MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 2, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0001

LpMab-23:
A Cancer-Specific Monoclonal Antibody
Against Human Podoplanin
Shinji Yamada,1,2 Satoshi Ogasawara,2 Mika K. Kaneko,1,2 and Yukinari Kato1–3

Human podoplanin (hPDPN), the ligand of C-type lectin-like receptor-2, is involved in cancer metastasis. Until
now, many monoclonal antibodies (mAbs) have been established against hPDPN. However, it is still difficult todevelop a cancer-specific mAb (CasMab) against hPDPN because the protein sequence of hPDPN expressed incancer cells is the same as that in normal cells. Herein, we report LpMab-23 of the mouse IgG1 subclass, a novel
CasMab against hPDPN. In an immunohistochemical analysis, LpMab-23 reacted with tumor cells of humanoral cancer, but did not react with normal cells such as lymphatic endothelial cells (LECs). In contrast, LpMab17, another anti-hPDPN mAb, reacted with both tumor cells and LECs. Furthermore, flow cytometric analysis
revealed that LpMab-23 reacted with hPDPN-expressing cancer cell lines (LN319, RERF-LC-AI/hPDPN,Y-MESO-14/hPDPN, and HSC3/hPDPN) but showed little reaction with normal cells (LECs and HEK-293T),
although another anti-hPDPN mAb, LpMab-7, reacted with both hPDPN-expressing cancer cells and normal
cells, indicating that LpMab-23 is a CasMab against hPDPN.
Keywords: podoplanin, monoclonal antibody, cancer-specific antibody, CasMab(GpMabs) against hPDPN.(23) One of these GpMabs, LpMab2, was shown to be a cancer-specific mAb (CasMab). LpMab-2
recognizes both an aberrant O-glycosylation and a Thr55Leu64 peptide from hPDPN. Because LpMab-2 reacts with
hPDPN-expressing cancer cells but not with normal cells, as
shown by flow cytometry and immunohistochemistry, it isexpected to be useful for molecular targeting therapy against
hPDPN-expressing cancers. Nevertheless, LpMab-2 is not very
useful in immunohistochemical analysis; therefore, another
CasMab against hPDPN is necessary. In this study, we report
LpMab-23, the second CasMab against hPDPN, which is very
useful in immunohistochemistry.

IntroductionHuman podoplanin (hPDPN) is expressed in many
cancers, including brain tumors, lung cancers, esophageal cancers, osteosarcomas, and malignant mesotheliomas.(1–13) C-type lectin-like receptor 2 is an endogenous
receptor of hPDPN and is involved in platelet aggregation.(14–16) Recently, hPDPN was reported to be a potential
biomarker for identifying the presence of early infiltrativeesophageal squamous cell carcinoma.(17) Three different
patterns can be distinguished in the expression of hPDPN in
the basal layer of squamous epithelium lesions as follows:complete type, noncontinuous type, or missing type. The
diagnosis of high-grade intraepithelial neoplasia can be madeif the basal layer shows noncontinuous or complete expression of hPDPN. In contrast, the diagnosis of early infiltration
can be made if hPDPN expression is completely missing. In
these studies, D2-40, one of the anti-hPDPN monoclonal
antibodies (mAbs), was used.
Although many anti-hPDPN mAbs have been reported,
almost all the mAbs such as D2-40 and NZ-1 react withthe peptides of hPDPN.(4,18–22) In contrast, we have used
our original technology to produce anti-glycopeptide mAbs

Materials and Methods
Hybridoma production

Female BALB/c mice (4 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). Animals were housed in a
specific pathogen-free environment. The Animal Care and
Use Committee of Tohoku University approved the animal
experiments described herein. BALB/c mice were immunized by intraperitoneal (i.p.) injection of 1 · 108 LN229/

Departments of 1Antibody Drug Development, and 2Regional Innovation, Tohoku University Graduate School of Medicine, Sendai,
Miyagi, Japan.
3New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan.

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LPMAB-23 AGAINST HUMAN PODOPLANIN

73

FIG. 1. Immunohistochemical analysis using anti-hPDPN mAbs to detect the expression of hPDPN in oral cancers. Tissue
sections were prepared from the oral squamous cell carcinoma. Sections were incubated with LpMab-23 (A–C) and LpMab17 (D–F) and made to react with the Envision+ kit. Color was developed using 3, 3-diaminobenzidine tetrahydrochloride,and the samples were then counterstained with hematoxylin. Sections were stained with hematoxylin and eosin as well(G–I). Cancer cells were detected by both LpMab-23 (A, B) and LpMab-17 (D, E). LpMab-23 showed little reaction with
LECs (A, C). Arrows: LECs. Scale bar = 100 mm. hPDPN, human podoplanin; LECs, lymphatic endothelial cells; mAbs,
monoclonal antibodies.

hPDPN cells together with Imject Alum (Thermo Fisher
Scientific, Inc., Waltham, MA). LN229/hPDPN cells were
produced previously.(23) After several additional immunizations, a booster injection was given i.p. 2 days before spleencells were harvested. The spleen cells were fused with P3U1
cells (ATCC, Manassas, VA) using PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in the
RPMI medium (Wako Pure Chemical Industries, Ltd., Osaka,
Japan) with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.).
Using enzyme-linked immunosorbent assay (ELISA), theculture supernatants were screened for binding to recombinant hPDPN purified from LN229/hPDPN cells.

Flow cytometry

Immunohistochemical analyses

Results and Discussion

This study examined oral cancer patients who underwent
surgery at the Sendai Medical Center as described previously.(24)
Informed consent for obtaining samples and for subsequent
data analyses was obtained from patients or the patient’s guardian, and 4-mm-thick histologic sections were deparaffinized inxylene and rehydrated. After antigen retrieval using the Envision
FLEX system (high pH; Dako, Glostrup, Denmark), sections
were incubated with 10 mg/mL of LpMab-23 or 1 mg/mL of
LpMab-17 for 1 hour at room temperature followed by treatment
with Envision+ kit (Dako) for 30 minutes. Color was developed
using 3, 3-diaminobenzidine tetrahydrochloride (DAB; Dako)for 2 minutes; subsequently, the sections were counterstainedwith hematoxylin (Wako Pure Chemical Industries Ltd.).

We immunized one mouse with the LN229/hPDPN, andthe harvested spleen cells were fused with P3U1. ELISA
screening was performed with supernatants from 960 hybridomas. Among 135 ELISA-positive wells, 19 wells reacted with LN229/hPDPN, but not with LN229 in flow
cytometry (data not shown). We performed single-cellcloning for 19 wells by limiting dilution and could obtain
14 hybridomas. Until now, six clones have been reported,
including LpMab-10, LpMab-12, LpMab-13, LpMab-17,
LpMab-19, and LpMab-21.(23–33) In this study, we report the
new clone, LpMab-23 (IgG1, kappa).
To investigate whether LpMab-23 is a CasMab, we performed immunohistochemical analyses using oral cancers.

Cell lines in this study (LN319 of glioblastoma, RERFLC-AI/hPDPN of lung cancer, Y-MESO-14/hPDPN of
mesothelioma, HSC3/hPDPN of oral cancer, lymphatic endothelial cell (LEC), and HEK-293T of renal epithelial cell)were cultured as described previously.(23) Cell lines were
harvested by brief exposure to 0.25% trypsin/1 mM EDTA
(Nacalai Tesque, Inc., Kyoto, Japan). After washing, the cells
were treated with LpMab-23 or LpMab-7 (1 mg/mL) for 30
minutes at 4C, followed by treatment with Oregon Green
488 goat anti-mouse IgG (Thermo Fisher Scientific, Inc.).
Fluorescence data were collected using a Cell Analyzer
EC800 (Sony Corp., Tokyo, Japan).

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YAMADA ET AL.

and HEK-293T of a renal epithelial cell) (Fig. 2). In contrast,
an anti-hPDPN mAb, LpMab-7, reacted with both hPDPNexpressing cancer cells and normal cells, indicating that
LpMab-23 is cancer specific.
Taken together, these results show that LpMab-23 might
be useful for antibody therapy against hPDPN-expressing
cancers. Reports have indicated that hPDPN is expressed inmany normal cells(34,35); therefore, anti-hPDPN mAbs might
trigger unexpected side effects. To ensure the safety and efficacy of anti-hPDPN mAbs, CasMabs such as LpMab-23
should be applied clinically to cancer patients.
Acknowledgments

We thank Takuro Nakamura, Miyuki Yanaka, Noriko
Saidoh, and Kanae Yoshida for their excellent technical assistance. We also thank Yuki Fujii, Hiroaki Uchida, andHideaki Tahara for their specialized advice. This work was
supported, in part, by the Basic Science and Platform Technology Program for Innovative Biological Medicine fromJapan Agency for Medical Research and development,
AMED (Y.K.), by the Translational Research Network Program from AMED (Y.K.), by Project for utilizing glycans in
the development of innovative drug discovery technologies
from AMED (Y.K.), by the Platform for Drug Discovery,
Informatics, and Structural Life Science (PDIS) from AMED
(Y.K.), by JSPS KAKENHI Grant No. 26440019 (M.K.K.)
and 16K10748 (Y.K.), and by the Regional Innovation
Strategy Support Program from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan(Y.K.). This work was performed, in part, under the Cooperative Research Program of Institute for Protein Research,
Osaka University, CR-16-05 and by the Grant for Joint Research Project of the Institute of Medical Science, the University of Tokyo. We thank Enago (www.enago.jp) for the
English language review.
Author Disclosure Statement

No competing financial interests exist.

FIG. 2. Flow cytometric analysis by anti-hPDPN mAbs.
Cells were treated with LpMab-23, LpMab-7 (1 mg/mL;red), or control phosphate-buffered saline (black) for 30
minutes at 4C, followed by treatment with anti-mouse IgGOregon green. Fluorescence data were collected using a Cell
Analyzer EC800.

As shown in Figure 1A and B, LpMab-23 reacted with tumor
cells, which were also detected by another anti-hPDPN mAb,
LpMab-17 (Fig. 1D, E).(24) In contrast, LpMab-23 showed
little reaction with LECs (Fig. 1A, C), which were recognizedby LpMab-17 (Fig. 1D, F). We further performed immunohistochemical analysis using 10 cases of oral cancers andobtained the same results (data not shown), indicating that
LpMab-23 is a CasMab against hPDPN.
Flow cytometric analysis showed that LpMab-23 reactedwith hPDPN-expressing cancer cell lines (LN319 of glioblastoma, RERF-LC-AI/hPDPN of lung cancer, Y-MESO14/hPDPN of mesothelioma, and HSC3/hPDPN of oral
cancer) but showed little reaction with normal cells (LECs

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Address correspondence to:
Dr. Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: January 17, 2017
Accepted: February 28, 2017