Oncotarget, Vol. 6, No. 34

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The chimeric antibody chLpMab-7 targeting human podoplaninsuppresses pulmonary metastasis via ADCC and CDC rather thanvia its neutralizing activity
Yukinari Kato1,*, Akiko Kunita2,*, Shinji Abe3,*, Satoshi Ogasawara1, Yuki Fujii1,
Hiroharu Oki1, Masashi Fukayama2, Yasuhiko Nishioka4, Mika K. Kaneko1
1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai, Miyagi 980-8575, Japan2

Department of Pathology, Graduate School of Medicine, the University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan3

 epartment of Clinical Pharmacy Practice Pedagogy, Institute of Biomedical Sciences, Tokushima University Graduate
D
School, Tokushima 770-8505, Japan4

 epartment of Respiratory Medicine and Rheumatology, Institute of Biomedical Sciences, Tokushima University Graduate
D
School, Tokushima 770-8503, Japan*

These authors have contributed equally to this work

Correspondence to:
Yukinari Kato, e-mail: yukinari-k@bea.hi-ho.ne.jp, yukinarikato@med.tohoku.ac.jp
Keywords: podoplanin, PDPN, metastasis, monoclonal antibody, ADCC/CDC
Received: June 07, 2015      Accepted: September 14, 2015      Published: September 25, 2015

ABSTRACT
Podoplanin (PDPN/Aggrus/T1α) binds to C-type lectin-like receptor-2 (CLEC-2)
and induces platelet aggregation. PDPN is associated with malignant progression,
tumor metastasis, and poor prognosis in several types of cancer. Although many antihuman PDPN (hPDPN) monoclonal antibodies (mAbs), such as D2-40 and NZ-1, havebeen established, these epitopes are limited to the platelet aggregation-stimulating(PLAG) domain (amino acids 29-54) of hPDPN. Recently, we developed a novel mouseanti-hPDPN mAb, LpMab-7, which is more sensitive than D2-40 and NZ-1, using theCancer-specific mAb (CasMab) method. The epitope of LpMab-7 was shown to be
entirely different from that of NZ-1, a neutralizing mAb against the PLAG domain
according to an inhibition assay and lectin microarray analysis. In the present study, we
produced a mouse-human chimeric anti-hPDPN mAb, chLpMab-7. ChLpMab-7 showed
high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent
cytotoxicity (CDC). Furthermore, chLpMab-7 inhibited the growth of hPDPN-expressing
tumors in vivo. Although chLpMab-7 recognizes a non-PLAG domain of hPDPN, it
suppressed the hematogenous metastasis of hPDPN-expressing tumors. These
results indicated that chLpMab-7 suppressed tumor development and hematogenous
metastasis in a neutralization-independent manner. In conclusion, hPDPN shows
promise as a target in the development of a novel antibody-based therapy.

(CAFs) has been associated with poor prognosis inpatients with cancer [17–21]. We previously compared themigration activities of PDPN-transfected osteosarcoma
cells and parental cells and found that PDPN-transfected
osteosarcoma cells exhibited a higher migration activity[14]. In solid tumors such as brain tumors, only a small andphenotypically distinct subset of cells can be responsible
for generating and sustaining tumors, and these cells are
considered cancer stem cells [22]. Because cancer stem
cells are thought to be resistant to conventional therapies
and are responsible for relapse, targeting cancer stem cells

INTRODUCTION
Podoplanin (PDPN/Aggrus/T1α) is a platelet
aggregation-inducing type I transmembrane O-glycoprotein[1–3]. The expression of human PDPN (hPDPN; 162amino acids) has been reported in many cancers, including
oral cancers, malignant brain tumors, esophageal cancers,lung cancers, malignant mesotheliomas, bladder cancers,
testicular tumors, and osteosarcomas [2, 4–16]. The
expression of hPDPN in cancer-associated fibroblasts
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PDPN has been reported to be a cancer stem cell marker[24]; therefore, immunotherapy using specific antibodies
against hPDPN may eradicate cancer stem cells in cancers.
PDPN has been reported to possess important
physiological functions. For the embryonic separationof blood-lymphatic blood vessels, interaction with
PDPN-C-type lectin-like receptor-2 (CLEC-2) andplatelet aggregation are critical [25]. We recently
performed crystallographic studies of hPDPN boundto CLEC-2 [26]. The interaction with CLEC-2 wasprimarily observed at Glu47 and Asp48 in the platelet
aggregation-stimulating (PLAG) domain (amino acids
29–54) and the α2–6 linked sialic acid at Thr52 of
hPDPN. The development of ectopic lymphoid follicles
is also dependent on PDPN, which is expressed in Th17
cells [27]. PDPN binds to CLEC-2 and rearranges theactin cytoskeleton in dendritic cells to promote efficientmobility along stromal surfaces [28]. The signaling
pathway triggered by the interaction of PDPN with
CLEC-2 controls the contractility of fibroblastic reticular
cells and lymph node microarchitecture [29]. CLEC2-expressing dendritic cells control the tension of the
PDPN-expressing fibroblastic reticular network andlymph node expansion [30].
In this study, we investigated whether a novel
mouse-human chimeric anti-hPDPN monoclonal antibody(mAb), chLpMab-7, shows ADCC and CDC activities
against hPDPN-possessing tumors, inhibits the growth of
hPDPN-expressing tumors, and suppresses hematogenous
metastases in a neutralization-independent manner.

moderately detected poly LacNAc binders (LEL, STL).
In contrast, NZ-1 strongly detected sialic acid ± core1
binders (ABA, Jacalin, ACA, MPA) and a sialo-mucinbinder (WGA) but weakly detected another sialo-mucinbinder (MAH). These results indicate that LpMab-7 canbe used for detecting different glycan profiles on hPDPN,
which has not been observed in anti-PLAG domain mAbs
such as NZ-1.

Production and characterization of a chimericanti-hPDPN antibody, chLpMab-7
We next generated a mouse-human chimericantibody (chLpMab-7) by fusing the VH and VL
regions of a mouse mAb (LpMab-7) with the CH and
CL regions of human IgG1, respectively. Both LpMab-7
and chLpMab-7 recognized hPDPN in LN229/hPDPN
and CHO/hPDPN cells, whereas neither mAb reactedwith the parental cells (Figure 2A and 2B), indicatingthat both mAbs specifically target hPDPN. The epitopeof chLpMab-7 is Arg79-Leu83 of hPDPN, which isconsistent with the epitope of LpMab-7 (Figure 2C).
The reaction of LpMab-7 and chLpMab-7 against
hPDPN was compared using hPDPN-expressing
cancer cell lines and normal cell lines. Previous
reports demonstrated that hPDPN is expressed in thehuman glioblastoma cell line LN319, the human lungsquamous cell carcinoma cell line PC-10, the human
mesothelioma cell line NCI-H226, the kidney cellline HEK-293T, lymphatic endothelial cells, and themesothelial cell line Met-5A [7, 9, 31, 33–36]. Both
LpMab-7 and chLpMab-7 similarly reacted with cancer
cell lines, such as LN319, PC-10, and NCI-H226 andnormal cell lines, including HEK-293T, lymphatic
endothelial cells, and Met-5A, as revealed by flow
cytometry (Figure 3). Because the binding affinity ofantibodies is critical for antibody-based cancer therapy,
the apparent dissociation constant (KD) was next
determined using flow cytometric analysis. As shownin Figure 4, the KD of chLpMab-7 was determined tobe 7.4 × 10−9 M against LN319 cells (Figure 4A) and5.1 × 10−9 M against CHO/hPDPN cells (Figure 4B)
by flow cytometry. In contrast, the KD of LpMab-7 wasdetermined to be 3.3 × 10−8 M against LN319 cells(Figure 4A) and 1.6 × 10−8 M against CHO/hPDPN
cells (Figure 4B), indicating that the binding affinity of
chLpMab-7 is greater than that of LpMab-7.

RESULTS
Characterization of the LpMab-7 epitope
We previously produced a novel anti-hPDPN
mAb, LpMab-7 (IgG1, kappa) [31], and identified theminimum epitope of LpMab-7 as Arg79-Leu83 of
hPDPN (162 amino acids), which is distinct from the
PLAG domain [32]. As shown in Figure 1A, the epitopeof LpMab-7 is not the PLAG domain, whereas that of
NZ-1, a neutralizing mAb, is the PLAG domain. In thepresent study, we further characterized the epitope of
LpMab-7 using inhibition assay and lectin microarray.
We first determined whether LpMab-7 inhibits hPDPNCLEC-2 interaction using ELISA. As a positive control,a rat anti-hPDPN mAb, NZ-1, was used. As shown in
Figure 1B, LpMab-7 did not block the binding of CLEC2-Fc to hPDPN-Fc, whereas NZ-1 significantly inhibitedthe interaction. Therefore, the epitope of LpMab-7 isentirely different from that of NZ-1. We further performed
an antibody-overlay lectin microarray analysis of hPDPN
using LpMab-7 and NZ-1 (Figure 1C). LpMab-7 faintly
detected sialic acid ± core1 binders (Jacalin, ACA,MPA), weakly detected a sialo-mucin binder (WGA) and
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ADCC and CDC are mediated by chLpMab-7
To develop targeted therapy against hPDPN,
we assessed whether chLpMab-7 can induce ADCC
against PDPN-expressing cell lines mediated byhuman mononuclear cells (MNC), including NK
cells and macrophages, as effector cells. As shownin Figure 5A, ADCC by chLpMab-7 was observed
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Figure 1: Characterization of the LpMab-7 epitope. A. Comparison of epitopes of anti-hPDPN mAbs. PLAG, platelet aggregation-

stimulating domain; SA, sialic acid; Gal, galactose; GalNAc, N-acetylgalactosamine. The minimum epitopes of NZ-1 and LpMab-7 are
MPGAEDD and RIEDL, respectively. B. The hPDPN-CLEC-2 interaction was blocked by NZ-1 but not by LpMab-7. Inhibition assay
was performed using ELISA. Purified hPDPN-Fc was immobilized at 1 μg/ml. After blocking, LpMab-7 or NZ-1 was added at 1 μg/ml.
The plates were incubated with biotinylated CLEC-2-Fc (1 μg/ml) followed by 1:1000-diluted peroxidase-conjugated streptavidin.
The enzymatic reaction was conducted with a 1-Step Ultra TMB-ELISA. The optical density was measured at 655 nm. C. Antibody-overlay
lectin microarray. Purified hPDPN was applied to a lectin array. After incubation, human IgG was applied to each well for blocking. Then,biotinylated LpMab-7 and NZ-1 were applied and Cy3-labeled streptavidin was added. The glass slide was scanned using a GlycoStation
Reader 1200.
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Figure 2: Flow cytometry of hPDPN-transfected cells using anti-hPDPN antibodies LpMab-7 and chLpMab-7. Cells

were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA. After washing with PBS, the cells were treated with LpMab-7 A. andchLpMab-7 B. followed by treatment with secondary antibodies. Fluorescence data were collected using a Cell Analyzer EC800. C. The
epitope mapping of chLpMab-7 by Western blot analysis. Total cell lysates of point mutants of hPDPN were electrophoresed on 5–20%
polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/ml mAbs (chLpMab-7
or NZ-8 against hPDPN, 1E5 against FLAG tag, AC-15 against β-actin) and then with peroxidase-conjugated secondary antibodies; themembrane was analyzed using a Sayaca-Imager.
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Figure 3: Flow cytometry of endogenous hPDPN-expressing cell lines using anti-hPDPN antibodies LpMab-7 andchLpMab-7. Cancer cells A. or normal cells B. were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA. After washing with
PBS, the cells were treated with LpMab-7 and chLpMab-7 followed by treatment with secondary antibodies. Fluorescence data werecollected using a Cell Analyzer EC800.

Anti-tumor effect of chLpMab-7 on primarytumor development and spontaneous lungmetastasis in nude mice inoculated with hPDPNexpressing cells

against LN319, PC-10, NCI-H226, and CHO/hPDPN
cells. ChLpMab-7 showed high ADCC activity against
LN319 glioblastoma cells (42.6%), CHO/hPDPN
cells (43.7%), PC-10 lung cancer cells (40.5%), and
NCI-H226 malignant mesothelioma cells (37.5%).
We next investigated whether chLpMab-7 can induce
CDC against hPDPN-expressing cell lines (Figure 5B).
ChLpMab-7 showed high CDC activity against LN319
glioblastoma cells (56.4%) and CHO/hPDPN cells(52.5%) but only moderate CDC activity against PC10 lung cancer cells (24.9%) and NCI-H226 malignant
mesothelioma cells (30.8%). Western blot analysis
showed that LN319 and CHO/hPDPN cells expressedhigher levels of hPDPN compared with PC-10 and
NCI-H226 cells (Figure 5C).

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To investigate the anti-tumor activity of
chLpMab-7 on primary tumor growth in vivo, CHO/
hPDPN cells were implanted subcutaneously into theflanks of nude mice. ChLpMab-7 or control human
IgG antibodies were injected into the peritoneal cavityof mice once a week for three weeks (n = 10 each).
Tumor formation was observed in all human IgG groupmice (tumor incidence: 100%: 10/10; Figure 6A).
In contrast, chLpMab-7 dramatically reduced thetumor development (tumor incidence: 10%: 1/10).

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Figure 4: Determination of the binding affinity using flow cytometry. LN319 and CHO/hPDPN cells (2 × 105) were resuspended

at 100 μl of serially diluted LpMab-7 A. or chLpMab-7 B. (0.02–100 μg/ml). Fluorescence data were collected using a Cell Analyzer
EC800. KD was obtained by fitting the binding isotherms using the built-in one-site binding models in Prism software.

Anti-tumor effect of chLpMab-7 against
endogenous hPDPN-expressing cells

Tumor volume and tumor weight were significantly
reduced by chLpMab-7 treatment (Figure 6B and 6C).
Furthermore, lung weight was reduced by chLpMab-7
treatment (Figure 6D). The primary tumor formation
was confirmed by HE staining and immunostaining(Figure 6E). Immunohistochemistry with an antihPDPN antibody (NZ-1) showed membranous staining
in tumor cells. Furthermore, HE staining revealed thespontaneous formation of lung micrometastases, which
was observed in 6 of 10 control mice (60%) and was
remarkably reduced to 10% (1/10) by chLpMab-7
treatment (Figure 6F and 6G). Lung micrometastasis
foci were confirmed by immunostaining against hPDPN.
The expression of hPDPN was maintained in the lungmetastasis (Figure 6G, inset). These results indicate thatthe administration of chLpMab-7 inhibited the primarytumor growth of CHO/hPDPN cells and reduced
spontaneous lung metastasis.
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To further investigate the anti-tumor effect of
chLpMab-7 on endogenous hPDPN-expressing cells
in vivo, PC-10 cells were implanted subcutaneously into
the flanks of nude mice. ChLpMab-7 or control human
IgG antibodies were injected into the peritoneal cavityof mice once a week (n = 6 each). Tumor formation was
evenly observed in both groups of mice for 29 days,
although chLpMab-7 was injected four times, indicatingthat the CDC activity of chLpMab-7 was not sufficient
to inhibit tumor growth in the PC-10 xenograft model(Figure 7A). ChLpMab-7 does not exhibit ADCC activity
in the mouse model; therefore, human NK cells were
injected around the tumors at Day 29 and Day 36 to induce
ADCC activity together with i.p. injection of chLpMab-7.
Tumor volume was significantly reduced by chLpMab-7
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Figure 5: ADCC and CDC activities of chLpMab-7. A. ADCC activities induced by human MNC against hPDPN-expressing celllines were determined using a 6 h 51Cr release assay at the E/T ratio of 100 in the presence of 1 μg/ml chLpMab-7 and human IgG. B. CDC
activities against hPDPN-expressing cell lines were demonstrated by 51Cr release assay. *P < 0.05, **P < 0.01 versus control (values aremeans ± SEM). C. Western-blot analysis. Total cell lysates were electrophoresed on 5–20% polyacrylamide gels and transferred onto a
PVDF membrane. After blocking, the membrane was incubated with 1 μg/ml mAbs (NZ-1 against hPDPN, 1E5 against FLAG tag, AC-15
against β-actin) and then with peroxidase-conjugated secondary antibodies; the membrane was analyzed using a Sayaca-Imager.
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Figure 6: Anti-tumor effect of chLpMab-7 on primary tumor development and spontaneous lung metastasis in nude
mice inoculated with hPDPN-expressing cells. CHO/hPDPN cells (3 × 106 cells/100 μl) were inoculated subcutaneously into

BALB/c nude mice. After one day, 100 μg of chLpMab-7 or human IgG antibodies was injected into the peritoneal cavity of mice. The
antibodies were injected once per week for three weeks (human IgG group: n = 10; chLpMab-7 group: n = 10). A. Comparison of thetumor size and tumor incidence in nude mice (day 30). B. Primary tumor growth in human IgG- or chLpMab-7-treated mice. ***p < 0.001;****p < 0.0001 by two-way ANOVA. C. Primary tumor weight of human IgG or chLpMab-7 treated mice. ****p < 0.0001. D. Lung weightof human IgG- or chLpMab-7 treated mice. *p < 0.05. E. HE staining and immunostaining with NZ-1 against the primary tumor formed byinjecting human IgG after inoculating CHO/hPDPN cells subcutaneously. Bars, 100 μm. F. Spontaneous metastasis of CHO/hPDPN cells
was inhibited by chLpMab-7 treatment. Incidence and number of metastases per lung following s.c. inoculation. *p < 0.05. G. HE stainingof the lung from human IgG or chLpMab-7-treated mice. The inset shows the higher magnification image of immunostaining of the lungs
for hPDPN. Bars, 500 μm.
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Figure 7: Anti-tumor effect of chLpMab-7 against PC-10 xenografts. PC-10 cells (5 × 106 cells/100 μl) were inoculated
subcutaneously into BALB/c nude mice. After one day, 100 μg of chLpMab-7 or human IgG antibodies was injected into the peritoneal
cavity of mice. The antibodies were injected once per week (human IgG group: n = 6; chLpMab-7 group: n = 6). Human NK cells were
injected around the tumors at Day 29 and Day 36. A. Primary tumor growth in human IgG- or chLpMab-7-treated mice. *p < 0.05;****p < 0.0001 by two-way ANOVA. B. Comparison of the tumor size and tumor incidence in nude mice (Day 53). C. Gross morphologyof the xenograft tumor. D. Primary tumor weight of human IgG- or chLpMab-7 treated mice. ** p < 0.01.
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the ADCC activity of chLpMab-7 was induced effectively(Figure 7A, 7B, and 7C). Tumor weight was significantly
reduced by chLpMab-7 treatment (Figure 7D). These
results indicate that the administration of chLpMab-7
inhibited the primary tumor growth of PC-10 cells.

expression, we used LN229 glioma cells, which exhibit
cancer-type glycan patterns, including aberrant sialylationand highly sulfated polylactosamine. We immunized
mice with LN229/hPDPN cells and developed LpMab-2,
a CasMab. At the same time, we could produce manyother anti-hPDPN mAbs. Some of these mAbs areglycopeptide-recognizing mAbs (GpMabs), such as
LpMab-3 and LpMab-9, and others are highly sensitivemAbs, such as LpMab-7. We named this advantageousmAb-producing method the “CasMab method.” LpMab-7
was shown to be more sensitive than NZ-1 and D2–40 byimmunohistochemistry [39].
We previously showed that the epitope of LpMab-7
is Arg79-Leu83 of hPDPN and that Ile80, Glu81, andLeu83 are the most important amino acids [32]. In thepresent study, we also demonstrated that the epitopeof LpMab-7 is entirely different from that of NZ-1, aneutralizing mAb against the PLAG domain, using an
inhibition assay. Unfortunately, the subclass of LpMab-7
is mouse IgG1, which does not induce ADCC and CDC;
therefore, LpMab-7 was converted to the human IgG1
subclass.
The binding affinity of chLpMab-7 was better
than that of the original mouse LpMab-7, althoughthose variable regions are the same. Our previous study
demonstrated that rat-human chimeric NZ-1, which was
designated as NZ-8, showed a lower affinity comparedwith NZ-1: the KD of NZ-1 was determined to be 7.2 × 10−9
M by flow cytometry or 4.1 × 10−10 M by ELISA, whereas
the KD of NZ-8 was determined to be 7.6 × 10−8 M byflow cytometry or 1.8 × 10−9 M by ELISA [40], although
the same constant regions of heavy chain (human IgG1)
and light chain (kappa) were used for both chLpMab-7
and NZ-8. We have characterized many human-mousechimeric mAbs and compared their affinities with thoseof the original mouse mAbs; however, almost the same
binding affinities have been obtained (data not shown).
Therefore, we have thus far been unable to determine thereason why the binding affinity of chLpMab-7 improved
after the conversion of the constant region from mouse
IgG1 to human IgG1.
ChLpMab-7 not only possesses high binding
affinity but also showed high ADCC and CDC activities
against hPDPN-expressing cancer cells, suggesting that
chLpMab-7 offers more advantages for antibody-based
molecular targeting therapy. LpMab-7 showed high
ADCC activities against all cancer cells; in contrast,
it showed higher CDC activities against the LN319
glioblastoma cell line than against PC-10 lung cancer
cells and NCI-H226 malignant mesothelioma cells, most
likely because i) LpMab-7 was produced by immunizing
mice with LN229/hPDPN glioblastoma cells; ii) the posttranslational modification, including the glycosylation of
hPDPN, is different among cancer cells [31]; or iii) the
expression level of hPDPN in LN319 is much higher
than that of the other cell lines. Indeed, Western blot

Suppression of experimental lung metastasis by
chLpMab-7
We next investigated whether chLpMab-7
antibodies could suppress hPDPN-induced pulmonary
metastasis in an experimental metastasis model. The
injection of CHO/hPDPN cells led to the developmentof multiple lung metastatic foci (5/5: 100%, Figure 8A).
ChLpMab-7 antibodies were administered concomitantlywith the cell injection (Day 0) or one day (Day 1) or 5days (Day 5) after cell injection. As a result, visible lungmetastases were dramatically reduced by the chLpMab-7
treatment (Figure 8A). Lung metastasis was completely
blocked by the chLpMab-7 treatment concomitantly withthe cell injection (Day 0, Figure 8B). Treatment with
chLpMab-7 after the cell injection (Day 1 or Day 5) also
blocked metastasis. Lung weight was significantly reducedby chLpMab-7 treatment (Figure 8C). In addition, bodyweight was significantly improved in all chLpMab-7treated groups (Figure 8D). HE staining revealed thatmany metastatic foci were present in the lungs of thehuman IgG group, whereas much fewer metastatic fociwere observed in the chLpMab-7 groups (Figure 8E).
Immunostaining for hPDPN confirmed the presence oflung metastasis (Figure 8E, inset).

DISCUSSION
Although anti-hPDPN mAbs with high sensitivity
and specificity are required to investigate the physiological
function of hPDPN in cancers and normal tissues, almost
all anti-hPDPN mAbs have been developed against
the PLAG domains of hPDPN because the neutralizing
activities of anti-hPDPN mAbs have been reported to beimportant for targeting therapy against hPDPN [9, 35,37]. Using anti-hPDPN mAbs recognizing the PLAG
domain, the same glycan profile, such as the sialylcore1 structure, was observed by antibody-overlay lectin
microarray using anti-PLAG mAbs [9, 38]. In contrast,
LpMab-7, an anti-non-PLAG hPDPN mAb, can be used
for detecting different glycan profiles of hPDPN, including
the poly LacNAc structure (Figure 1C), indicating that
LpMab-7 may be advantageous for analyzing the novel
pathophysiological function of hPDPN in cancers andnormal tissues.
We recently established a platform to produce
cancer-specific mAbs (CasMabs) [31]. Briefly, themembranous proteins were transfected into cancer celllines, which express cancer-specific glycans. For hPDPN
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Figure 8: Suppression of experimental lung metastasis by chLpMab-7. CHO/hPDPN cells (5 × 105 cells/100 μl) were injected
intravenously into nude mice. Animals were assigned to 4 groups, which received chLpMab-7 or human IgG concomitantly with the cell
injection (Day 0), chLpMab-7 treatment 1 day after tumor cell injection (Day 1), or chLpMab-7 treatment 5 days after cell injection (Day 5).
A. Visible lung metastasis. B. Incidence and number of visible metastases per lung. C. Lung weight. D. Body weight. ***p < 0.001;****p < 0.0001. E. HE staining of the lung from each mouse. The inset shows the higher magnification image of immunostaining of thelungs for hPDPN. Bars, 1 mm.
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analysis showed that LN319 and CHO/hPDPN cells
express higher levels of hPDPN compared with PC-10
and NCI-H226 cells (Figure 5C). Although PC-10 cells
express a much higher level of hPDPN than NCI-H226
cells (Figure 5C), lower CDC activity was observed in PC10 cells compared with NCI-H226 cells, indicating that
ADCC/CDC activities may depend on several interrelated
factors. We investigated whether chLpMab-7 exerts effects
on cancer cell growth, sphere forming property, adhesion,and migration in vitro. However, chLpMab-7 produced noeffect on cancer cell growth (Supplementary Figure S1A),
sphere forming property (Supplementary Figure S1B),
adhesion (Supplementary Figure S2), or migration in vitro(Supplementary Figure S3).
It has been demonstrated that all anti-hPDPN
mAbs suppress hPDPN-induced pulmonary metastasis by
inhibiting platelet aggregation [13, 33, 41, 42]. Previous
studies showed that not only whole IgGs but also F(ab’)2
fragments almost completely suppressed the formationof pulmonary metastasis by hPDPN-expressing cells.
In contrast, whole IgGs, such as NZ-1 and NZ-8, clearly
demonstrated ADCC and CDC activities and inhibitedthe growth of hPDPN-expressing cells in vivo, indicatingthat the suppression of pulmonary metastasis might bedependent on both neutralizing activity and ADCC/CDC
activities. However, it has not been determined that ADCC
or CDC activities are sufficient for suppressing hPDPNinduced pulmonary metastasis because almost all antihPDPN mAbs have been produced against the anti-PLAG
domain (amino acids 29–54) of hPDPN. In this study, weinvestigated whether an anti-non-PLAG hPDPN domain
mAb can inhibit the pulmonary metastasis of hPDPNexpressing cells in a neutralization-independent manner.
The injection of chLpMab-7, which recognizes Arg79Leu83 of hPDPN, significantly blocked the formationof pulmonary metastasis even 5 days after the injectionof cancer cells, suggesting that the pulmonary metastasis of
hPDPN-expressing cells can be inhibited in a neutralizationindependent manner. In mouse models, chLpMab-7
can exhibit only CDC activity because mouse NK cells
or macrophages do not bind to human IgG1. Although
chLpMab-7 demonstrated a high anti-tumor activity in both
xenograft and experimental metastasis models of CHO/
hPDPN cells, only chLpMab-7 did not show an anti-tumor
effect in a PC-10 xenograft. After human NK cell injection
around a PC-10 xenograft together with i.p. injection ofchLpMab-7, the tumor volume was significantly reduced,
indicating that both ADCC and CDC activities were much
more toxic against hPDPN-expressing tumors.
In this study, we did not seek to minimize theimportance of the PDPN-CLEC-2 interaction for thetumor development and cancer metastasis. PDPN canbind to CLEC-2, the only endogenous receptor of
PDPN, and induce platelet aggregation. Several growth
factors, such as TGF-β and PDGF from activatedplatelets, may contribute to the tumor development in the
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microenvironment. Therefore, neutralizing mAbs mightalso be effective by targeting PDPN-expressing tumors.

MATERIALS AND METHODS
Cell lines and stable transfectants
Chinese hamster ovary (CHO)-K1, LN229,
NCI-H226, HEK-293T, and Met-5A cells were obtained
from the American Type Culture Collection (ATCC,
Manassas, VA). Human lymphatic endothelial cells(LECs) were obtained from Cambrex (Walkersville, MD).
PC-10 cells were purchased from Immuno-Biological
Laboratories Co., Ltd. (Gunma, Japan). The humanglioblastoma cell line LN319 was donated by Dr. Webster
K. Cavenee (Ludwig Institute for Cancer Research,
San Diego, CA). CHO/hPDPN and LN229/hPDPN
were established as described previously [2, 31]. CHO/
hPDPN, PC-10, and NCI-H226 cells were cultured in
RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan)
supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Thermo Fisher Scientific Inc., Waltham,MA), 2 mM L-glutamine (Thermo Fisher Scientific Inc.),
100 units/ml penicillin, 100 μg/ml streptomycin, and 25
μg/ml amphotericin B (Nacalai Tesque, Inc.) at 37°C
in a humidified atmosphere of 5% CO2 and 95% air.
Geneticin (0.5 mg/ml; Wako Pure Chemical Industries,
Ltd., Osaka, Japan) was added for CHO/hPDPN cells.
HEK-293T, LN229, LN229/hPDPN, LN319, and Met5A cells were cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM) (Nacalai Tesque, Inc.) supplemented
with 10% heat-inactivated FBS, 2 mM L-glutamine, 100
units/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml
amphotericin B at 37°C in a humidified atmosphere of 5%
CO2 and 95% air. MITO + serum extender (Thermo Fisher
Scientific Inc.) was added for Met-5A cells. Geneticin (0.5mg/ml) was added for LN229/hPDPN cells. LEC cells
were cultured in endothelial cell medium EGM-2MV
supplemented with 5% FBS (Cambrex).

Animals
Female BALB/c nude (nu/nu) mice (5–7-weekold) were purchased from Charles River Japan, Inc.
(Kanagawa, Japan). Animals were housed under pathogenfree conditions. The Animal Care and Use Committees of
the University of Tokyo approved the animal experiments
described herein.

Antibodies
LpMab-7, a mouse anti-hPDPN mAb (IgG1, kappa),
was developed as described previously [31]. Human IgGs
were purchased from Beckman Coulter, Inc. (Fullerton,
CA). For the generation of mouse-human chimeric antihPDPN (chLpMab-7), the appropriate VH and VL cDNAs
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Urtica dioica agglutinin; PWM, Pokeweed mitogen;
PNA, peanut agglutinin; WFA, Wisteria floribunda
agglutinin; ACA, Amaranthus caudatus agglutinin;
MPA, Maclura pomifera agglutinin; HPA, Helix pomatia
agglutinin; VVA, Vicia villosa agglutinin; DBA, Dolichos
biflorus agglutinin; SBA, Soybean agglutinin; PTL I,
Psophocarpus tetragonolobus lectin I; MAH, Maackia
amurensis hemagglutinin; WGA, wheat germ agglutinin;
GSL-I, Griffonia simplicifolia lectin I.

of a mouse LpMab-7 antibody and CH and CL of human
IgG1 were subcloned into the pCAG-Ble or pCAGNeo vectors (Wako Pure Chemical Industries, Ltd.),
respectively. Antibody expression vectors were transfected
into CHO-K1 cells using Lipofectamine LTX reagent(Thermo Fisher Scientific Inc.). Stable transfectants of
CHO-K1/chLpMab-7 were selected by culturing thetransfectants in medium containing 0.5 mg/ml geneticin
and 0.5 mg/ml zeocin (InvivoGen, San Diego, CA). NZ-1,
a rat anti-hPDPN mAb (IgG2a, lambda) was produced as
described previously [9]. 1E5, a mouse anti-FLAG mAb,
and AC-15, a mouse anti-β-actin mAb, were purchasedfrom Wako Pure Chemical Industries, Ltd., and SigmaAldrich Corp. (St. Louis, MO), respectively.

Flow cytometry
Cells were harvested by brief exposure to 0.25%
Trypsin/1 mM EDTA (Nacalai Tesque, Inc.). Afterwashing with PBS, the cells were treated with LpMab-7
and chLpMab-7 (1 μg/ml) for 30 min at 4°C followed
by incubation with Oregon green-conjugated anti-mouse
IgG or FITC-conjugated anti-human IgG (Thermo Fisher
Scientific Inc.). Fluorescence data were collected using a
Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

Neutralization assays of PDPN and CLEC-2
Inhibition assays were performed using enzymelinked immunosorbent assay (ELISA). Purified hPDPN-Fc
was immobilized on Nunc Maxisorp 96-well immunoplates(Thermo Fisher Scientific Inc.) at 1 μg/ml for 30 min. After
blocking with SuperBlock T20 (PBS) Blocking Buffer,
LpMab-7 or NZ-1 was added at 1 μg/ml for 30 min.
The plates were incubated with biotinylated CLEC-2Fc (1 μg/ml) followed by 1:1000-diluted peroxidaseconjugated streptavidin (GE Healthcare, Piscataway, NJ).
The enzymatic reaction was conducted with a 1-Step Ultra
TMB-ELISA (Thermo Fisher Scientific Inc.). The opticaldensity was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories Inc., Philadelphia, PA).

Determination of binding affinity by flow cytometry
Binding affinity was determined as described
previously [43]. Briefly, cells (2 × 105) were resuspended
in 100 μl of serially diluted antibodies (0.02–100 μg/ml),
followed by secondary antibodies (Thermo Fisher ScientificInc.). Fluorescence data were collected using a Cell
Analyzer EC800. The apparent dissociation constants (KD)were obtained by fitting the binding isotherms using builtin one-site binding models in Prism software (GraphPad
Software, San Diego, CA).

Lectin microarray

Preparation of effector cells

CHO/hPDPN cells were solubilized using 1%
Triton-X100 in PBS (PBST), and hPDPN was purified
using a FLAG-tag system (Sigma-Aldrich Corp.). Then,
100 μl of purified hPDPN (31.25–2000 ng/ml) wasapplied to a lectin array (LecChip ver1.0; GlycoTechnica,
Hokkaido, Japan), including triplicate spots of 45 lectins
in each of seven divided incubation baths on the glass
slide. After incubation at 20°C for 17 h, 4 μl of human
IgG (5 mg/ml; Sigma-Aldrich Corp.) was applied to eachwell. After incubation at 20°C for 30 min, the glass slide
was washed three times with PBST; 60 μl of biotinylated
LpMab-7 and NZ-1 (1 μg/ml) in PBS were applied to thearray and incubated at 20°C for 3 h. After washing threetimes with PBST, Cy3-labeled streptavidin (Thermo Fisher
Scientific Inc.) was added to the array and incubated at
20°C for 30 min. The glass slide was scanned using a
GlycoStation Reader 1200 (GlycoTechnica). Abbreviationof lectins: GNA, Galanthus nivalis agglutinin; HHL,
Hippeastrum hybrid lectin; ACG, Agrocybe cylindracea
galectin; TxLCI, Tulipa gesneriana lectin; BPL,
Bauhinia purpurea alba lectin; TJA-II, Trichosanthes
japonica agglutinin; EEL, Euonymus europaeus lectin;
ABA, Agaricus bisporus agglutinin; LEL, Lycopersiconesculentum lectin; STL, Solanum tuberosum lectin; UDA,
www.impactjournals.com/oncotarget

Human peripheral blood mononuclear cells (MNCs)
were obtained from the peripheral blood of healthy donors
as described previously [44]. The human study wasapproved by the ethics committee of the University ofTokushima, and written informed consent was obtained
from all study participants. Human natural killer cells
were purchased from Takara Bio Inc. (Shiga, Japan).

Antibody-dependent cellular cytotoxicity(ADCC) assay
ADCC was evaluated using a 51Cr release assay
as described previously [45]. Target cells were labeledwith 0.1 μCi of 51Cr-sodium chromate at 37°C for 1 h.
51
Cr-labeled target cells were placed in 96-well plates
in triplicate, to which effector cells and antibodies(chLpMab-7 and human IgG) were added. After a
6 h incubation, the radioactivity present in 100 μlof supernatant was measured in a gamma counter(PerkinElmer, Waltham, MA). The percentage ofcytotoxicity was calculated using the following formula:% Specific lysis = (E – S)/(M – S) × 100, where E is the36015

Oncotargetrelease in the test sample, S is the spontaneous release, andM is the maximum release.

concentration of 1 μg/ml, and secondary biotin-conjugatedanti-rat antibody (Vector Laboratories, Peterborough, UK)
was used at 1:200 dilution.

Complement-dependent cytotoxicity (CDC) assay

Anti-tumor activity of anti-hPDPN antibodies on
PC-10 cells

CDC was determined by 51Cr release assay [45].
Target cells were labeled with 0.1 μCi of 51Cr-sodium
chromate at 37°C for 1 h. After incubation, the cells were
washed with medium three times. 51Cr-labeled cells wereadded to 96-well plates and incubated with baby rabbit
complement and chLpMab-7 and human IgG for 6 h.
51
Cr release in the supernatant from each well (100 μl)
was measured using a gamma counter. The percentage ofcytotoxicity was calculated as above.

PC-10 cells were suspended using trypsin, washedand suspended with PBS, adjusted to a density of 5 × 107
cells/ml, and inoculated subcutaneously into BALB/c nude
mice at a dose of 100 μl/animal. After one day, 100 μl of
1 mg/ml chLpMab-7 or human IgG were respectively
injected into the peritoneal cavity of mice, and theantibodies were injected once per week for 50 days (control
group: n = 6; chLpMab-7 group: n = 6). Human NK cells(5 × 105) were injected around the tumors at Day 29 andDay 36. The tumor diameter was measured at intervals of
three to four days and calculated using the same formula
as above. On Day 53 after cell implantation, the mice wereeuthanized and primary tumor tissues were harvested.

Western blot analyses
Cell lysates (10 μg) were boiled in SDS sample
buffer (Nacalai Tesque, Inc.). The proteins were
electrophoresed on 5–20% polyacrylamide gels (Wako
Pure Chemical Industries Ltd.) and transferred onto a
PVDF membrane (EMD Millipore Corp., Billerica, MA).
After blocking with 4% skim milk (Nacalai Tesque, Inc.),
the membrane was incubated with primary antibodies, thenwith peroxidase-conjugated secondary antibodies (Dako;
1/1000 diluted), and developed with Pierce Western
Blotting Substrate Plus (Thermo Fisher Scientific Inc.) or
ImmunoStar LD Chemiluminescence Reagent (Wako Pure
Chemical Industries Ltd.) using a Sayaca-Imager (DRC
Co. Ltd., Tokyo, Japan).

Experimental lung metastasis
CHO/hPDPN cells were harvested, washed, andresuspended in PBS (5 × 106 cells/ml). The cells wereincubated with chLpMab-7 or human IgG and inoculated
intravenously (5 × 105 cells/mouse) into the lateral tailvein of female BALB/c-nu/nu mice. In some groups,
chLpMab-7 antibodies were inoculated either 1 day or
5 days after cell injection into the tail vein. After 17 days,
the mice were euthanized and the surface lung metastatic
foci was counted and measured. The lung tissues from
CHO/hPDPN-bearing mice were processed for HE staining.

Anti-tumor activity of anti-hPDPN antibodies on
CHO/hPDPN cells

Statistical analyses

CHO/hPDPN cells were suspended using trypsin,washed and suspended with PBS, adjusted to a densityof 3 × 107 cells/ml, and inoculated subcutaneously into
BALB/c nude mice at a dose of 100 μl/animal. After one
day, 100 μl of 1 mg/ml chLpMab-7 or human IgG wererespectively injected into the peritoneal cavity of mice,and the antibodies were injected once per week for three
weeks (control group: n = 10; chLpMab-7 group: n = 10).
The tumor diameter was measured at intervals of three tofour days and was calculated using the following formula:tumor volume = W2 × L/2, where W = short diameter and
L = long diameter. On Day 30 after cell implantation, themice were euthanized and the lungs and primary tumor
tissues harvested for hematoxylin and eosin (HE) staining.

All data are shown as means ± SEM. Student’s t test,
Mann-Whitney U-test, one-way ANOVA followed by TukeyKramer multiple comparisons, and two-way ANOVA were
performed as indicated. P values < 0.05 were considered
statistically significant. All statistical tests were two-sided.

ACKNOWLEDGMENTS AND FUNDING
We thank Xing Liu, Ryusuke Honma, Yuta
Tsujimoto, Takuro Nakamura, Noriko Saidoh, Kanae
Yoshida, Kimiko Takeshita, and Kei Sakuma for their
excellent technical assistance. This work was supported
in part by the Platform for Drug Discovery, Informatics,
and Structural Life Science (PDIS) from Japan Agency
for Medical Research and development, AMED (Y.K.);
by the Basic Science and Platform Technology Programfor Innovative Biological Medicine from AMED (Y.K.);
by the Regional Innovation Strategy Support Programfrom the Ministry of Education, Culture, Sports, Scienceand Technology (MEXT) of Japan (Y.K.); and by JSPS
KAKENHI Grant Number 25462242 (Y.K.), Grant

Immunohistochemical analyses
All tissue samples were fixed in formalin andembedded in paraffin, and sections were cut at 3 μm
thickness. Immunostaining was performed according tostandard techniques using an autostainer (BenchMark
XT; Ventana Medical Systems, Inc., Tucson, AZ, USA).
The anti-hPDPN antibody NZ-1 [9] was used at awww.impactjournals.com/oncotarget

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Number 25460189 (S.A.), Grant Number 24390210(Y.N.), and Grant Number 26440019 (M.K.K.).

11. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T,
Matsutani M. Increased expression of podoplanin inmalignant astrocytic tumors as a novel molecular marker
of malignant progression. Acta Neuropathol (Berl). 2006;
111:483–488.

CONFLICTS OF INTEREST
The authors have no conflict of interest.

12. 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,
et al. A novel targeting therapy of malignant mesothelioma using anti-podoplanin antibody. J Immunol. 2013;
190:6239–6249.

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