernatants were screened through flow
cytometry to select anti-gpPDPN mAb-producing hybridomas. PMab-314 was established by limiting dilution. CHO,
Chinese hamster ovary; gpPDPN, giant panda podoplanin.

FIG. 2. Flow cytometry of gpPDPN-overexpressed cells using PMab-314. CHO/PA16-gpPDPN (A) and CHO-K1(B) cells were treated with 0.01–10 mg/mL of PMab-314 (red line) or blocking buffer (negative control, black line), followed
by treatment with Alexa Fluor 488-conjugated anti-mouse IgG. NZ-1 is used as a positive control to detect PA16-gpPDPN
expression on CHO/PA16-gpPDPN.
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OUCHIDA ET AL.

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FIG. 3. The determination of the binding affinity ofPMab-314 against CHO/PA16-gpPDPN cells by flow cytometry. The dots are the geometric mean of fluorescenceintensity of CHO/PA16-gpPDPN at each concentration. The
solid lines show the fitting curve calculated by GraphPad
PRISM 6.

mice were fused with P3U1 cells by using polyethylene
glycol. Hybridomas producing antibodies against CHO/
PA16-gpPDPN cells were selected by flow cytometry(Fig. 1B). After limiting dilution of hybridomas, PMab-314(mouse IgG1, kappa) was established.
Flow cytometry using PMab-314

We examined the reactivity of PMab-314 to gpPDPN byflow cytometry. PMab-314 reacted to CHO/PA16-gpPDPN
cells in a dose-dependent manner (Fig. 2A). In contrast,
PMab-314 did not bind to CHO-K1 cells even at 10 lg/mL(Fig. 2B). These data indicated that PMab-314 specificallydetects gpPDPN.
Next, we determined the KD of PMab-314 with CHO/
PA16-gpPDPN cells by flow cytometry. After the calculationof the geometric mean at each concentration and fitting onesite binding models, the KD value of PMab-314 for gpPDPN
was determined as 1.3 · 10-8 M (Fig. 3).
Western blotting using PMab-314

Finally, we examined the application of PMab-314 inwestern blotting. Lysates of CHO-K1 and CHO/PA16gpPDPN cells were probed. PMab-314 and NZ-1 detected the
48-kDa band of gpPDPN in lysates from CHO/PA16-gpPDPN
cells, whereas any bands were not detected in lysates of CHOK1 cells (Fig. 4).
Discussion

CKD exhibits irreversible and progressive loss of functionof the kidney. Previous reports showed that CKD was acommon cause of mortality in captive animals such as
bears.43 In giant panda, a case report describes clinical features of azotemia, polyuria, and anemia.4 The histopathological analysis of kidney showed that glomeruli showed
diffuse cystic changes with capillary tufts atrophy, andBowman’s capsules were moderately thickened.4 Furthermore, there was marked interstitial fibrosis which replaced
approximately half of glomeruli and tubules.4 Since PDPN is

FIG. 4. Western blot analysis using PMab-314. Cell lysates(10 mg) of CHO-K1 and CHO/PA16-gpPDPN cells were
electrophoresed, after which proteins were transferred topolyvinylidene difluoride membranes. After blocking, membranes were subsequently incubated with PMab-314, an antiPA16 tag mAb (NZ-1), or an anti-IDH1 mAb (RcMab-1).
expressed in podocytes in the glomeruli, PMab-314 could
contribute to the analysis of podocytes in the kidney.
Similarities between tumors developed in giant pandas andhumans have been reported. PDAC was discovered in a
captive adult female giant panda.5 Immunohistochemical
staining showed that ductal adenocarcinoma cells in the giant
panda expressed cytokeratins 7 and 19, which are expressed
as 90% of PDAC in humans.44,45 Cancer-associated fibroblasts (CAFs) are one of the most significant components inthe tumor microenvironment of PDAC.46 PDPN expression
in CAFs enhances tumor progression of PDAC in humans.47
In 2023, a captive adult male giant panda was diagnosed withtesticular seminoma and underwent surgical management.6
The authors diagnosed testicular seminoma due to the elevation of testicular tumor markers including a-fetoprotein
and lactate dehydrogenase, which are markers of testiculartumor in humans.48 We previously reported that PDPN is an
important diagnostic marker that distinguishes seminoma
from embryonal carcinoma in human testicular germ celltumors.49 PMab-314 could also contribute to the diagnosis of
testicular seminoma in giant panda.
Authors’ Contributions

T.O., G.L., M.Y., T.N., S.H., and T.T. performed the experiments. M.K.K. and Y.K. designed the experiments. T.O.
and T.T. analyzed the data. T.O., H.S., and Y.K. wrote themanuscript. All authors have read and agreed to the publishedversion of the manuscript.

ANTI-PANDA PDPN MAB
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported, in part, by Japan Agency for
Medical Research and Development (AMED) under Grant
Nos.: JP23ama121008 (to Y.K.), JP23am0401013 (to Y.K.),
JP23bm1123027 (to Y.K.), and JP23ck0106730 (to Y.K.),
and by the Japan Society for the Promotion of Science ( JSPS)
Grants-in-Aid for Scientific Research (KAKENHI) Grant
Nos.: 23K19494 (to T.O.), 21K20789 (to T.T.), 22K06995(to H.S.), 21K07168 (to M.K.K.), and 22K07224 (to Y.K.).

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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku, Sendai
Miyagi 980-8575
JapanE-mail: yukinari.kato.e6@tohoku.ac.jp
Received: February 1, 2024
Accepted: February 24, 2024