MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 3, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0014

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ChLpMab-23:
Cancer-Specific Human–Mouse Chimeric
Anti-Podoplanin Antibody Exhibits Antitumor Activity
via Antibody-Dependent Cellular Cytotoxicity
Mika K. Kaneko,1,2 Takuro Nakamura,1,2 Akiko Kunita,3 Masashi Fukayama,3 Shinji Abe,4,5
Yasuhiko Nishioka,5 Shinji Yamada,1,2 Miyuki Yanaka,1,2 Noriko Saidoh,1,2 Kanae Yoshida,2Yuki Fujii,2 Satoshi Ogasawara,2 and Yukinari Kato1,2,6

Podoplanin is expressed in many cancers, including oral cancers and brain tumors. The interaction between
podoplanin and its receptor C-type lectin-like receptor 2 (CLEC-2) has been reported to be involved in cancer
metastasis and tumor malignancy. We previously established many monoclonal antibodies (mAbs) against human
podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-23 (IgG1, kappa), one of the mouse antipodoplanin mAbs, was shown to be a CasMab. However, we have not shown the usefulness of LpMab-23 for
antibody therapy against podoplanin-expressing cancers. In this study, we first determined the minimum epitope of
LpMab-23 and revealed that Gly54–Leu64 peptide, especially Gly54, Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60,
and Leu64 of podoplanin, is a critical epitope of LpMab-23. We further produced human–mouse chimeric LpMab23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC)
and antitumor activity. In flow cytometry, chLpMab-23 showed high sensitivity against a podoplanin-expressing
glioblastoma cell line, LN319, and an oral cancer cell line, HSC-2. chLpMab-23 also showed ADCC activity
against podoplanin-expressing CHO cells (CHO/podoplanin). In xenograft models with HSC-2 and CHO/podoplanin, chLpMab-23 exerts antitumor activity using human natural killer cells, indicating that chLpMab-23 could
be useful for antibody therapy against podoplanin-expressing cancers.
Keywords: podoplanin, monoclonal antibody, ADCC, CDC, antitumor activity
IntroductionP

odoplanin is expressed in many cancers, including
oral cancers and brain tumors.(1–13) C-type lectin-likereceptor 2 (CLEC-2) is an endogenous receptor of podoplanin and is involved in platelet aggregation.(14–22) Recently,
podoplanin was reported to be a potential biomarker for
identifying the presence of early infiltrative esophageal
squamous cell carcinoma.(23) Previously, podoplanin has also
been reported as a marker of tumor-initiating cells (TICs) in ahuman squamous cell carcinoma cell line, A431.(24) The
same group recently showed that high clonal expansion capacity of podoplanin-positive TICs populations was the resultof reduced cell death caused by podoplanin-mediated signaling, indicating that podoplanin may be a therapeutic target
in squamous cell carcinoma treatment.(25)Transforming growth factor-b (TGF-b) regulates many
physiological events including tumorigenesis.(26) We previously found that TGF-b induced podoplanin in human fibrosarcoma HT1080 cells and enhanced the platelet aggregating
ability of HT1080.(27) TGF-b type I receptor inhibitor(SB431542) and short hairpin Smad4 inhibited the podoplanin
induction by TGF-b, suggesting that TGF-b is a physiologicalregulator of podoplanin in tumor cells. Recently, anothergroup confirmed this phenomenon using in vivo analysis.(28)
Podoplanin-mediated epithelial–mesenchymal transition (EMT)

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Pathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
4
Department of Clinical Pharmacy Practice Pedagogy, Graduate School of Biomedical Sciences, Tokushima University, Tokushima,
Japan.
5
Department of Respiratory Medicine and Rheumatology, Graduate School of Biomedical Sciences, Tokushima University, Tokushima,
Japan.
6
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2
3

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CHLPMAB-23 AGAINST PDPN

reportedly resulted in increased invasiveness and extravasationof tumor cells, and treatment of mice with a TGF-b-neutralizing
antibody inhibited podoplanin-mediated hematogenous metastasis in vivo, indicating that podoplanin promoted hematogenous
metastasis in part by releasing TGF-b from platelets that wasessential for EMT of tumor cells.
Many anti-podoplanin monoclonal antibodies (mAbs) havebeen reported, and almost all of them, such as D2-40, 18H5,
and NZ-1, react with the peptides of podoplanin.(19,29–35)
We have used our original technology to produce antiglycopeptide mAbs (GpMabs) against podoplanin.(36–40) Oneof these GpMabs, LpMab-2, was shown to be a cancer-specificmAb (CasMab).(36) LpMab-2 recognizes both an aberrant Oglycosylation and a Thr55–Leu64 peptide from podoplanin.
Because LpMab-2 reacts with podoplanin-expressing cancer
cells but not with normal cells, as shown by flow cytometry
and immunohistochemistry, it is expected to be useful formolecular targeting therapy against podoplanin-expressing
cancers.(41) Another anti-podoplanin CasMab is LpMab-23.(42)In this study, we produced human–mouse chimeric LpMab23 (chLpMab-23) and investigated whether chLpMab-23 exerts antibody-dependent cellular cytotoxicity (ADCC) andantitumor activity.
Materials and Methods
Cell lines

Chinese hamster ovary cell lines (CHO)-K1, LN229, andP3U1 were obtained from the American Type Culture Collection (Manassas, VA). HSC-2 was obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka,
Japan). The LN319 cell line was provided by Prof. KazuhikoMishima (Saitama Medical University, Saitama, Japan).(43)
CHO-S was purchased from Thermo Fisher Scientific, Inc.
(Waltham, MA). Core fucose-knockout CHO-S cells (PDIS5) was established using CRISPR-Cas9 in our previous
study.(41) CHO-K1 and LN229 cells were transfected withpodoplanin plasmids using Lipofectamine 2000 (Thermo
Fisher Scientific, Inc., Waltham, MA) according to the manufacturer’s instructions.(36)The CHO-K1, CHO-K1/podoplanin, and P3U1 cells were
cultured in Roswell Park Memorial Institute (RPMI) 1640
medium containing L-glutamine (Nacalai Tesque, Inc., Kyoto,
Japan) and 10% heat-inactivated fetal bovine serum (FBS)(Thermo Fisher Scientific, Inc.). HSC-2 cells were cultured in
Eagle’s minimal essential medium (Nacalai Tesque, Inc.) or
Dulbecco’s modified Eagle’s medium (DMEM) containing Lglutamine and 10% heat-inactivated FBS. The LN319, LN229,
and LN229/podoplanin cells were cultured in DMEM containing L-glutamine and 10% heat-inactivated FBS. Cells
were kept at 37C in a humidified atmosphere containing 5%
CO2. All media contained 100 U/mL of penicillin, 100 mg/mLof streptomycin, and 25 mg/mL of amphotericin B (Nacalai
Tesque, Inc.).
Antibodies

LpMab-23, a mouse anti-podoplanin mAb (IgG1, kappa),
was developed as previously described.(42) Human IgG waspurchased from Beckman Coulter, Inc. (Fullerton, CA) or
Sigma-Aldrich Corp. (St. Louis, MO). To generate human–
mouse chimeric anti-podoplanin (chLpMab-23), appropriate

105

VH and VL cDNAs of mouse LpMab-23 and CH and CL ofhuman IgG1 were subcloned into pCAG-Ble and pCAG-Neo
vectors (Wako Pure Chemical Industries, Ltd., Osaka, Japan),respectively. Antibody expression vectors were transfected
into CHO-K1 or PDIS-5(41) using the Lipofectamine LTX
reagent (Thermo Fisher Scientific, Inc.). Stable transfectantsof CHO-K1/chLpMab-23 or PDIS-5/chLpMab-23 cells wereselected by cultivating the transfectants in a medium containing 0.5 mg/mL of both geneticin and zeocin (InvivoGen,
San Diego, CA). CHO-K1/chLpMab-23 and PDIS-5/
chLpMab-23 cells were cultivated in CHO-S-SFM II medium (Thermo Fisher Scientific, Inc.). ChLpMab-23 waspurified using Protein G-Sepharose (GE Healthcare UK Ltd,
Buckinghamshire, England).
Production of point mutants

The amplified human podoplanin cDNA was subcloned
into a pcDNA3 vector (Thermo Fisher Scientific, Inc.), and a
FLAG epitope tag was added at the C-terminus. Amino acidsin podoplanin were substituted with alanine using a QuikChange Lightning site-directed mutagenesis kit (Agilent
Technologies, Inc., Santa Clara, CA) with oligonucleotides
containing the desired mutations. CHO-K1 or CHO-S cells
were transfected with the plasmids using a Gene Pulser Xcell
electroporation system (Bio-Rad Laboratories, Inc., Berkeley, CA). The point mutant cells of CHO-K1 were cultured in
RPMI 1640 medium containing L-glutamine and 10% heatinactivated FBS as described above. The point mutant cells of
CHO-S were cultured in CHO-S-SFMII medium.
Flow cytometry

The cell lines were harvested after brief exposure to
0.25% trypsin/1 mM ethylenediaminetetraacetic acid (Nacalai
Tesque, Inc.). After washing with 0.1% bovine serum albuminin phosphate-buffered saline (PBS), the cells were treatedwith primary mAbs for 30 min at 4C, followed by treatment
with fluorescein isothiocyanate (FITC)-conjugated goat antihuman or antimouse IgG (Thermo Fisher Scientific, Inc.).
Fluorescence data were acquired using Cell Analyzer EC800(Sony Corp., Tokyo, Japan).
Determination of the binding affinity
using flow cytometry

LN319 (2 · 105 cells) were resuspended with 100 mL ofserially diluted chLpMab-23 (0.02–50 mg/mL) or NZ-12(0.01–20 mg/mL) followed by secondary antihuman IgG.
Fluorescence data were collected using EC800. The dissociation constants (KD) were obtained by fitting the binding
isotherms using the built-in one-site binding models in
GraphPad PRISM 6 (GraphPad software, Inc., La Jolla, CA).
Western blot analyses

Cell lysates (10 mg) were boiled in SDS sample buffer(Nacalai Tesque, Inc.). The proteins were electrophoresed on
5%–20% polyacrylamide gels (Wako Pure Chemical Industries Ltd.) and were transferred onto a polyvinylidene difluoride membrane (Merck KGaA, Darmstadt, Germany).
After blocking with 4% skim milk (Nacalai Tesque, Inc.), themembrane was incubated with chLpMab-23, NZ-12, or anti-bactin (clone AC-15; Sigma-Aldrich Corp.) and then with

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peroxidase-conjugated antihuman IgG (Sigma-Aldrich Corp.)
and was developed with the ImmunoStar LD Chemiluminescence Reagent (Wako Pure Chemical Industries Ltd.) using a
Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

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Antibody-dependent cellular cytotoxicity

Effector cells were prepared as previously described.(7)Human peripheral blood mononuclear cells (MNCs) wereobtained from leukocytes, which were separated from theperipheral blood of healthy donors. The study with humansubjects was approved by the Ethics Committee of the Tokushima University. ADCC was determined using the 51Crrelease assay.(7) Target cells were incubated with 0.1 mCi of
51
Cr-sodium chromate at 37C for 1 hour. After washing
three times with 10% FBS-supplemented RPMI 1640, 51Crlabeled target cells were seeded in 96-well plates in triplicate.
Human peripheral blood MNCs and chLpMab-23 or control
human IgG were added to the cells. After 6 hours of incubation, 51Cr released from cells into the supernatant (100 mL)
was measured using a gamma counter (PerkinElmer, Waltham,MA). The percentage of cytotoxicity was calculated using
the following formula:% specific lysis = (E-S)/(M-S) · 100,
where E is the release in the test sample, S is the spontaneous
release, and M is the maximum release.
Antitumor activity of anti-podoplanin antibodies

CHO/podoplanin cells and HSC-2 cells were trypsinized
and washed with PBS. The cell density was adjusted with
PBS to 5.0 · 107 cells/mL (CHO/podoplanin) or 1.0 · 107
cells/mL (HSC-2), and 100 mL/animal of the cell suspension
was subcutaneously inoculated into BALB/c nude mice.
After 1 day, 100 mL of 1 mg/mL of chLpMab-23 and human
IgG were injected into the peritoneal cavity of mice. Additional antibodies were injected once a week for several
weeks. Human natural killer (NK) cells (5.0 · 105 cells; Takara Bio, Inc., Shiga, Japan) were injected around the tumors
several times. The tumor diameter was measured every 3–
7 days and was calculated using the following formula:volume = W2 · L/2, where W is the short diameter and L is thelong diameter. The mice were euthanized 21 (CHO/podoplanin) or 35 days (HSC-2) after cell implantation.
Statistical analyses

All data were expressed as means – SEMs. Student’s t-test,
Mann–Whitney U-test, one-way analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisons,and two-way ANOVA were performed as appropriate. pValues <0.05 were considered to be significant. All statisticaltests were two-sided.
Results
Epitope mapping of LpMab-23

We performed flow cytometry using several point mutantsof human podoplanin to determine the epitope of LpMab-23.
Seventeen podoplanin point mutants against Val50–Thr66
were transiently expressed in CHO-K1 cells. As shown in
Figure 1A, LpMab-23 did not detect G54A, T55A, S56A,
E57A, D58A, R59A, Y60A, or L64A. In contrast, LpMab-7,(30)a positive control mAb against human podoplanin, recognized

KANEKO ET AL.

all point mutants. These results indicate that the minimum
epitope of LpMab-23 is Gly54–Leu64, which is very similar tothat of LpMab-2 (Fig. 1B). Flow cytometry and western blot
analyses using several glycan-deficient podoplanin cells revealed that the epitope of LpMab-23 is independent of glycans(data not shown), whereas the epitope of LpMab-2 includes Oglycan of Thr55/Ser56.(36) Here, we produced a human–mousechimeric antibody from LpMab-23 and investigated whether
chLpMab-23 possesses ADCC and antitumor activity.
Production of chLpMab-23

We first checked the sensitivity of chLpMab-23 using flow
cytometry. As shown in Figure 2A, chLpMab-23 reaction
against human glioblastoma cell line, LN319, was observed
at a very low concentration of 10 ng/mL. A positive control
NZ-12, the most sensitive chimeric anti-podoplanin antibody,(44) reacted with LN319 in the same way. Both antibodies reacted with LN319 in a dose-dependent manner. We
next performed a kinetic analysis of the interaction ofchLpMab-23 with LN319 using flow cytometry. As shown in
Figure 2B, KD of chLpMab-23 was determined to be 1.2 · 10-8
M. The KD of NZ-12 was also determined to be 1.2 · 10-8 M.
NZ-12 was derived from the rat anti-podoplanin mAb, NZ-1,
which possesses the highest binding affinity(8); this showedthat chLpMab-23 also possesses high binding affinity against
podoplanin. Western blot analysis showed that chLpMab-23
did not detect T55A and S56A (Fig. 2C), which is consistent
with the results of flow cytometry using LpMab-23 (Fig. 1A).
chLpMab-12,(30) a human–mouse chimeric antibody produced
from another GpMab, LpMab-12 against human podoplanin,
did not recognize T52A, which is consistent with our previous
study.(37) chLpMab-7, a human–mouse chimeric antibody
produced from LpMab-7 against human podoplanin,(45) and
NZ-12 reacted with all point mutants. Because the podoplanin
expression level in S56A mutant is comparatively low, thesignals of chLpMab-12, chLpMab-7, and NZ-12 were lower
than those of the other mutants. These results indicate that the
epitope of chLpMab-23 was confirmed to include Thr55 andSer56.
ADCC of chLpMab-23 against
podoplanin-expressing cells

Next, we investigated whether chLpMab-23 possesses
ADCC activity against podoplanin-expressing cells. chLpMab23 reacted with CHO/podoplanin in a dose-dependent manner(Fig. 3A). A positive control NZ-12 showed a similar reaction.
Western blot analysis showed that chLpMab-23 specificallydetected podoplanin, which is expressed in LN229 and CHOK1 (Fig. 3B). NZ-12 also helped detect podoplanin in the sameway. As shown in Figure 3C, NZ-12 showed ADCC activity
against CHO/podoplanin cells. In the same way, chLpMab-23
exerted ADCC against the CHO/podoplanin cells but notagainst CHO-K1 cells, indicating that ADCC against CHO/
podoplanin is specific against podoplanin.
Antitumor activity of chLpMab-23

To study the antitumor activity of chLpMab-23 on primarytumor growth in vivo, CHO/podoplanin cells were subcutaneously implanted into the flanks of nude mice. To upregulate the ADCC activity of chLpMab-23, PDIS-5 cells

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CHLPMAB-23 AGAINST PDPN

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FIG. 1. Epitope mapping of LpMab-23 using point mutants of human podoplanin in flow cytometry (A) Seventeen
podoplanin point mutants were transiently expressed in CHO-K1 cells. Cells were treated with LpMab-23 (1 mg/mL; red),
LpMab-7 (1 mg/mL; blue), or control phosphate-buffered saline (PBS) (black) for 30 min at 4C, followed by treatment with
anti-mouse IgG–Oregon green. Fluorescence data were collected using a Cell Analyzer EC800. (B) Schematic illustration of
the epitope of anti-podoplanin mAbs including LpMab-23 (red) and LpMab-7 (blue). mAbs, monoclonal antibodies.

(FUT-8-knockout cell line) were used for producing
chLpMab-23. Core fucose-negative-type chLpMab-23,
which was produced using PDIS-5, and control human IgG
were injected four times into the peritoneal cavity of mice,and human NK cells were injected twice around the tumors.
Tumor formation was observed in mice from the control andtreated groups. However, chLpMab-23 significantly reduced
tumor development compared with control human IgG on
day 21 (Fig. 3D).
We further investigated whether chLpMab-23 also inhibited the tumor growth of an oral cancer cell line, HSC-2.
chLpMab-23 reacted with HSC-2 in a dose-dependent manner (Fig. 4A). NZ-12 also reacted with HSC-2 in a dosedependent manner. The reaction of NZ-12 is much moresensitive compared with that of chLpMab-23. For in vivo
analysis, we used normal-type chLpMab-23, which was
produced in CHO-K1 cells. HSC-2 cells were subcutaneously

implanted into the flanks of nude mice. chLpMab-23 andcontrol human IgG were injected seven times into the peritoneal cavity of mice, and human NK cells were injected
three times around the tumors. As shown in Figure 4B, tumor
formation was observed in mice from the control and treatedgroups. chLpMab-23 significantly reduced tumor development of HSC-2 compared with control human IgG on day 35(Fig. 4C). These results indicate that administration ofchLpMab-23 with the NK cells inhibited the primary tumor
growth in vivo.
Discussion

In a previous study, through an immunohistochemical
analysis, we showed that LpMab-23 reacted with tumor cellsof human oral cancer but not with normal cells such as
lymphatic endothelial cells (LECs).(42) Furthermore, flow

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KANEKO ET AL.

FIG. 2. Characterization of chLpMab-23 (A) LN319 cells were treated with chLpMab-23 or NZ-12 (10 mg/mL–10 ng/mL,red) and PBS (black) for 30 min at 4C, followed by treatment with antihuman IgG–FITC. Fluorescence data were collectedusing Cell Analyzer EC800. (B) Determination of the binding affinity of chLpMab-23 or NZ-12 using flow cytometry. (C)Six podoplanin point mutants were stably expressed in CHO-S cells. Western blot analysis with chLpMab-23, chLpMab-12,
chLpMab-7, or NZ-12. The membrane was incubated with 10 mg/mL of primary antibodies and then with peroxidaseconjugated antihuman IgG; the membrane was detected using a Sayaca-Imager. AC-15 (anti-b-actin) was used as internal
control. FITC, fluorescein isothiocyanate.

cytometric analysis revealed that LpMab-23 reacted withpodoplanin-expressing cancer cell lines but showed little
reaction with normal cells including LECs and HEK-293T.
Although the epitope of another CasMab, LpMab-2, wasdetermined to be Thr55–Leu64 peptide from podoplanin, theepitope of LpMab-23 has not been clarified. In this study, we
performed flow cytometry using several point mutants ofhuman podoplanin and revealed that Gly54–Leu64 peptide
from podoplanin is the LpMab-23 epitope (Fig. 1A). Furthermore, eight amino acids namely Gly54, Thr55, Ser56,
Glu57, Asp58, Arg59, Tyr60, and Leu64 of podoplanin areimportant for LpMab-23. In contrast, seven amino acids
namely Thr55, Ser56, Glu57, Asp58, Arg59, Tyr60, andLeu64 of podoplanin are important for LpMab-2,(36) indicating that epitopes of the two different CasMabs LpMab-2
and LpMab-23 are very similar (Fig. 1B).
We had not shown that LpMab-23 possesses ADCC
against podoplanin-expressing cancer cells because LpMab-

23 is a mouse IgG1 subclass, which does not exert ADCC. In
this study, we developed a human–mouse chimeric antibody,
chLpMab-23, from a mouse mAb, LpMab-23. chLpMab-23
reacted with LN319, CHO/podoplanin, and HSC-2 in a dosedependent manner (Figs. 2A, 3A, and 4A). chLpMab-23
showed ADCC against the CHO/podoplanin cells (Fig. 3C)
but not against the CHO-K1 cells, indicating that ADCC
against CHO/podoplanin is specific against podoplanin.
chLpMab-23 also showed ADCC against the LN319 glioblastoma cell line (data not shown). However, chLpMab-23
did not exert ADCC against malignant mesothelioma cell line
NCI-H226 or lung cancer cell line PC-10 (data not shown)because podoplanin expression in those cell lines may becomparatively low. Furthermore, we investigated thecomplement-dependent cytotoxicity (CDC) of chLpMab-23
against several podoplanin-expressing cancer cells. However, chLpMab-23 showed no CDC against podoplaninexpressing cancer cells (data not shown). In our previous

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FIG. 3. ADCC and antitumor effects of chLpMab-23 against CHO/podoplanin (A) CHO/podoplanin cells were treatedwith chLpMab-23 or NZ-12 (10 mg/mL–10 ng/mL, red) and PBS (black) for 30 min at 4C, followed by treatment withantihuman IgG–FITC. Fluorescence data were collected using Cell Analyzer EC800. (B) Western blot analysis with
chLpMab-23 or NZ-12. The membrane was incubated with 10 mg/mL of chLpMab-23 and then with peroxidase-conjugatedantihuman IgG; the membrane was detected using a Sayaca-Imager. AC-15 (anti-b-actin) was used as internal control. (C)
ADCC of chLpMab-23 or NZ-12 against CHO and CHO/podoplanin using human MNCs was evaluated using a 6-hour
51
Cr-release assay in the presence of 10 mg/mL of antibodies with an E/T ratio of 100. **p < 0.01 with Student’s t-test. (D)Antitumor effects of chLpMab-23 on tumor development of CHO/podoplanin xenograft. CHO/podoplanin cells (5 · 106
cells/100 mL) were subcutaneously inoculated into BALB/c nude mice. After 1 day, 100 mg of core fucose-negative
chLpMab-23 or control human IgG was injected into the peritoneal cavity of the mice. The antibodies were further injected
three times (days 4, 11, and 18; control group: n = 6; chLpMab-23 group: n = 6). NK cells were injected around the tumor ondays 4 and 11. The tumor diameter was measured at 3–4-day intervals and was calculated using the following formula:tumor volume = W2 · L/2, where W = short diameter and L = long diameter. ****p < 0.001 with two-way ANOVA. ADCC,
antibody-dependent cellular cytotoxicity; ANOVA, analysis of variance; MNCs, mononuclear cells; NK, natural killer.
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FIG. 4. Antitumor effects of chLpMab-23 on tumor development of HSC-2 xenograft, (A) HSC-2 cells were treated with
chLpMab-23 or NZ-12 (10 mg/mL–10 ng/mL, red) and PBS (black) for 30 min at 4C, followed by treatment with antihuman
IgG–FITC. Fluorescence data were collected using Cell Analyzer EC800. (B) HSC-2 cells (1 · 106 cells/100 mL) were
subcutaneously inoculated into BALB/c nude mice. After 1 day, 100 mg of chLpMab-23 or control human IgG was injected
into the peritoneal cavity of the mice. The antibodies were further injected six times (days 10, 14, 18, 22, 26, and 30; control
group: n = 8; chLpMab-23 group: n = 8). NK cells were injected around the tumor on days 10, 18, and 26. The tumor
diameter was measured at 3–4 day intervals and was calculated using the following formula: tumor volume = W2 · L/2,
where W = short diameter and L = long diameter. ***p < 0.005 with two-way ANOVA. (C) Comparison of the tumor size(day 35).
study, we showed that ADCC of anti-podoplanin mAbs ismuch more important for antitumor activity.(7,44) Therefore,
we moved on to the investigation of antitumor activity using
chLpMab-23.
To study the antitumor activity of chLpMab-23 on the
primary tumor growth of CHO/podoplanin cells in vivo, corefucose-negative-type chLpMab-23 was used because corefucose-negative mAbs show higher ADCC activity.(46)chLpMab-23 significantly reduced tumor development of
CHO/podoplanin on day 21 (Fig. 3D). We previously usednormal-type (core fucose-positive-type) chLpMab-23 for thesame xenograft model of CHO/podoplanin cells; however,chLpMab-23 did not reduce tumor development of CHO/
podoplanin xenograft. We further showed that normal-type
chLpMab-23 significantly reduced tumor development of
HSC-2 (Fig. 4B). The different results for the CHO/podoplanin and HSC-2 xenograft models may depend on thegrowth speed of each xenograft or the sensitivity of humanNK cells. We did not compare the antitumor activity of corefucose-negative-type and normal-type chLpMab-23 in the
HSC-2 xenograft model. In the near future, we should determine the best format of chLpMab-23 for antibody therapy
against podoplanin-expressing tumors.

Taken together, chLpMab-23 could be useful for antibody
therapy against podoplanin-expressing cancers. The CasMabs and chLpMab-23 developed by us could be applied tonovel antitumor reagents to provide strict specificity against
tumor cells.
Acknowledgments

We thank Kimiko Takeshita for the excellent technicalassistance. We also thank Junichi Takagi (Osaka University),
Takeshi Murata (Chiba University), Hiroaki Uchida (The
University of Tokyo), and Hideaki Tahara (The University ofTokyo) for their specialized advice. This work was supported
in part by the Basic Science and Platform Technology Program for Innovative Biological Medicine from Japan Agency
for Medical Research and development, 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 Number 26440019 (M.K.K.) and 16K10748(Y.K.), and by the Regional Innovation Strategy Support
Program from the Ministry of Education, Culture, Sports,

CHLPMAB-23 AGAINST PDPN

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 CR-17-05 and by the Grant for Joint Research
Project of the Institute of Medical Science, the University ofTokyo. The authors would like to thank Enago (www.enago.
jp) for the English language review.
Author Disclosure Statement

No competing financial interests exist.

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3. Kunita A, Kashima TG, Ohazama A, Grigoriadis AE, andFukayama M: Podoplanin is regulated by AP-1 and promotes platelet aggregation and cell migration in osteosarcoma. Am J Pathol 2011;179:1041–1049.
4. 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.
5. 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.
6. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T, andMatsutani M: Increased expression of podoplanin in malignant
astrocytic tumors as a novel molecular marker of malignant
progression. Acta Neuropathol (Berl) 2006;111:483–488.
7. 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.
8. 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.
9. 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.
10. Kato Y, Sasagawa I, Kaneko M, Osawa M, Fujita N, andTsuruo T: Aggrus: A diagnostic marker that distinguishes
seminoma from embryonal carcinoma in testicular germ
cell tumors. Oncogene 2004;23:8552–8556.
11. Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, and Osawa
M: Enhanced expression of Aggrus (T1alpha/podoplanin),111

a platelet-aggregation-inducing factor in lung squamouscell carcinoma. Tumor Biol 2005;26:195–200.
12. 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.
13. Kaneko MK, Kato Y, Kitano T, and Osawa M: Conservation of a platelet activating domain of Aggrus/podoplanin
as a platelet aggregation-inducing factor. Gene 2006;378:
52–57.
14. Herzog BH, Fu J, Wilson SJ, Hess PR, Sen A, McDaniel
JM, Pan Y, Sheng M, Yago T, Silasi-Mansat R, McGee S,
May F, Nieswandt B, Morris AJ, Lupu F, Coughlin SR,
McEver RP, Chen H, Kahn ML, and Xia L: Podoplaninmaintains high endothelial venule integrity by interactingwith platelet CLEC-2. Nature 2013;502:105–109.
15. Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima
K, Yatomi Y, Yamazaki Y, Narimatsu H, and Ozaki Y:
Involvement of the snake toxin receptor CLEC-2, in
podoplanin-mediated platelet activation, by cancer cells. J
Biol Chem 2007;282:25993–26001.
16. 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.
17. Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK,
Kato Y, and Yamaguchi Y: A platform of C-type lectin-likereceptor CLEC-2 for binding O-glycosylated podoplanin
and nonglycosylated rhodocytin. Structure 2014;22:1711–
1721.
18. Suzuki-Inoue K, Fuller GL, Garcia A, Eble JA, Pohlmann
S, Inoue O, Gartner TK, Hughan SC, Pearce AC, Laing
GD, Theakston RD, Schweighoffer E, Zitzmann N, Morita
T, Tybulewicz VL, Ozaki Y, and Watson SP: A novel Sykdependent mechanism of platelet activation by the C-type
lectin receptor CLEC-2. Blood 2006;107:542–549.
19. Ogasawara S, Kaneko MK, Price JE, and Kato Y: Characterization of anti-podoplanin monoclonal antibodies:
Critical epitopes for neutralizing the interaction between
podoplanin and CLEC-2. Hybridoma 2008;27:259–267.
20. Chaipan C, Steffen I, Tsegaye TS, Bertram S, Glowacka I,
Kato Y, Schmokel J, Munch J, Simmons G, GerardySchahn R, and Pohlmann S: Incorporation of podoplanin
into HIV released from HEK-293T cells, but not PBMC, isrequired for efficient binding to the attachment factor
CLEC-2. Retrovirology 2010;7:47.
21. Astarita JL, Cremasco V, Fu J, Darnell MC, Peck JR,
Nieves-Bonilla JM, Song K, Kondo Y, Woodruff MC,
Gogineni A, Onder L, Ludewig B, Weimer RM, Carroll
MC, Mooney DJ, Xia L, and Turley SJ: The CLEC-2podoplanin axis controls the contractility of fibroblastic
reticular cells and lymph node microarchitecture. Nat Immunol 2015;16:75–84.
22. Acton SE, Astarita JL, Malhotra D, Lukacs-Kornek V,
Franz B, Hess PR, Jakus Z, Kuligowski M, Fletcher AL,
Elpek KG, Bellemare-Pelletier A, Sceats L, Reynoso ED,
Gonzalez SF, Graham DB, Chang J, Peters A, Woodruff M,
Kim YA, Swat W, Morita T, Kuchroo V, Carroll MC, Kahn
ML, Wucherpfennig KW, and Turley SJ: Podoplanin-richstromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2. Immunity
2012;37:276–289.

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23. Chen G, Xu R, Yue B, Mei X, Li P, Zhou X, Huang S,
Gong L, and Zhang S: The expression of podoplaninprotein is a diagnostic marker to distinguish the early Infiltration of esophageal squamous cell carcinoma. Oncotarget 2017;8:19013–19020.
24. Atsumi N, Ishii G, Kojima M, Sanada M, Fujii S, andOchiai A: Podoplanin, a novel marker of tumor-initiating
cells in human squamous cell carcinoma A431. Biochem
Biophys Res Commun 2008;373:36–41.
25. Miyashita T, Higuchi Y, Kojima M, Ochiai A, and Ishii G:
Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity ofsquamous cell carcinoma cells. Sci Rep 2017;7:39971.
26. Morikawa M, Derynck R, and Miyazono K: TGF-beta andthe TGF-beta family: Context-dependent roles in cell andtissue physiology. Cold Spring Harb Perspect Biol 2016
May 2;8: pii: a021873.
27. Suzuki H, Kato Y, Kaneko MK, Okita Y, Narimatsu H, andKato M: Induction of podoplanin by transforming growth
factor-beta in human fibrosarcoma. FEBS Lett 2008;582:
341–345.
28. Takemoto A, Okitaka M, Takagi S, Takami M, Sato S,
Nishio M, Okumura S, and Fujita N: A critical role ofplatelet TGF-beta release in podoplanin-mediated tumour
invasion and metastasis. Sci Rep 2017;7:42186.
29. Kaneko MK, Oki H, Ogasawara S, Takagi M, and Kato Y:
Anti-podoplanin monoclonal antibody LpMab-7 detects
metastatic legions of osteosarcoma. Monoclon Antib Immunodiagn Immunother 2015;34:154–161.
30. Oki H, Kaneko MK, Ogasawara S, Tsujimoto Y, Liu X,
Sugawara M, Takakubo Y, Takagi M, and Kato Y: Characterization of a monoclonal antibody LpMab-7 recognizing non-PLAG domain of podoplanin. Monoclon AntibImmunodiagn Immunother 2015;34:174–180.
31. Ogasawara S, Oki H, Kaneko MK, Hozumi Y, Liu X,
Honma R, Fujii Y, Nakamura T, Goto K, Takagi M, andKato Y: Development of monoclonal antibody LpMab-10
recognizing non-glycosylated PLAG1/2 domain including
Thr34 of human podoplanin. Monoclon Antib Immunodiagn Immunother 2015;34:318–326.
32. Kato Y, Ogasawara S, Oki H, Honma R, Takagi M, Fujii Y,
Nakamura T, Saidoh N, Kanno H, Umetsu M, Kamata S,
Kubo H, Yamada M, Sawa Y, Morita K, Harada H, Suzuki
H, and Kaneko MK: Novel monoclonal antibody LpMab17 developed by CasMab technology distinguishes human
podoplanin from monkey podoplanin. Monoclon AntibImmunodiagn Immunother 2016;35:109–116.
33. Sekiguchi T, Takemoto A, Takagi S, Takatori K, Sato S,
Takami M, and Fujita N: Targeting a novel domain in
podoplanin for inhibiting platelet-mediated tumor metastasis. Oncotarget 2016;7:3934–3946.
34. Takagi S, Oh-hara T, Sato S, Gong B, Takami M, andFujita N: Expression of Aggrus/podoplanin in bladdercancer and its role in pulmonary metastasis. Int J Cancer
2014;134:2605–2614.
35. Takagi S, Sato S, Oh-hara T, Takami M, Koike S, Mishima
Y, Hatake K, and Fujita N: Platelets promote tumor growthand metastasis via direct interaction between Aggrus/
podoplanin and CLEC-2. PLoS One 2013;8:e73609.
36. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
37. Kato Y, Ogasawara S, Oki H, Goichberg P, Honma R, Fujii
Y, and Kaneko MK: LpMab-12 established by CasMab

KANEKO ET AL.

technology specifically detects sialylated O-glycan on
Thr52 of platelet aggregation-stimulating domain of human
podoplanin. PLoS One 2016;11:e0152912.
38. Ogasawara S, Kaneko MK, and Kato Y: LpMab-19 recognizes sialylated O-glycan on Thr76 of human podoplanin.
Monoclon Antib Immunodiagn Immunother 2016;35:245–
253.
39. Kaneko MK, Nakamura T, Honma R, Ogasawara S, Fujii
Y, Abe S, Takagi M, Harada H, Suzuki H, Nishioka Y, andKato Y: Development and characterization of antiglycopeptide monoclonal antibodies against human podoplanin, using glycan-deficient cell lines generated by
CRISPR/Cas9 and TALEN. Cancer Med 2017;6:382–396.
40. Oki H, Ogasawara S, Kaneko MK, Takagi M, Yamauchi M,
and Kato Y: Characterization of monoclonal antibody
LpMab-3 recognizing sialylated glycopeptide of podoplanin. Monoclon Antib Immunodiagn Immunother 2015;34:
44–50.
41. Kaneko MK, Yamada S, Nakamura T, Abe S, Nishioka Y,
Kunita A, Fukayama M, Fujii Y, Ogasawara S, and Kato Y:
Antitumor activity of chLpMab-2, a human–mouse chimeric cancer-specific antihuman podoplanin antibody, via
antibody-dependent cellular cytotoxicity. Cancer Med
2017;6:768–777.
42. Yamada S, Kaneko MK, and Kato Y: LpMab-23: A cancerspecific monoclonal antibody against human podoplanin.
Monoclon Antib Immunodiagn Immunother 2017;36:72–76.
43. Hayatsu N, Ogasawara S, Kaneko MK, Kato Y, and Narimatsu H: Expression of highly sulfated keratan sulfate
synthesized in human glioblastoma cells. Biochem Biophys
Res Commun 2008;368:217–222.
44. Abe S, Kaneko MK, Tsuchihashi Y, Izumi T, Ogasawara S,
Okada N, Sato C, Tobiume M, Otsuka K, Miyamoto L,
Tsuchiya K, Kawazoe K, Kato Y, and Nishioka Y: Antitumor effect of novel anti-podoplanin antibody NZ-12
against malignant pleural mesothelioma in an orthotopic
xenograft model. Cancer Sci 2016;107:1198–1205.
45. Kato Y, Kunita A, Abe S, Ogasawara S, Fujii Y, Oki H,
Fukayama M, Nishioka Y, and Kaneko MK: The chimericantibody chLpMab-7 targeting human podoplanin suppresses pulmonary metastasis via ADCC and CDC rather
than via its neutralizing activity. Oncotarget 2015;6:36003–
36018.
46. Niwa R, Natsume A, Uehara A, Wakitani M, Iida S, Uchida
K, Satoh M, and Shitara K: IgG subclass-independent improvement of antibody-dependent cellular cytotoxicity byfucose removal from Asn297-linked oligosaccharides. J
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Address correspondence to:
Dr. Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: February 26, 2017
Accepted: April 5, 2017