MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 3, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0015

Short Communication

Development of an Anti-Elephant Podoplanin Monoclonal
Antibody PMab-265 for Flow Cytometry

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

Hideki Hosono,1,* Teizo Asano,1,* Junko Takei,1 Masato Sano,1Tomohiro Tanaka,1 Mika K. Kaneko,1 and Yukinari Kato1,2,i

The development of specific antibodies is essential to understand a wide variety of biological phenomena andpathophysiological analyses. Podoplanin (PDPN), a type I transmembrane glycoprotein, is known as a diagnostic marker. Anti-PDPN monoclonal antibodies (mAbs) against many species, such as human, mouse, rat, rabbit,
dog, bovine, cat, tiger, horse, pig, goat, alpaca, Tasmanian devil, bear, whale, and sheep, have been establishedin recent studies. However, sensitive and specific mAbs against elephant PDPN (elePDPN) have not been
established. Thus, this study established a novel mAb against African savanna elephant (Loxodonta africana)
PDPN using the Cell-Based Immunization and Screening method. elePDPN-overexpressed Chinese hamster
ovary-K1 (CHO/elePDPN) cells were immunized, and mAbs were screened against elePDPN using flow cytometry. One of the mAbs, PMab-265 (IgM, k), specifically detected CHO/elePDPN cells by flow cytometry. Thesefindings suggested the potential usefulness of PMab-265 for the functional analyses of elePDPN.
Keywords: elephant podoplanin, PDPN, CBIS, flow cytometry

IntroductionElephants are known to be extremely cancer resistant;however, about 3% of elephants develop cancer.(1,2) The
analysis of elephant cancers will help in the elucidation of
the mechanism of cancer development. For pathophysiological analysis, specific and sensitive antibodies against
elephant cells are essential.
Podoplanin (PDPN) is a type I transmembrane glycoprotein
expressed in many cell types, including lymphatic endothelial
cells.(3) Therefore, PDPN has been used as a lymphatic endothelial cell marker to distinguish lymphatic endothelial
cells from vascular endothelial cells in pathophysiologicalstudies.(4) In humans, PDPN expression has been reported
in several tumors, including malignant brain tumors,(5–8) oral
squamous cell carcinomas,(9) lung cancers,(10) esophageal
cancers,(11) malignant mesotheliomas,(12,13) osteosarcomas,(14–16) chondrosarcomas,(15) and testicular tumors.(17)Moreover, PDPN expression is associated with malignant
progression and cancer metastasis.(5,18,19) Therefore, antiPDPN monoclonal antibodies (mAbs) are useful for investigating PDPN expression in various cancers.
Previously, we have developed anti-PDPN mAbs forcat,(20) tiger,(21) horse,(22) pig,(23) goat,(24) alpaca,(25) Tasmaniandevil,(26) bear,(27) whale,(28) and sheep(29) using the CellBased Immunization and Screening (CBIS) method.(30–32)
These mAbs have all been proven useful in flow cytometry, Western blotting, and immunohistochemical analyses.
However, sensitive and specific mAbs for elephant PDPN
(elePDPN) have not yet been developed. In this study, we
immunized mice with MAP16-tagged elePDPN-overexpressed
Chinese hamster ovary-K1 (CHO/MAP16-elePDPN) cells,
and established anti-elePDPN mAbs, which can be used forflow cytometry.
Materials and Methods
Cell lines

CHO-K1 and P3X63Ag8U.1 (P3U1) cells were obtained
from the American Type Culture Collection (Manassas, VA).
African savanna elephant fibroblast-like cells (LACF-NaNaI)
were obtained from the RIKEN BioResource Center (Ibaraki,
Japan).(33) The cDNA encoding the full-length open reading
frame (ORF) of elePDPN (accession no. XM_010593105.2)
was obtained by polymerase chain reaction using cDNA
derived from LACF-NaNaI cells. The elePDPN ORF with
N-terminal MAP16 tag (MAP16-elePDPN) or RIEDL tag(RIEDL-elePDPN) was subcloned into a pCAG-Ble vector

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
ORCID ID (https://orcid.org/0000-0001-5385-8201).
*Contributed equally to this study.
2
i

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142(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 Zeocin(InvivoGen, San Diego, CA). CHO/MAP16-elePDPN cells
were subsequently used as an immunogen, whereas CHO/RIEDL-elePDPN cells were used for further analyses. P3U1,
CHO-K1, CHO/MAP16-elePDPN, and CHO/RIEDLelePDPN cells were cultured in RPMI-1640 (Nacalai Tesque,Inc., Kyoto, Japan) supplemented with 10% heat-inactivatedfetal bovine serum (FBS; Thermo Fisher Scientific, Inc.),
100 U/mL penicillin, 100 lg/mL streptomycin, and0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.), and incubated at 37C in a humidified atmosphere containing 5%
CO2. LACF-NaNaI cells were cultured in minimum essential
medium (Nacalai Tesque, Inc.) supplemented with 10% heatinactivated FBS (Thermo Fisher Scientific, Inc.), 100 U/mL
penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL amphotericin B (Nacalai Tesque, Inc.), 5 lg/mL plasmocin(InvivoGen), and 1 mM L-sodium pyruvate (Nacalai Tesque,Inc.) and incubated at 37C in a humidified atmosphere
containing 5% CO2.

HOSONO ET AL.
Results and Discussion

To develop anti-elePDPN mAbs, the CBIS method was
employed using stable transfectants for immunization andflow cytometry (Fig. 1). Two mice were immunized with
CHO/MAP16-elePDPN cells that overexpressed elePDPN
with an N-terminal MAP16 tag. Hybridomas were seeded
into 96-well plates, and CHO/RIEDL-elePDPN-positive and
CHO-K1-negative wells were identified. The first screening
identified strong signals from CHO/RIEDL-elePDPN cells
and weak or no signals from CHO-K1 cells in 4 of 1054
wells (0.38%). After limiting dilution and several additional
screenings, PMab-265 (IgM, k), which reacted most strongly
with CHO/RIEDL-elePDPN, was finally established.
Next, flow cytometric analyses were performed using
PMab-265 with CHO/RIEDL-elePDPN, CHO-K1, and LACFNaNaI cells. LACF-NaNaI cells are fibroblast-like cells derived from African savanna elephants.(33) Cells were treatedwith PMab-265 (Fig. 2, black line) or without PMab-265(Fig. 2, gray shading). PMab-265 recognized CHO/RIEDLelePDPN cells (Fig. 2A) but not CHO-K1 cells (Fig. 2B).
PMab-265 also recognized endogenous elePDPN in LACFNaNaI cells (Fig. 2C). These results demonstrated that

Hybridoma production

Female BALB/c mice (6 weeks 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. The CBIS method was used to develop mAbs
against elePDPN. Two BALB/c mice were immunized with
CHO/MAP16-elePDPN cells (1 · 108) intraperitoneally with
Imject Alum (Thermo Fisher Scientific, Inc.). The procedureincluded three additional immunizations with CHO/MAP16elePDPN cells (1 · 108), followed by a final booster injectionof CHO/MAP16-elePDPN cells (1 · 108) 2 days before harvest of splenic cells. Subsequently, splenocytes were fusedwith P3U1 cells using polyethylene glycol 1500 (Roche
Diagnostics, Indianapolis, IN). The hybridomas were thengrown in RPMI-1640 supplemented with 10% heatinactivated FBS (Thermo Fisher Scientific, Inc.), 100 U/mLof penicillin, 100 lg/mL streptomycin, and 0.25 lg/mL
amphotericin B (Nacalai Tesque, Inc.), 5 lg/mL plasmocin (InvivoGen), and hypoxanthine/aminopterin/thymidine(Thermo Fisher Scientific, Inc.). The culture supernatants
were screened for anti-elePDPN antibody production using
flow cytometry.
Flow cytometry

CHO/RIEDL-elePDPN and parental CHO-K1 cells were
harvested after a brief exposure to 0.25% trypsin in 1 mM
ethylenediaminetetraacetic acid (Nacalai Tesque, Inc.). Afterwashing with 0.1% bovine serum albumin (BSA; Nacalai
Tesque, Inc.) in phosphate-buffered saline (PBS; Nacalai
Tesque, Inc.), cells were treated with anti-elePDPN mAb(1 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). Fluorescence data were collected using BD FACSLyric (Becton,
Dickinson and Company, Franklin Lakes, NJ).

FIG. 1. Production of anti-elePDPN mAbs. Schematic
representation of the CBIS method. (1) CHO/MAP16elePDPN cells were immunized into BALB/c mice by intraperitoneal injection. (2) Splenocytes were fused withmyelomas for hybridoma production. (3) Screening of antielePDPN mAb-producing hybridomas was performed byflow cytometry using CHO/RIEDL-elePDPN and parental
CHO-K1 cells. (4) Anti-elePDPN mAb-producing hybridomas were cloned using the limiting dilution method. CBIS,
Cell-Based Immunization and Screening.

DEVELOPMENT OF AN ANTI-ELEPHANT PDPN MAB

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PMab-265 is applicable for detecting both overexpressed
elePDPN and endogenous elePDPN using flow cytometry.
Although elephants are extremely cancer-resistant, about
3% of them develop cancers.(1,2) Since PMab-265 is an
elePDPN-specific mAb, it may be useful for the pathophysiological analysis of lymphangiogenesis in elephant cancers.
This study succeeded in developing anti-elePDPN using the CBIS method. The CBIS method was previously
successful in developing several mAbs against membraneproteins, such as CD20,(34) CD44,(32) and TROP2.(35) Thismethod helps establish mAbs against membrane proteinsbecause purified proteins are not required as immunogens andfor screening purposes. Moreover, the biological structure
and modification of proteins, such as glycosylation andfolding, could be retained; therefore, mAbs established using
the CBIS method could detect endogenous proteins.
This study showed that PMab-265 is useful for detecting
endogenous elePDPN by flow cytometric analysis; however,
it is not clear whether PMab-265 is applicable for the otheranalyses, such as immunohistochemistry and Western blotting. Future studies need to assess the usability of PMab-265
in immunohistochemistry and Western blotting analyses ofelephant tissues.
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(to Y.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 (toM.K.K.), 20K16322 (to M.S.), and 19K07705 (to Y.K.).
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FIG. 2. Detection of elePDPN by flow cytometry using
PMab-265. CHO/RIEDL-elePDPN (A), CHO-K1 (B), and
LACF-NaNaI (C) cells were treated with PMab-265 (blackline) at a concentration of 1 mg/mL or 0.1% BSA in PBS(gray shading) for 30 minutes, followed by incubation withsecondary antibodies. BSA, bovine serum albumin; PBS,
phosphate-buffered saline.

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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 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 31, 2021
Accepted: April 17, 2021