MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 3, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0011

Development of Monoclonal Antibody PMab-269
Against California Sea Lion Podoplanin

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Tomohiro Tanaka,1 Teizo Asano,1 Masato Sano,1 Junko Takei,1,2 Hideki Hosono,1 Ren Nanamiya,1Takuro Nakamura,1 Miyuki Yanaka,1 Hiroyuki Harada,2 Masato Fukui,3 Hiroyoshi Suzuki,4 Kazuyuki Uchida,5Takayuki Nakagawa,6 Yukinari Kato,1,7,i and Mika K. Kaneko1

The development of protein-specific antibodies is essential for understanding a wide variety of biologicalphenomena. Parasitic and viral infections and cancers are known to occur within California sea lion (Zalophus
californianus) populations. However, sensitive and specific monoclonal antibodies (mAbs) for the pathophysiological analysis of California sea lion tissues have not yet been developed. A type I transmembrane
glycoprotein, podoplanin (PDPN), is a known diagnostic marker of lymphatic endothelial cells. We havepreviously developed several anti-PDPN mAbs in various mammalian species, with applications in flow cytometry, Western blotting, and immunohistochemistry. In this study, we established a novel mAb against
California sea lion PDPN (seaPDPN), clone PMab-269 (mouse IgG1, kappa), using a Cell-Based Immunizationand Screening method. PMab-269 is specifically detected in seaPDPN-overexpressed Chinese hamster ovary(CHO)-K1 cells using flow cytometry and Western blotting. Moreover, PMab-269 clearly identified pulmonary
type I alveolar cells, renal podocytes, and colon lymphatic endothelial cells in California sea lion tissues using
immunohistochemistry. These findings demonstrate the usefulness of PMab-269 for the pathophysiologicalanalysis of lung, kidney, and lymphatic tissues of the California sea lion.
Keywords: California sea lion, podoplanin, seaPDPN, monoclonal antibody, CBIS

Introduction

V

enous thromboembolism is a life-threatening complication that often occurs in patients with advanced
cancer.(1,2) Aberrant activation and aggregation of platelets,
and resultant thromboembolisms, have been reported to be
associated with cancer progression and metastasis.(3)Podoplanin (PDPN)/Aggrus/PA2.26 is a type I transmembrane mucin-like sialoglycoprotein involved in platelet aggregation and cancer metastasis through C-type
lectin-like receptor-2 (CLEC-2).(4–7) In many cancers, including mesotheliomas,(8,9) oral squamous cell carcinomas(OSCCs),(7,10) and malignant brain tumors,(11–13) PDPN expression is significantly upregulated and the protein is aberrantly glycosylated.(14–16) In normal tissue, PDPN expression
is used as a marker of lymphatic endothelial cells, pulmonary

type I alveolar cells, and renal podocytes.(17,18) It also plays apivotal role during embryonic development.(19) Activation of
the PDPN-CLEC-2 signaling axis can also trigger infectioninduced thrombosis.(20) Higher expression of PDPN significantly reduces the 5-year survival rate of OSCC patients andis linked to advanced-stage tumors.(21) PDPN has also been
identified in circulating tumor cells (CTCs), which are defined as a subpopulation of cancer cells harboring highmetastatic potential.(22,23) These PDPN-positive CTCs areassociated with poorer prognoses in head and neck squamouscell carcinoma patients. Given its potential role in carcinogenesis, PDPN has been exploited as a therapeutic target,with remarkable antitumor effects seen with anti-PDPN
monoclonal antibodies (mAbs).(24,25) Recently, anti-PDPN
mAbs, such as NZ-1, have been used in chimeric antigen
receptor T cell therapy.(26,27) PDPN is also expressed by

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Bunkyo-ku, Japan.
3
Zenoaq Resource Co., Ltd., Koriyama, Japan.
4
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
Laboratories of 5Veterinary Pathology and 6Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The Universityof Tokyo, Bunkyo-ku, Japan.
7
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
i
ORCID ID (https://orcid.org/0000-0001-5385-8201).
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DEVELOPMENT OF ANTI-CALIFORNIA SEA LION PDPN MAB

several cell types within the tumor microenvironment, including inflammatory macrophages, Th17 cells, and cancerassociated fibroblasts (CAFs).(28–30) PDPN-positive CAFs,
which are observed in melanoma and non-small-cell lungcancer patients, are correlated with lymph node metastases
and poorer prognoses.(31,32)
We have previously developed anti-PDPN mAbs for several
mammalian species, including humans,(14,33,34) mice,(35)rats,(36) rabbits,(37) dogs,(38) cats,(39) pigs,(40,41) bovines,(42)Tasmanian devils,(43) alpacas,(44) tigers,(45) goats,(46) horses,(47,48) bears,(49,50) whales,(51) and sheep,(52,53) using CellBased Immunization and Screening (CBIS) methods.(54–56) In
this study, we successfully developed a novel mAb against
California sea lion PDPN (seaPDPN), which can be used forflow cytometry, Western blotting, and immunohistochemistry.

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Materials and Methods
Cell lines

Chinese hamster ovary (CHO)-K1 and P3X63Ag8U.1(P3U1) cells were obtained from the American Type Culture

FIG. 1. Schematic model of the CBIS method. seaPDPNoverexpressed stable transfectants were used as an immunogen. Anti-seaPDPN mAb-secreting hybridomas werescreened for using flow cytometry of parental cells andstable transfectants. CBIS, Cell-Based Immunization andScreening; mAb, monoclonal antibody; seaPDPN, sea lion
podoplanin.

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Collection (ATCC, Manassas, VA). Synthesized DNA (Eurofins Genomics KK, Tokyo Japan) encoding seaPDPN (accession no.: XM_027576686) with N-terminal MAP tag(57)or 2 · RIEDL tag(58) was subcloned into a pCAG-Ble 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 using cell sorter(SH800; Sony Biotechnology Corp., Tokyo, Japan) andcultivation in media containing 0.5 mg/mL of Zeocin(InvivoGen, San Diego, CA). CHO/MAP-seaPDPN was
subsequently used as an immunogen, whereas CHO/
2 · RIEDL-seaPDPN was used for further analyses.
P3U1, CHO-K1, CHO/MAP-seaPDPN, CHO/2 · RIEDLseaPDPN, CHO/human PDPN (hPDPN),(5) CHO/mouse
PDPN (mPDPN),(5) CHO/rat PDPN (rPDPN),(36) CHO/
rabbit PDPN (rabPDPN),(37) CHO/dog PDPN (dPDPN),(38)CHO/bovine PDPN (bovPDPN),(42) CHO/cat PDPN
(cPDPN),(39) CHO/pig PDPN (pPDPN),(41) CHO/horse
PDPN (horPDPN),(47) CHO/tiger PDPN (tigPDPN),(45)CHO/alpaca PDPN (aPDPN),(44) CHO/bear PDPN
(bPDPN),(59) CHO/Tasmanian devil PDPN (tasPDPN),(43)CHO/goat PDPN (gPDPN),(46) CHO/whale PDPN
(wPDPN),(51) and CHO/sheep PDPN (sPDPN)(60) were cultured in Roswell Park Memorial Institute (RPMI) 1640
complete media: RPMI 1640 (Nacalai Tesque, Inc., Kyoto,
Japan) supplemented with 10% heat-inactivated fetal bovine

FIG. 2. Detection of seaPDPN using PMab-269 throughflow cytometry. (A) CHO/seaPDPN and CHO-K1 cells weretreated with PMab-269 (red line) at a concentration of
5 mg/mL. (B) CHO/seaPDPN and CHO-K1 cells were treated with an anti-RIEDL tag mAb (LpMab-7; blue line) at aconcentration of 10 mg/mL. Primary mAbs or 0.1% BSA inPBS (black line) were applied for 30 minutes, followed byincubation with secondary antibodies. Fluorescence data
were collected using the SA3800 Cell Analyzer. BSA, bovine serum albumin; CHO, Chinese hamster ovary; PBS,
phosphate-buffered saline.

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FIG. 3. Crossreactivity of PMab-269 with PDPNs of other species. (A) PDPN stable transfectants from other species weretreated with PMab-269 (red line) at a concentration of 5 mg/mL or 0.1% BSA in PBS (black line) for 30 minutes, followed
by incubation with secondary antibodies. (B) PDPN stable transfectants of other species were treated with each positive
control (blue line) at a concentration of 5 mg/mL or 0.1% BSA in PBS (black line) for 30 minutes, followed by incubation
with secondary antibodies. Fluorescence data were collected using the SA3800 Cell Analyzer.

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serum (Thermo Fisher Scientific, Inc.), 100 U/mL of penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin
B (Nacalai Tesque, Inc.), and incubated at 37C in a humidified atmosphere containing 5% CO2.

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Antibodies

Mouse anti-hPDPN mAb LpMab-12 (IgG1, kappa),(61) rat
anti-mPDPN mAb PMab-1 (IgG2a, kappa),(35) mouse antirPDPN mAb PMab-2 (IgG1, kappa),(36) mouse anti-rabPDPN
mAb PMab-32 (IgG1, kappa),(37) mouse anti-dPDPN mAb
PMab-38 (IgG1, kappa),(38) mouse anti-bovPDPN mAb PMab44 (IgG1, kappa),(42) mouse anti-cPDPN mAb PMab-52 (IgM,
kappa),(39) mouse anti-pPDPN mAb PMab-213 (IgG2b, kappa),(40) mouse anti-horPDPN mAb PMab-219 (IgG2a, kappa),(62)mouse anti-tasPDPN mAb PMab-233 (IgG1, kappa),(43) mouseanti-gPDPN mAb PMab-235 (IgG1, kappa),(46) mouse antiaPDPN mAb PMab-225 (IgG2b, kappa),(44) mouse anti-tigPDPN
mAb PMab-231 (IgG2a, kappa),(45) mouse anti-bPDPN mAb
PMab-247 (IgG1, kappa),(59) mouse anti-wPDPN mAb PMab237 (IgG1, kappa),(51) and mouse anti-sPDPN mAb PMab-256(IgG1, kappa)(52) were developed as previously described.
Hybridoma production

Female BALB/c mice (6-week-old) were purchased from
CLEA Japan (Tokyo, Japan). The animals were housed under
specific pathogen-free conditions. The Animal Care and Use
Committee of Tohoku University approved all animal experiments. We used a CBIS method to develop mAbs against
seaPDPN. A BALB/c mouse was immunized with CHO/
MAP-seaPDPN cells (1 · 108) intraperitoneally with Imject
Alum (Thermo Fisher Scientific, Inc.). The procedure included
three additional immunizations with CHO/MAP-seaPDPN
cells (1 · 108) followed by a final booster injection of CHO/
MAP-seaPDPN cells (1 · 108) 2 days before the harvest of
splenic cells. Subsequently, splenic cells were fused with
P3U1 cells using polyethylene glycol 1500 (PEG1500; Roche
Diagnostics, Indianapolis, IN). The hybridomas were thengrown in RPMI complete media supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo
Fisher Scientific, Inc.). The culture supernatants were screened
for anti-seaPDPN antibodies production using flow cytometry.

127

electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred
onto polyvinylidene difluoride (PVDF) membranes( Merck KGaA, Darmstadt, Germany). After blocking with
4% skim milk (Nacalai Tesque, Inc.), PVDF membranes
were incubated with 5 lg/mL of PMab-269, 1 lg/mL ofanti-RIEDL tag (clone LpMab-7), or 1 lg/mL of anti-bactin (clone AC-15; Sigma-Aldrich Corp., St. Louis, MO),
followed by incubation with peroxidase-conjugated antimouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc., Santa Clara, CA). Blots were developed
using ImmunoStar LD (FUJIFILM Wako Pure Chemical
Corporation) and imaged with a Sayaca-Imager (DRC Co.
Ltd., Tokyo, Japan).
Immunohistochemical analysis

Normal California sea lion tissues obtained from routine
necropsies performed at the Laboratory of Veterinary Pathology, the University of Tokyo, fixed in 10% neutralbuffered formalin, were processed to make formalin-fixedparaffin-embedded (FFPE) tissue sections.(63) Histologicalsections (4 lm thickness) were autoclaved in citrate buffer(pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) or EnVision FLEX Target Retrieval Solution High pH (Agilent
Technologies, Inc.) for 20 minutes. After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.), sections were incubated with PMab-269(5 lg/mL) for 1 hour at room temperature and treated using EnVision+ Kit (Agilent Technologies, Inc.) for 30 minutes. Color was developed using 3,3¢-diaminobenzidine
tetrahydrochloride (DAB; Agilent Technologies, Inc.) for

Flow cytometry

CHO/2 · RIEDL-seaPDPN and parental CHO-K1 cells(2 · 105/mL) were harvested after a brief exposure to 0.25%
trypsin in 1 mM ethylenediaminetetraacetic acid (EDTA;
Nacalai Tesque, Inc.). After washing with 0.1% bovine serum
albumin (Nacalai Tesque, Inc.) in phosphate-buffered saline(PBS; Nacalai Tesque, Inc.), cells were treated with antiPDPN mAbs (5 lg/mL) or an anti-RIEDL tag (clone LpMab7; 10 lg/mL) for 30 minutes at 4C, followed by Alexa Fluor
488-conjugated anti-mouse IgG (1:1000, product no. 4408;
Cell Signaling Technology, Inc., Danvers, MA) or Alexa Fluor
488-conjugated anti-rat IgG (1:1000; product no. 4416; Cell
Signaling Technology, Inc.). Fluorescence data were collectedusing the SA3800 Cell Analyzer (Sony Biotechnology Corp.).
Western blot analysis

Cell lysates (10 lg) were boiled in sodium dodecyl sulfate(SDS) sample buffer (Nacalai Tesque, Inc.). Proteins were

FIG. 4. Detection of seaPDPN by Western blot analysis.
Cell lysates of CHO-K1 and CHO/seaPDPN (10 mg) were
electrophoresed and transferred onto PVDF membranes. The
membranes were incubated with 5 mg/mL of PMab-269, 1 mg/mLof an anti-RIEDL tag mAb (LpMab-7), and an anti-b-actin mAb(AC-15) and subsequently with peroxidase-conjugatedanti-mouse immunoglobulins. PVDF, polyvinylidene
difluoride.

128

2 minutes, and counterstaining was performed with hematoxylin and eosin (FUJIFILM Wako Pure Chemical
Corporation).
Results
Establishment of anti-seaPDPN mAbs

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To develop anti-seaPDPN mAbs, we used the CBIS
method, using stable transfectants for immunization and flow
cytometry (Fig. 1). A mouse was immunized with CHO/
MAP-seaPDPN cells, which overexpress seaPDPN with an
N-terminal MAP tag. Hybridomas were seeded into 96-wellplates, and CHO/2 · RIEDL-seaPDPN (hereinafter referred
to as CHO/seaPDPN)-positive and CHO-K1-negative wells
were identified. After limiting dilution and selection using
Western blotting and immunohistochemistry, PMab-269(mouse IgG1, kappa) was isolated.
Flow cytometric analyses

We performed flow cytometric analyses using PMab-269
with CHO/seaPDPN and CHO-K1 cells. PMab-269 recog-

TANAKA ET AL.

nized CHO/seaPDPN but not CHO-K1 cells (Fig. 2A). As
expected, an anti-RIEDL tag mAb (LpMab-7) could also
detect CHO/seaPDPN cells (Fig. 2B). These results demonstrate that PMab-269 is suitable the detection of seaPDPN
using flow cytometry.
We evaluated crossreactivity of PMab-269 with otheranimal PDPNs, which are overexpressed in CHO-K1 cells.
PMab-269 crossreacted with rat, dog, cat, alpaca, and tiger
PDPNs (Fig. 3A). The expression levels of each of these
PDPNs were confirmed using their specific anti-PDPN mAbs
(Fig. 3B).
Western blot analyses

We investigated whether PMab-269 can be used forWestern blot analyses by analyzing CHO-K1 and CHO/
seaPDPN cell lysates. As shown in Figure 4, PMab-269 could
detect seaPDPN as a 40 kDa band (2 · RIEDL+seaPDPN) in
CHO/seaPDPN cell lysates, whereas no major bands weredetected in the parental CHO-K1 cells. Anti-RIEDL tag(LpMab-7) was used as a positive control and could also
detect a band of the same molecular weight in CHO/seaPDPN

FIG. 5. Immunohistochemical analysis of California sea lion lung tissue. Histological sections of California sea lion lungwere directly autoclaved in citrate buffer and incubated with 5 mg/mL of PMab-269 (A, B) or blocking buffer (C, D),followed by use of the EnVision+ Kit. (E, F) H&E staining. Scale bar = 100 mm. H&E, hematoxylin and eosin.

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DEVELOPMENT OF ANTI-CALIFORNIA SEA LION PDPN MAB

129

FIG. 6. Immunohistochemical analysis of California sea lion kidney tissue. Histological sections of California sea lion
kidney were directly autoclaved in EnVision FLEX Target Retrieval Solution High pH and incubated with 5 mg/mL of
PMab-269 (A, B) or blocking buffer (C, D), followed by use of the EnVision+ Kit. (E, F) H&E staining. Scalebar = 100 mm.

cell lysates. The 40 kDa major band represents a full-length
form, whereas the lower band (35 kDa), which was detectedby both PMab-269 and LpMab-7, is presumably a truncatedisoform.(5,14) Additionally, two weaker, smaller bands (35–28 kDa) and two larger bands (100–63 kDa) were detected,
which may represent additional truncated and aggregatedisoforms, respectively. These results indicate that PMab-269
can detect seaPDPN in Western blot analyses.
Immunohistochemical analyses

Lastly, we investigated whether PMab-269 can be used for
immunohistochemical analyses using FFPE California sea
lion tissue sections. We examined normal lung, kidney, andcolon tissue from California sea lions, all of which reportedly
express PDPN in various species, including humans,(33)mice,(35) rats,(36) rabbits,(37) dogs,(38) cats,(39) bovines,(42)pigs,(40) Tasmanian devils,(43) alpacas,(44) tigers,(45)whales,(51) goats,(46) horses,(47) bears,(49) and sheep.(52,53) Asshown in Figure 5, PMab-269 strongly and specifically

identified type I alveolar cells within the lung. PMab-269 also
identified renal glomeruli and Bowman’s capsules (Fig. 6).
Furthermore, lymphatic endothelial cells of colon were also
detected by PMab-269 (Fig. 7). These results indicate that
PMab-269 is useful in the detection of seaPDPN-positive
cells in FFPE tissue.
Discussion

Animal models of pathological conditions are essential for
elucidating pathogenesis and developing effective therapeutic
agents. Although California sea lion (Zalophus californianus) is
a nonlaboratory animal, infectious diseases, such as parasites andviruses, and several cancers, including metastatic carcinoma ofurogenital origin, intestinal T cell lymphoma, and lingualsquamous cell carcinoma, have been observed among their
populations.(64–67) In addition to the carcinogenesis observed inwomen infected with the papillomavirus, a relationship between
carcinogenesis and herpesvirus and bacterial infections has
similarly been observed in California sea lion.(68) Immune

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TANAKA ET AL.

FIG. 7. Immunohistochemical analyses of California sea lion colon tissue. Histological sections of California sea lioncolon were directly autoclaved in citrate buffer and incubated with 5 mg/mL of PMab-269 (A, B) or blocking buffer (C, D),followed by use of the EnVision+ Kit. (E, F) H&E staining. Scale bar = 100 mm.
system analyses and genetic research of California sea lions
could thus improve our understanding of human pathology.(66,69)
Although pathological analyses, such as immunohistochemistry, have been previously reported, specific antibodies
against California sea lion proteins are rarely used.(63,70) In
previous studies, the expression of California sea lion proteins,
including the estrogen receptor, progesterone receptor, amyloidb and tau, have been evaluated through immunohistochemistryby exploiting antibodies raised in other species. However, suchapproaches may limit sensitivity and specificity. In this study,
we developed a novel mAb (PMab-269) against seaPDPN using
the CBIS method (Fig. 1). PMab-269 could detect seaPDPN byflow cytometry (Fig. 2), Western blotting (Fig. 4), and immunohistochemistry (Figs. 5–7). In future studies, PMab-269 could
be used to obtain pathophysiological data for California sea
lions.
Authors’ Contributions

T.T., T.A., M.S., J.T., H.Ho, R.N, T.N., M.Y., and M.Y.
performed experiments; M.K.K., H.Ha, M.F., H.S., K.U., andT.N. designed the experiments; T.T. and Y.K. wrote the article.

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
Numbers: JP21am0401013 (to Y.K.) and JP21am0101078 (toY.K.), and by the Japan Society for the Promotion of Science(JSPS) Grants-in-Aid for Scientific Research (KAKENHI)
grant nos. 21K15523 (to T.A.), 21K07168 (to M.K.K.),
20K16322 (to M.S.), and 19K07705 (to Y.K.).
References

1. Gay LJ, and Felding-Habermann B: Contribution of platelets to tumour metastasis. Nat Rev Cancer 2011;11:123–
134.
2. Key NS, Khorana AA, Mackman N, McCarty OJ, White
GC, Francis CW, McCrae KR, Palumbo JS, Raskob GE,
Chan AT, and Sood AK: Thrombosis in cancer: Research
priorities identified by a National Cancer Institute/National

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

DEVELOPMENT OF ANTI-CALIFORNIA SEA LION PDPN MAB

Heart, Lung, and Blood Institute Strategic Working Group.
Cancer Res 2016;76:3671–3675.
3. Menter DG, Tucker SC, Kopetz S, Sood AK, Crissman JD,
and Honn KV: Platelets and cancer: A casual or causal
relationship: revisited. Cancer Metastasis Rev 2014;33:
231–269.
4. Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A,
Ogasawara S, Matsuura N, Hasegawa Y, Suzuki-Inoue K,
Inoue O, Ozaki Y, and Narimatsu H: Molecular analysis of
the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.
5. Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M,
and Tsuruo T: Molecular identification of Aggrus/T1alphaas a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem 2003;278:51599–51605.
6. Kunita A, Kashima TG, Morishita Y, Fukayama M, Kato
Y, Tsuruo T, and Fujita N: The platelet aggregationinducing factor aggrus/podoplanin promotes pulmonary
metastasis. Am J Pathol 2007;170:1337–1347.
7. Martin-Villar E, Scholl FG, Gamallo C, Yurrita MM,
Munoz-Guerra M, Cruces J, and Quintanilla M: Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamouscell carcinomas. Int J Cancer 2005;113:899–910.
8. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
9. Abe S, Morita Y, Kaneko MK, Hanibuchi M, Tsujimoto Y,
Goto H, Kakiuchi S, Aono Y, Huang J, Sato S, Kishuku M,
Taniguchi Y, Azuma M, Kawazoe K, Sekido Y, Yano S,
Akiyama S, Sone S, Minakuchi K, Kato Y, and Nishioka Y:
A novel targeting therapy of malignant mesothelioma using
anti-podoplanin antibody. J Immunol 2013;190:6239–6249.
10. Retzbach EP, Sheehan SA, Nevel EM, Batra A, Phi T,
Nguyen ATP, Kato Y, Baredes S, Fatahzadeh M, Shienbaum AJ, and Goldberg GS: Podoplanin emerges as afunctionally relevant oral cancer biomarker and therapeutictarget. Oral Oncol 2018;78:126–136.
11. Kato Y, Vaidyanathan G, Kaneko MK, Mishima K, Srivastava N, Chandramohan V, Pegram C, Keir ST, Kuan
CT, Bigner DD, and Zalutsky MR: Evaluation of antipodoplanin rat monoclonal antibody NZ-1 for targeting
malignant gliomas. Nucl Med Biol 2010;37:785–794.
12. Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A,
Fujita N, Tsuruo T, Nishikawa R, Hirose T, and Matsutani
M: Podoplanin expression in primary central nervous system germ cell tumors: A useful histological marker for the
diagnosis of germinoma. Acta Neuropathol (Berl) 2006;
111:563–568.
13. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T,
and Matsutani M: Increased expression of podoplanin inmalignant astrocytic tumors as a novel molecular marker ofmalignant progression. Acta Neuropathol (Berl) 2006;111:
483–488.
14. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
15. Kaneko MK, Kato Y, Kameyama A, Ito H, Kuno A, Hirabayashi J, Kubota T, Amano K, Chiba Y, Hasegawa Y,
Sasagawa I, Mishima K, and Narimatsu H: Functional glycosylation of human podoplanin: glycan structure of platelet
aggregation-inducing factor. FEBS Lett 2007;581:331–336.
16. Kaneko M, Kato Y, Kunita A, Fujita N, Tsuruo T, andOsawa M: Functional sialylated O-glycan to platelet ag-

131gregation on Aggrus (T1alpha/podoplanin) molecules expressed in Chinese Hamster Ovary cells. J Biol Chem 2004;
279:38838–38843.
17. Breiteneder-Geleff S, Matsui K, Soleiman A, Meraner P,
Poczewski H, Kalt R, Schaffner G, and Kerjaschki D: Podoplanin, novel 43-kd membrane protein of glomerularepithelial cells, is down-regulated in puromycin nephrosis.
Am J Pathol 1997;151:1141–1152.
18. Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R,
Amann G, Kriehuber E, Diem K, Weninger W, Tschachler
E, Alitalo K, and Kerjaschki D: Angiosarcomas express
mixed endothelial phenotypes of blood and lymphatic
capillaries: Podoplanin as a specific marker for lymphatic
endothelium. Am J Pathol 1999;154:385–394.
19. Astarita JL, Acton SE, and Turley SJ: Podoplanin: Emerging functions in development, the immune system, andcancer. Front Immunol 2012;3:283.
20. Hitchcock JR, Cook CN, Bobat S, Ross EA, FloresLangarica A, Lowe KL, Khan M, Dominguez-Medina CC,
Lax S, Carvalho-Gaspar M, Hubscher S, Rainger GE,
Cobbold M, Buckley CD, Mitchell TJ, Mitchell A, Jones
ND, Van Rooijen N, Kirchhofer D, Henderson IR, Adams
DH, Watson SP, and Cunningham AF: Inflammation drives
thrombosis after Salmonella infection via CLEC-2 onplatelets. J Clin Invest 2015;125:4429–4446.
21. Kreppel M, Scheer M, Drebber U, Ritter L, and Zöller JE:
Impact of podoplanin expression in oral squamous cell
carcinoma: Clinical and histopathologic correlations.
Virchows Arch 2010;456:473–482.
22. Hsieh JC, Lin HC, Huang CY, Hsu HL, Wu TM, Lee CL,
Chen MC, Wang HM, and Tseng CP: Prognostic value ofcirculating tumor cells with podoplanin expression in patients with locally advanced or metastatic head and necksquamous cell carcinoma. Head Neck 2015;37:1448–1455.
23. Mir Seyed Nazari P, Riedl J, Pabinger I, and Ay C: The roleof podoplanin in cancer-associated thrombosis. Thromb
Res 2018;164 Suppl 1:S34–S39.
24. Kaneko MK, Nakamura T, Kunita A, Fukayama M, Abe S,
Nishioka Y, Yamada S, Yanaka M, Saidoh N, Yoshida K,
Fujii Y, Ogasawara S, and Kato Y: ChLpMab-23: Cancerspecific human-mouse chimeric anti-podoplanin antibody
exhibits antitumor activity via antibody-dependent cellularcytotoxicity. Monoclon Antib Immunodiagn Immunother2017;36:104–112.
25. Kaneko MK, Ohishi T, Nakamura T, Inoue H, Takei J,
Sano M, Asano T, Sayama Y, Hosono H, Suzuki H, Kawada M, and Kato Y: Development of core-fucosedeficient humanized and chimeric anti-human podoplaninantibodies. Monoclon Antib Immunodiagn Immunother2020;39:167–174.
26. Waseda M, and Kaneko S: Podoplanin as an attractive
target of CAR T cell therapy. Cells 2020;9:1971.
27. Shiina S, Ohno M, Ohka F, Kuramitsu S, Yamamichi A,
Kato A, Motomura K, Tanahashi K, Yamamoto T, Watanabe R, Ito I, Senga T, Hamaguchi M, Wakabayashi T,
Kaneko MK, Kato Y, Chandramohan V, Bigner DD, andNatsume A: CAR T cells targeting podoplanin reduce orthotopic glioblastomas in mouse brains. Cancer Immunol
Res 2016;4:259–268.
28. Krishnan H, Rayes J, Miyashita T, Ishii G, Retzbach EP,
Sheehan SA, Takemoto A, Chang YW, Yoneda K, Asai J,
Jensen L, Chalise L, Natsume A, and Goldberg GS: Podoplanin: An emerging cancer biomarker and therapeutictarget. Cancer Sci 2018;109:1292–1299.

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

132

29. Suzuki J, Aokage K, Neri S, Sakai T, Hashimoto H, Su Y,
Yamazaki S, Nakamura H, Tane K, Miyoshi T, Sugano M,
Kojima M, Fujii S, Kuwata T, Ochiai A, Tsuboi M, andIshii G: Relationship between podoplanin-expressing
cancer-associated fibroblasts and the immune microenvironment of early lung squamous cell carcinoma. Lung
Cancer 2021;153:1–10.
30. Zhou Q, Wang Z, Zeng H, Zhang H, Liu Z, Huang Q,
Wang J, Chang Y, Bai Q, Liu L, Zhu Y, Xu L, Dai B, Guo
J, Xia Y, Wang Y, and Xu J: Identification and validation ofpoor prognosis immunoevasive subtype of muscle-invasive
bladder cancer with tumor-infiltrating podoplanin(+) cellabundance. Oncoimmunology 2020;9:1747333.
31. Kan S, Konishi E, Arita T, Ikemoto C, Takenaka H, Yanagisawa A, Katoh N, and Asai J: Podoplanin expression incancer-associated fibroblasts predicts aggressive behavior
in melanoma. J Cutan Pathol 2014;41:561–567.
32. Irvine AF, Waise S, Green EW, Stuart B, and Thomas GJ:
Characterising cancer-associated fibroblast heterogeneity in
non-small cell lung cancer: A systematic review and metaanalysis. Sci Rep 2021;11:3727.
33. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K,
Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H, Mishima K, and Osawa M: Inhibition of tumor cell-induced
platelet aggregation using a novel anti-podoplanin antibody
reacting with its platelet-aggregation-stimulating domain.
Biochem Biophys Res Commun 2006;349:1301–1307.
34. Yamada S, Kaneko MK, and Kato Y: LpMab-23: A cancerspecific monoclonal antibody against human podoplanin.
Monoclon Antib Immunodiagn Immunother 2017;36:
72–76.
35. Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, and Sawa
Y: Immunohistochemical examination of novel rat monoclonal antibodies against mouse and human podoplanin.
Acta Histochem Cytochem 2012;45:227–237.
36. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Takagi M,
Kaneko MK, and Kato Y: Development of sensitive
monoclonal antibody PMab-2 against rat podoplanin.
Monoclon Antib Immunodiagn Immunother 2015;34:396–
403.
37. Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura
T, Kaneko MK, Takagi M, and Kato Y: Establishment of anovel monoclonal antibody PMab-32 against rabbit podoplanin. Monoclon Antib Immunodiagn Immunother 2016;35:41–47.
38. Honma R, Kaneko MK, Ogasawara S, Fujii Y, Konnai S,
Takagi M, and Kato Y: Specific detection of dog podoplanin expressed in renal glomerulus by a novel monoclonal antibody PMab-38 in immunohistochemistry.
Monoclon Antib Immunodiagn Immunother 2016;35:212–
216.
39. Yamada S, Itai S, Nakamura T, Yanaka M, Saidoh N,
Chang YW, Handa S, Harada H, Kagawa Y, Ichii O,
Konnai S, Kaneko MK, and Kato Y: PMab-52: Specific andsensitive monoclonal antibody against cat podoplanin for
immunohistochemistry. Monoclon Antib Immunodiagn
Immunother 2017;36:224–230.
40. Kato Y, Yamada S, Furusawa Y, Itai S, Nakamura T, Yanaka M, Sano M, Harada H, Fukui M, and Kaneko MK:
PMab-213: A monoclonal antibody for immunohistochemical analysis against pig podoplanin. Monoclon AntibImmunodiagn Immunother 2019;38:18–24.
41. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T,
Yanaka M, Fukui M, Harada H, Mizuno T, Sakai Y, Takasu

TANAKA ET AL.

M, Kaneko MK, and Kato Y: PMab-210: A monoclonal
antibody against pig podoplanin. Monoclon Antib Immunodiagn Immunother 2019;38:30–36.
42. Honma R, Ogasawara S, Kaneko M, Fujii Y, Oki H, Nakamura T, Takagi M, Konnai S, and Kato Y: PMab-44
detects bovine podoplanin in immunohistochemistry.
Monoclon Antib Immunodiagn Immunother 2016;35:186–
190.
43. Furusawa Y, Yamada S, Itai S, Nakamura T, Takei J, Sano
M, Harada H, Fukui M, Kaneko MK, and Kato Y: Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil
podoplanin. Biochem Biophys Rep 2019;18:100631.
44. Kato Y, Furusawa Y, Yamada S, Itai S, Takei J, Sano M,
and Kaneko MK: Establishment of a monoclonal antibody
PMab-225 against alpaca podoplanin for immunohistochemical analyses. Biochem Biophys Rep 2019;18:100633.
45. Furusawa Y, Kaneko MK, Nakamura T, Itai S, Fukui M,
Harada H, Yamada S, and Kato Y: Establishment of a
monoclonal antibody PMab-231 for tiger podoplanin.
Monoclon Antib Immunodiagn Immunother 2019;38:89–95.
46. Furusawa Y, Yamada S, Nakamura T, Sano M, Sayama Y,
Itai S, Takei J, Harada H, Fukui M, Kaneko MK, and Kato
Y: PMab-235: A monoclonal antibody for immunohistochemical analysis against goat podoplanin. Heliyon 2019;5:e02063.
47. Furusawa Y, Yamada S, Itai S, Sano M, Nakamura T, Yanaka M, Handa S, Mizuno T, Maeda K, Fukui M, Harada H,
Kaneko MK, and Kato Y: Establishment of monoclonal
antibody PMab-202 against horse podoplanin. Monoclon
Antib Immunodiagn Immunother 2018;37:233–237.
48. Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, andKaneko MK: Anti-horse podoplanin monoclonal antibody
PMab-219 is useful for detecting lymphatic endothelial
cells by immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2018;37:272–274.
49. Takei J, Furusawa Y, Yamada S, Nakamura T, Sayama Y,
Sano M, Konnai S, Kobayashi A, Harada H, Kaneko MK,
and Kato Y: PMab-247 detects bear podoplanin in immunohistochemical analysis. Monoclon Antib Immunodiagn
Immunother 2019;38:171–174.
50. Takei J, Yamada S, Konnai S, Ishinazaka T, Shimozuru M,
Kaneko MK, and Kato Y: PMab-241 specifically detects
bear podoplanin of lymphatic endothelial cells in the lungof brown bear. Monoclon Antib Immunodiagn Immunother2019;38:282–284.
51. Kato Y, Furusawa Y, Itai S, Takei J, Nakamura T, Sano M,
Harada H, Yamada S, and Kaneko MK: Establishment ofan anticetacean podoplanin monoclonal antibody PMab237 for immunohistochemical analysis. Monoclon AntibImmunodiagn Immunother 2019;38:108–113.
52. Kato Y, Furusawa Y, Sano M, Takei J, Nakamura T, Yanaka
M, Okamoto S, Handa S, Komatsu Y, Asano T, Sayama Y,
and Kaneko MK: Development of an anti-sheep podoplanin
monoclonal antibody PMab-256 for immunohistochemical
analysis of lymphatic endothelial cells. Monoclon AntibImmunodiagn Immunother 2020;39:82–90.
53. Kaneko MK, Sano M, Takei J, Asano T, Sayama Y, Hosono H, Kobayashi A, Konnai S, and Kato Y: Development
and characterization of anti-sheep podoplanin monoclonal
antibodies PMab-253 and PMab-260. Monoclon AntibImmunodiagn Immunother 2020;39:144–155.
54. Itai S, Fujii Y, Nakamura T, Chang YW, Yanaka M, Saidoh
N, Handa S, Suzuki H, Harada H, Yamada S, Kaneko MK,

Downloaded by Yukinari Kato from www.liebertpub.com at 06/23/21. For personal use only.

DEVELOPMENT OF ANTI-CALIFORNIA SEA LION PDPN MAB

and Kato Y: Establishment of CMab-43, a sensitive andspecific anti-CD133 monoclonal antibody, for immunohistochemistry. Monoclon Antib Immunodiagn Immunother2017;36:231–235.
55. Yamada S, Itai S, Nakamura T, Yanaka M, Chang YW,
Suzuki H, Kaneko MK, and Kato Y: Monoclonal antibody
L1Mab-13 detected human PD-L1 in lung cancers. Monoclon Antib Immunodiagn Immunother 2018;37:110–115.
56. Yamada S, Itai S, Nakamura T, Yanaka M, Kaneko MK,
and Kato Y: Detection of high CD44 expression in oral
cancers using the novel monoclonal antibody, C44Mab-5.
Biochem Biophys Rep 2018;14:64–68.
57. Fujii Y, Kaneko MK, and Kato Y: MAP tag: A novel
tagging system for protein purification and detection.
Monoclon Antib Immunodiagn Immunother 2016;35:293–
299.
58. Asano T, Kaneko MK, and Kato Y: RIEDL tag: A novel
pentapeptide tagging system for transmembrane protein
purification. Biochem Biophys Rep 2020;23:100780.
59. Furusawa Y, Takei J, Sayama Y, Yamada S, Kaneko MK,
and Kato Y: Development of an anti-bear podoplanin
monoclonal antibody PMab-247 for immunohistochemical
analysis. Biochem Biophys Rep 2019;18:100644.
60. Kato Y, Yamada S, Itai S, Kobayashi A, Konnai S, andKaneko MK: Immunohistochemical detection of sheep
podoplanin using an antibovine podoplanin monoclonal
antibody PMab-44. Monoclon Antib Immunodiagn Immunother 2018;37:265–268.
61. Kato Y, Ogasawara S, Oki H, Goichberg P, Honma R, Fujii
Y, and Kaneko MK: LpMab-12 established by CasMab
technology specifically detects sialylated O-glycan on
Thr52 of platelet aggregation-stimulating domain of human
podoplanin. PLoS One 2016;11:e0152912.
62. Furusawa Y, Yamada S, Itai S, Nakamura T, Yanaka M,
Sano M, Harada H, Fukui M, Kaneko MK, and Kato Y:
PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin. Biochem Biophys
Rep 2019;18:100616.
63. Takaichi Y, Chambers JK, Takahashi K, Soeda Y, Koike R,
Katsumata E, Kita C, Matsuda F, Haritani M, Takashima A,
Nakayama H, and Uchida K: Amyloid b and tau pathology
in brains of aged pinniped species (sea lion, seal, andwalrus). Acta Neuropathol Commun 2021;9:10.
64. McAloose D, and Newton AL: Wildlife cancer: A conservation perspective. Nat Rev Cancer 2009;9:517–526.

133

65. Sato T, Higuchi T, Shibuya H, Ohba S, Nogami S, Shirai
W, Watanabe H, and Honda S: Lingual squamous cell
carcinoma in a California sea lion (Zalophus californianus).
J Zoo Wildl Med 2002;33:367–370.
66. Browning HM, Acevedo-Whitehouse K, Gulland FM, Hall
AJ, Finlayson J, Dagleish MP, Billington KJ, Colegrove K,
and Hammond JA: Evidence for a genetic basis of urogenital carcinoma in the wild California sea lion. Proc BiolSci 2014;281:20140240.
67. Colegrove KM, Wellehan JF, Jr., Rivera R, Moore PF,
Gulland FM, Lowenstine LJ, Nordhausen RW, and Nollens
HH: Polyomavirus infection in a free-ranging California
sea lion (Zalophus californianus) with intestinal T-cell
lymphoma. J Vet Diagn Invest 2010;22:628–632.
68. Browning HM, Gulland FM, Hammond JA, Colegrove
KM, and Hall AJ: Common cancer in a wild animal: The
California sea lion (Zalophus californianus) as an emerging
model for carcinogenesis. Philos Trans R Soc Lond B BiolSci 2015;370:20140228.
69. Peñı́n I, Figueroa-Cabañas ME, Guerrero-de la Rosa F,
Soto-Garcı́a LA, Álvarez-Martı́nez R, Flores-Morán A, andAcevedo-Whitehouse K: Transcriptional profiles of California sea lion peripheral NK and CD(+8) T cells reflectecological regionalization and infection by oncogenic
viruses. Front Immunol 2019;10:413.
70. Colegrove KM, Gulland FM, Naydan DK, and Lowenstine
LJ: Tumor morphology and immunohistochemical expression of estrogen receptor, progesterone receptor, p53, andKi67 in urogenital carcinomas of California sea lions (Zalophus californianus). Vet Pathol 2009;46:642–655.

Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
MiyagiJapan
E-mail: yukinarikato@med.tohoku.ac.jp
Received: March 16, 2021
Accepted: April 16, 2021