MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 35, Number 5, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0031

LpMab-19 Recognizes Sialylated O-Glycan
on Thr76 of Human Podoplanin
Satoshi Ogasawara, Mika K. Kaneko, and Yukinari Kato

Human podoplanin (hPDPN) is expressed in lymphatic vessels, pulmonary type-I alveolar cells, and renal
glomerulus. The hPDPN/C-type lectin-like receptor-2 (CLEC-2) interaction is involved in platelet aggregationand cancer metastasis. High expression of hPDPN in cancer cells or cancer-associated fibroblasts (CAFs) leads
to a poor prognosis for cancer patients. In our previous research, we reported on several anti-hPDPN monoclonal antibodies (mAbs), including LpMab-2, LpMab-3, LpMab-7, LpMab-9, LpMab-12, LpMab-13, and
LpMab-17 of mouse IgG1 subclass, which were produced using CasMab technology. Here we produced a novelanti-hPDPN mAb LpMab-19 of mouse IgG2b subclass. Flow cytometry revealed that the epitope of LpMab-19
includes O-glycan, which is attached to Thr76 of hPDPN. We further identified the minimum epitope of
LpMab-19 as Thr76–Arg79 of hPDPN. Immunohistochemistry revealed that LpMab-19 is useful for detecting
not only normal cells, including lymphatic vessels, but also glioblastoma and oral squamous cell carcinoma
cells. LpMab-19 could be useful for investigating the physiological function of O-glycosylated hPDPN.
Keywords: podoplanin, PDPN, CasMab, glycopeptide, monoclonal antibody
IntroductionTo determine the physiological function of membrane proteins in normal tissues and cancers, it is necessary to have highly sensitive and specific monoclonal
antibodies (mAbs). Human podoplanin (hPDPN), known as aplatelet aggregation-inducing (PLAG) factor (gp44/Aggrus),is involved in cancer metastasis.(1–10) We have produced manyanti-hPDPN mAbs,(11–21) including NZ-1, which was produced
by immunizing rats with the synthetic peptide (hpp3851) of
hPDPN, and it has high specificity, sensitivity, and binding
affinity for hPDPN.(21–23) The high binding affinity of NZ-1
has also been confirmed by other studies.(24,25) NZ-1 washighly internalized into glioma cell lines and also well accumulated into tumors in vivo; therefore, NZ-1 is a suitable
candidate for therapy against malignant gliomas.(23,26) NZ-1
has antibody-dependent cellular cytotoxicity and complementdependent cytotoxicity against hPDPN-expressing tumor
cells.(27) Furthermore, NZ-1 inhibited the tumor cell-induced
platelet aggregation and tumor metastasis by its neutralizing
activity.(28) NZ-1 possesses good applications in many experiments, such as western blot, flow cytometry, immunohistochemical analysis, and immunoprecipitation.
We recently established the CasMab technology, which
produces cancer-specific mAbs, although one protein in thecancer and normal cells has the same amino acid sequence.(11)We used LN229/hPDPN cells, not purified recombinant proteins, for immunization to develop novel anti-hPDPN mAbs.

Although we have produced many anti-hPDPN mAbs using
CasMab technology,(11–17,19,20) almost all mAbs, including
LpMab-2, LpMab-3, LpMab-7, LpMab-9, LpMab-12, LpMab13, and LpMab-17, were determined to belong to the IgG1
subclass. Only the clone LpMab-10 was classified as IgG3
subclass; however, mouse IgG3 subclass is often aggregated.(29)
Furthermore, almost all anti-hPDPN mAbs react with PLAG
domains (PLAG1–PLAG3) of hPDPN.(21,22,30–33) A novel
PLAG domain of hPDPN was recently reported as PLAG4 (81EDLPT-85), and anti-PLAG4 mAbs were produced.(34) We
need to produce further anti-hPDPN mAbs, which target different epitopes of hPDPN, to fully understand the pathophysiological function of hPDPN.
Here we produced a novel anti-hPDPN mAb, LpMab-19(mouse IgG2b, kappa), the epitope of which is a O-glycosylated
glycopeptide of hPDPN.
Materials and Methods
Cell lines

LN229, HEK-293T, Chinese hamster ovary (CHO)-K1,
and P3U1 were obtained from the American Type Culture
Collection (ATCC, Manassas, VA). LN319 was donated by
Prof. Kazuhiko Mishima (Saitama Medical University, Saitama, Japan).(35) CHO-S was purchased from Thermo Fisher
Scientific, Inc. (Waltham, MA). LN229 was previouslytransfected with hPDPN plasmids (LN229/hPDPN) using
Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.

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to the manufacturer’s instructions.(11) The HEK-293T/
hPDPN-knockout (KO) cell line (PDIS-2) and the LN319/
hPDPN-KO cell line (PDIS-6) were generated by transfection
using CRISPR/Cas plasmids (Target ID: HS0000333287) thattarget PDPN (Sigma-Aldrich, St. Louis, MO). PDIS-2 and
PDIS-6 cells were screened using the NZ-1 mAb.(12,15,18–20)
CHO-K1 and P3U1 were cultured in RPMI 1640 medium,including l-glutamine (Nacalai Tesque, Inc., Kyoto, Japan)
supplemented with 10% heat-inactivated fetal bovine serum(FBS; Thermo Fisher Scientific, Inc.). CHO-S was cultured
using CHO-S-SFM II (Thermo Fisher Scientific, Inc.).
LN229, LN229/hPDPN, LN319, HEK-293T, PDIS-2, and
PDIS-6 were cultured in Dulbecco’s modified Eagle’s medium, including l-glutamine (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated FBS at 37C in ahumidified atmosphere of 5% CO2 and 95% air. Antibiotics,
including 100 units/mL of penicillin, 100 mg/mL of streptomycin, and 25 mg/mL of amphotericin B (Nacalai Tesque,Inc.), were added to all media.
Production of deletion mutants

The amplified hPDPN complementary DNA (cDNA) was
subcloned into a pCAG-Ble(Zeo) vector (Wako Pure Chemical Industries Ltd. [Osaka, Japan]) with an MAP tag, which
was detected by PMab-1(36,37) and added at the N-terminus
using In-Fusion HD cloning kit (Clontech Laboratories Inc.,
Mountain View, CA). Deletion mutants of hPDPN were
produced using several primers. Sense primers used were as
follows: AGAAGACAAAAAGCTTGCCAGCACAGGCC
AGCC for dN23, AGAAGACAAAAAGCTTGAAGGCG
GCGTTGCCAT for dN37, AGAAGACAAAAAGCTTGCCGAAGATGATGTGGTG for dN46, AGAAGACAAAAAGCTTACCAGCGAAGACCGCTA for dN55, AGAAGACAAAAAGCTTACAACTCTGGTGGCAACA for dN64, AG
AAGACAAAAAGCTTGTAACAGGCATTCGCATC for
dN75, AGAAGACAAAAAGCTTACTTCAGAAAGCACAGTCC for dN85, AGAAGACAAAAAGCTTCAAAGTCC
AAGCGCCAC for dN95, AGAAGACAAAAAGCTTGCCACCAGTCACTCCAC for dN105.
The following antisense primer was used: TCTAGAG
TCGCGGCCGCTTACTTGTCGTCATCGT.
CHO-K1 cells were transfected with the plasmids using a
Lipofectamin LTX (Thermo Fisher Scientific, Inc.). Deletionmutants were cultured in RPMI 1640 medium including
l-glutamine supplemented with heat-inactivated FBS.
Stable transfectants of CHO-K1/ssMAP-hPDPNdN mutants were selected by cultivating the transfectants in a
medium containing 0.5 mg/mL of zeocin (InvivoGen, Inc.,San Diego, CA).
Production of point mutants

The amplified hPDPN cDNA was subcloned into a
pcDNA3 vector (Thermo Fisher Scientific, Inc.), and a FLAG
epitope tag was added at the C-terminus. Amino acids in
hPDPN were substituted with alanine or glycine using a
QuikChange Lightning site-directed mutagenesis kit (Agilent
Technologies, Inc., Santa Clara, CA) with oligonucleotides
containing the desired mutations. CHO-S or CHO-K1 cells
were transfected with the plasmids using a Gene Pulser Xcell
electroporation system (Bio-Rad Laboratories, Inc., Berke-

OGASAWARA ET AL.

ley, CA). Point mutants of CHO-K1 and CHO-S were cultured
in RPMI 1640 medium including l-glutamine supplemented
with heat-inactivated FBS and CHO-S-SFM II, respectively.
Hybridoma production

Four-week-old female BALB/c mice (CLEA Japan, Tokyo, Japan) were immunized by intraperitoneal (i.p.) injection of 1 · 108 LN229/hPDPN cells together with Imject
Alum (Thermo Fisher Scientific, Inc.).(11) After several additional immunizations, a booster i.p. injection was given
2 days before mice were euthanized by cervical dislocation,and spleen cells were harvested. The spleen cells were fusedwith P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). Hybridomas were grown in RPMI 1640
medium including l-glutamine with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo
Fisher Scientific, Inc.). Culture supernatants were screenedusing enzyme-linked immunosorbent assay (ELISA) for
binding to recombinant hPDPN purified from LN229/hPDPN
cells. Proteins were immobilized on Nunc Maxisorp 96-wellimmunoplates (Thermo Fisher Scientific, Inc.) at 1 mg/mL for
30 minutes. After blocking with 1% bovine serum albumin(BSA) in 0.05% Tween20/phosphate-buffered saline (PBS;
Nacalai Tesque, Inc.), the plates were incubated with culture
supernatant followed by 1:2000 diluted peroxidase-conjugatedantimouse IgG (Agilent Technologies, Inc.). The enzymatic
reaction was produced with a 1-Step Ultra TMB-ELISA(Thermo Fisher Scientific, Inc.). Optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad
Laboratories, Inc.).
Flow cytometry

Cell lines were harvested by brief exposure to 0.25%
trypsin/1 mM EDTA (Nacalai Tesque, Inc.). After washingwith 0.1% BSA in PBS, cells were treated with primary mAbs
for 30 minutes at 4C, followed by treatment with Oregon
Green 488 goat antimouse IgG or antirat IgG (Thermo Fisher
Scientific, Inc.). Fluorescence data were collected using a
Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).
Immunohistochemical analyses

The use of human cancer tissues was reviewed and approved
by Sendai Medical Center Review Board.(19) Written informed
consent was obtained for the human cancer tissue samples usedin this study. Four-micrometer-thick histologic sections weredeparaffinized in xylene and rehydrated. After antigen retrieval procedure (autoclave using citrate buffer, pH 6.0;
Agilent Technologies, Inc.), sections were incubated with
1 mg/mL of LpMab-19 for 1 hour at room temperature followed by treatment with Envision+ kit (Agilent Technologies,Inc.) for 30 minutes. Color was developed using 3,3diaminobenzidine tetrahydrochloride (Agilent Technologies,Inc.) for 5 minutes, and then the sections were counterstainedwith hematoxylin (Wako Pure Chemical Industries Ltd.).
Results and Discussion

The expression of hPDPN has been reported in many
cancers, such as oral cancer, lung cancer, esophageal cancer,
malignant mesothelioma, malignant glioma, testicular tumor,

LPMAB-19 AGAINST HPDPN

bladder cancer, and osteosarcoma.(8,21,23,26,38–46) hPDPN is a
PLAG factor, which is involved in cancer metastasis.(8–10)
hPDPN-expressing cancer-associated fibroblasts are associated with poor prognosis of several cancers.(47–52) We previously identified C-type lectin-like receptor-2 (CLEC-2) asan endogenous receptor of hPDPN,(28,53) and recently performed comparative crystallographic studies on hPDPN in
association with CLEC-2.(54) The interaction with CLEC-2
was mainly observed at Glu47 and Asp48 in the PLAG3
domain and the a2–6-linked sialic acid at Thr52 of hPDPN.
Clone LpMab-12, our recently produced mAb, specificallydetects this sialylated Thr52.(18)247

Herein we used the CasMab technology for production ofanti-hPDPN mAbs.(11) LN229/hPDPN cells were immunized
into mice, and culture supernatants were screened using
ELISA for binding to recombinant hPDPN, which was purified from LN229/hPDPN cells.(11) Finally, LpMab-19(mouse IgG2b, kappa), the first IgG2b of mouse anti-hPDPN
mAbs using CasMab technology, was produced after limiting
dilution.
LpMab-19 reacted with LN229/hPDPN, and did not with
LN229 (hPDPN-negative cell) in flow cytometry (Fig. 1A).
LpMab-19 recognized endogenous hPDPN, which is expressed in LN319 (a glioblastoma cell line), whereas it did

FIG. 1. In flow cytometry, LpMab-19 specifically detects hPDPN. (A) LN229, LN229/hPDPN, LN319, LN319/hPDPNKO cell (PDIS-6), HEK-293T, and HEK-293T/hPDPN-KO cell (PDIS-2) were treated with LpMab-19 (1 mg/mL; red) orcontrol PBS (black) for 30 minutes at 4C, followed by treatment with antimouse IgG-Oregon green. (B) LN229, LN229/
hPDPN, LN319, LN319/hPDPN-KO cell (PDIS-6), HEK-293T, and HEK-293T/hPDPN-KO cell (PDIS-2) were treatedwith LpMab-13 (1 mg/mL; blue) or control PBS (black) for 30 minutes at 4C, followed by treatment with antimouse IgGOregon green. Fluorescence data were collected using a Cell Analyzer EC800. hPDPN, human podoplanin; PBS, phosphatebuffered saline.

248

not react with LN319/hPDPN-KO cell (PDIS-6) (Fig. 1A).
In addition, LpMab-19 detected hPDPN of HEK-293T (anepithelial cell line of kidney), but lost the reaction with
HEK-293T/hPDPN-KO cell (PDIS-2) (Fig. 1A). As a positive
control, an anti-hPDPN mAb LpMab-13 was used (Fig. 1B).

OGASAWARA ET AL.

LpMab-13 detected LN229/hPDPN, LN319, and HEK-293T,
whereas it lost the reaction with PDIS-6 and PDIS-2.
We herein produced several deletion mutants of hPDPN,
which were expressed in CHO-K1 cell lines. LpMab-19 detected the delta N-terminus (dN)23 that starts from Ala23.

FIG. 2. Epitope mapping of LpMab-19 using deletion mutants of hPDPN in flow cytometry. Each hPDPN deletion mutant(dN23, dN37, dN46, dN55, dN64, dN75, dN85, dN95, and dN105) was treated with LpMab-19 (A, 1 mg/mL; red), PMab-1(anti-MAP tag; B, 1 mg/mL; green), or control PBS (A, B; black) for 30 minutes at 4C, followed by treatment with
antimouse IgG or antirat IgG-Oregon green. Fluorescence data were collected using a Cell Analyzer EC800.

FIG. 3. Epitope mapping of LpMab-19 using point mutants of Ser/Thr residues of hPDPN in flow cytometry. Stable CHOS transfectants expressing hPDPN point mutants (T65A, T66A, T70A, S71A, S74A, T76A, T85A, S86A, and S88A) weretreated with LpMab-19 (A, 1 mg/mL; red), LpMab-13 (B, 1 mg/mL; blue), or control PBS (A, B; black) for 30 minutes at
4C, followed by treatment with antimouse IgG-Oregon green. Fluorescence data were collected using a Cell Analyzer
EC800. CHO, Chinese hamster ovary.
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Similarly, LpMab-19 detected dN37, dN46, dN55, dN64, anddN75. In contrast, LpMab-19 lost the reaction with dN85,
dN95, and dN105, indicating that the N-terminus of the
LpMab-19 epitope exists between the 75th and 85th aminoacids (Fig. 2A). All deletion mutants of hPDPN possess the
N-terminal MAP tag and were detected by the anti-MAP tag
mAb (clone: PMab-1) (Fig. 2B).
Next, we produced stable CHO-S cell lines expressing
point mutants of hPDPN: T65A, T66A, T70A, S71A, S74A,
T76A, T85A, S86A, and S88A (Fig. 3A). The epitope of
LpMab-19 was thought to include glycans because LpMab-19
did not react with synthetic peptides of hPDPN (data not shown).

OGASAWARA ET AL.

LpMab-19 lost the reaction with the T76A mutant, whereas theother point mutants were detected by LpMab-19 (Fig. 3A).
All point mutants targeting Ser/Thr residues were detected by
LpMab-13 (Fig. 3B). Therefore, the epitope of LpMab-19
includes sialic acid on O-glycan, which is attached to Thr76.
We further determined the LpMab-19 epitope using alanine scanning around Thr76 of hPDPN. Nine hPDPN point
mutants against Ser74–Thr85 were transiently expressed in
CHO-K1 cells. As shown in Figure 4A, LpMab-19 did notdetect T76A, I78A, and R79A. In contrast, LpMab-13 recognized all point mutants (Fig. 4B), indicating that the minimum epitope of LpMab-19 was Thr76–Arg79.

FIG. 4. Epitope mapping of LpMab-19 using point mutants of hPDPN in flow cytometry. Nine hPDPN point mutants
against Ser74-Thr85 were transiently expressed in CHO-K1 cells. Cells were treated with LpMab-19 (A, 1 mg/mL; red),
LpMab-13 (B, 1 mg/mL; blue), or control PBS (A, B; black) for 30 minutes at 4C, followed by treatment with antimouse
IgG-Oregon green. Fluorescence data were collected using a Cell Analyzer EC800. (C) Schematic illustration of the epitopeof anti-hPDPN mAbs. mAbs, monoclonal antibodies.

LPMAB-19 AGAINST HPDPN

251

thank Yuki Fujii and Ryusuke Honma for their suggestion
and advice. This work was supported, in part, by the Platformfor Drug Discovery, Informatics, and Structural Life Science(PDIS) from Japan Agency for Medical Research and development, AMED (Y.K.), by the Regional Innovation
Strategy Support Program from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan(Y.K.), by JSPS KAKENHI Grant Number 26440019(M.K.K.) and Grant Number 16K10748 (Y.K.), and by the
Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED (Y.K.). This work
was performed, in part, under the Cooperative Research
Program of Institute for Protein Research, Osaka University,
CR-15–05 and CR-16–05 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.
References

FIG. 5. Immunohistochemical analysis by LpMab-19
against oral squamous cell carcinoma. Sections were incubatedwith 1 mg/mL of LpMab-19 (A, B, E, F) or LpMab-13 (C, D,
G, H), followed by Envision+ kit, and color was developed
using DAB and counterstained with hematoxylin. Scale bar:
100 mm. DAB, 3,3-diaminobenzidine tetrahydrochloride.
It has been reported that hPDPN is expressed in many
cancers, such as malignant brain tumor, mesothelioma, oral
cancer, lung cancer, esophageal cancer, testicular tumor, andosteosarcoma.(13,38,44) Here we compared the reactivity of
LpMab-19 and LpMab-13 in an immunohistochemical analysis using oral squamous cell carcinoma. LpMab-19 stainedcancer cells (Fig. 5A, B) and LECs (Fig. 5E, F) in a membrane/
cytoplasmic-staining pattern without an antigen retrieval
procedure. LpMab-19 did not react with vascular endothelial
cells (Fig. 5E, F). The staining pattern of LpMab-19 is similar
to LpMab-13, indicating that LpMab-19 is very useful for
immunohistochemistry.
Therefore, LpMab-19 could be useful for investigating theexpression and function of hPDPN in cancers and normal tissues. In future, different epitope-possessing anti-hPDPN mAbs
should be produced for uncovering the function of hPDPN.
Acknowledgments

We thank Takuro Nakamura, Noriko Saidoh, and Kanae
Yoshida for their excellent technical assistance. We also

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Address correspondence to:
Dr. Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2–1 Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: June 18, 2016
Accepted: August 1, 2016