Chimeric anti-podoplanin antibody suppresses tumor
metastasis through neutralization andantibody-dependent cellular cytotoxicity
Mika Kato Kaneko,1,8 Akiko Kunita,2,8 Shinji Abe,3,8 Yuta Tsujimoto,1,4,8 Masashi Fukayama,2 Kaoru Goto,4
Yoshihiko Sawa,5 Yasuhiko Nishioka6 and Yukinari Kato1,7
1
Molecular Tumor Marker Research Team, Global COE Program, Yamagata University Faculty of Medicine, Yamagata; 2Department of Pathology, Graduate
School of Medicine, The University of Tokyo, Tokyo; 3Practice Room for Clinical Pharmacy, Institute of Health Biosciences, The University of Tokushima
Graduate School, Tokushima; 4Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata; 5Department ofMorphological Biology, Fukuoka Dental College, Fukuoka; 6Department of Respiratory Medicine and Rheumatology, The University of Tokushima Graduate
School, Tokushima, Japan(Received April 23, 2012 ⁄ Revised July 9, 2012 ⁄ Accepted July 16, 2012 ⁄ Accepted manuscript online July 20, 2012)

Podoplanin is a platelet aggregation-inducing factor associatedwith tumor metastasis, malignant progression, and cancer stem
cells. We produced a rat–human chimeric anti-podoplanin mAb,
NZ-8, from rat anti-podoplanin mAb (NZ-1). Although both NZ-1
and NZ-8 possess high binding affinities and high neutralizing
activities of platelet aggregation, the antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity of NZ-8
were much higher than NZ-1. Furthermore, both NZ-1 and NZ-8
inhibited the growth of podoplanin-expressing tumors in vivo.
Both NZ-1 and NZ-8 also suppressed hematogenous metastasis of
podoplanin-expressing tumors. These results suggest that antipodoplanin mAbs suppressed hematogenous metastasis by bothneutralization and antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity activities. Targeting therapy to
podoplanin-expressing tumors should be useful as a novel immunotherapy. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02385.x,
2012)

P

odoplanin is a platelet aggregation-inducing factor, and its
expression has been reported in many tumors including
malignant brain tumors, mesotheliomas, and squamous cell
carcinoma.(1–10) Importantly, recent investigations have suggested that expression of podoplanin is associated with tumor
metastasis, malignant progression, and epithelial–mesenchymaltransition.(11–18) Podoplanin expression has also been reported
to be associated with clinical outcome.(19–21) Recently, we
have shown podoplanin expression in atherosclerotic lesion.(22)In solid tumors such as brain tumors, only a small and phenotypically distinct subset of cells could be responsible for generating and sustaining tumors, and thus be considered as cancer
stem cells or tumor-initiating cells (TICs).(23) Because TICs arethought to be resistant to conventional therapies, and are responsible for relapse, targeting TICs could be a promising approachto cancer therapy.(24) Podoplanin has been reported to be a TIC
marker;(25) therefore, immunotherapy using specific antibodies
reactive to podoplanin may eradicate TICs in cancers.
We previously produced an anti-podoplanin antibody,
NZ-1.(5) NZ-1 should have not only high specificity and sensitivity but also high binding-affinity against podoplanin to be
applied for radioimmunotherapy or immunotoxin therapy. Previous studies showed that NZ-1 is a suitable candidate for therapy against malignant gliomas because NZ-1 was highlyinternalized into glioma cell lines, and also well accumulated
into tumors in vivo.(26) Moreover, NZ-1 inhibited tumor cellinduced platelet aggregation and tumor metastasis by its neutralizing activity.(12) However, it has not been clarified whether

doi: 10.1111/j.1349-7006.2012.02385.x© 2012 Japanese Cancer Association

NZ-1 possesses antibody-dependent cellular cytotoxicity(ADCC) or complement-dependent cytotoxicity (CDC) against
podoplanin-expressing tumor cells.
In this study, we produced rat–human chimeric anti-podoplanin antibody (NZ-8) from rat anti-podoplanin neutralizing
antibody (NZ-1), and characterized NZ-8 activity in flow
cytometry, Western blot, platelet aggregation, and ADCC/CDC
analyses in vitro. Next, we investigated the antitumor and antimetastatic activities of the anti-podoplanin mAbs in vivo.
Materials and Methods
Cell lines and stable transfectants. Chinese hamster ovary,glycan-deficient CHO cell lines (Lec1, Lec2, and Lec8), andhuman malignant mesothelioma cell line H226 cells wereobtained from ATCC (Manassas, VA, USA).(27) Human lymphatic endothelial cells were obtained from Cambrex (Walkersville, MD, USA). The CHO cells transfected with human
podoplanin (CHO/hPDPN) were established as described previously.(1) Both CHO/hPDPN cells and H226 cells were cultured
in RPMI-1640 medium (Wako Pure Chemical Industries,
Osaka, Japan) supplemented with 10% heat-inactivated FBS(Life Technologies, Carlsbad, CA, USA), 2 mM L-glutamine(Life Technologies), 100 U/mL penicillin, 100 lg/mL streptomycin (Life Technologies), and 1 mg/mL geneticin (G418;
Wako Pure Chemical Industries) for CHO/hPDPN at 37°C in ahumidified atmosphere of 5% CO2 and 95% air. The humanglioblastoma cell lines LN319 and D397, donated by Dr. Webster K. Cavenee (Ludwig Institute for Cancer Research, San
Diego, CA, USA) and Dr. Darell D. Bigner (Duke University
Medical Center, Durham, NC, USA), respectively, were cultured in DMEM (Wako Pure Chemical Industries) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine,
100 U/mL penicillin, and 100 lg/mL streptomycin. Humanlymphatic endothelial cells were cultured in endothelial cell
medium EGM-2MV supplemented with 5% FBS (Cambrex).
Animals. Female BALB/c nude (nu/nu) mice (7 weeks old)
were purchased from Charles River Japan (Kanagawa, Japan).
Male Wistar rats, 6 weeks old, were obtained from CLEA Japan(Osaka, Japan). Animals were housed under pathogen-free conditions. The Animal Care and Use Committees of Yamagata
University (Yamagata, Japan), the University of Tokyo (Tokyo,
Japan), and the University of Tokushima (Tokushima, Japan)
approved the animal experiments described herein.

7
To whom correspondence should be addressed.
E-mail: yukinari-k@bea.hi-ho.ne.jp
8
These authors contributed equally to this work.

Cancer Sci | 2012

Antibodies. A rat anti-human podoplanin mAb (IgG2a),
NZ-1, was developed as described previously.(5) Rat IgG andhuman IgG were purchased from Beckman Coulter (Fullerton,
CA, USA). For the generation of rat–human chimera antihuman podoplanin (NZ-8), the appropriate VH and VL cDNAs
of a rat NZ-1 antibody and CH and CL of human IgG1 were
subcloned into pcDNA3.3/Neo or pcDNA3.1/Zeo vectors (LifeTechnologies), respectively. Antibody expression vectors
were transfected into CHO cells using Lipofectamine 2000
reagent (Life Technologies). Stable transfectants of CHO/NZ-8
were selected by cultivating the transfectants in medium
containing 1 mg/mL geneticin and 0.5 mg/mL zeocin (LifeTechnologies).
Flow cytometry. LN319, D397, and H226 cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA
(Wako Pure Chemical Industries). After washing with PBS,
the cells were treated with NZ-1 or NZ-8 (1 lg/mL) for
30 min at 4°C followed by treatment with Oregon green-conjugated anti-rat IgG or FITC-conjugated anti-human IgG (LifeTechnologies). Fluorescence data were collected using a
FACSCalibur flow cytometer (BD Biosciences, Braintree, MA,
USA).
Determination of binding-affinity by flow cytometry. Bindingaffinity was determined as described previously.(28) Briefly,
LN319 cells (2 9 105) were resuspended at 100 lL of seriallydiluted antibodies (0.01–20 lg/mL), followed by secondary
antibodies (Life Technologies). Fluorescence data were collected using a FACSCalibur flow cytometer (BD Biosciences).
The dissociation constants (KD) were obtained by fitting thebinding isotherms using the built-in one-site binding models in
Prism software (GraphPad Software Inc., La Jolla, CA, USA).
Determination of binding-affinity by ELISA. The ELISA wascarried out as described previously.(29) Briefly, the synthetic
peptide EGGVAMPGAEDDVV (hpp3851), corresponding toamino acids 38–51 of human podoplanin was immobilized at
1 lg/mL. The plates were incubated with serially diluted antibodies (150 pg/mL–2.5 lg/mL), followed by 1:1000 diluted
peroxidase-conjugated anti-mouse IgG (Dako, Glostrup, Denmark). The dissociation constants (KD) were obtained by fitting
the binding isotherms using the built-in one-site binding
models in Prism software.
Western blot analyses. Cultured cell pellets were lysed with
PBS with 1% TritonX-100 for 30 min on ice. Cell lysates containing 5 lg total protein were boiled in SDS sample buffer(50 mM Tris, 2% SDS, 5% glycerol, 10% 2-mercaptoethanol,
pH 6.8). They were electrophoresed on 5–20% polyacrylamidegels (Wako Pure Chemical Industries), and were transferred to a
PVDF membrane (Bio-Rad Laboratories, Philadelphia, PA,
USA). After blocking with 4% skim milk in PBS, the membrane
was incubated with NZ-1 or NZ-8 (1 lg/mL) then with peroxidase-conjugated secondary antibodies (1:1000 dilution; Dako)and developed with ECL-plus reagents (GE Healthcare, Buckinghamshire, UK) using Sayaka-Imager (DRC, Tokyo, Japan).
Preparation of effector cells. Rat splenocytes were harvested
from Wistar rat spleens.(30) After depletion of red blood cells,
splenocytes were used as effector cells. Human peripheral
blood mononuculear cells (MNC) were obtained from leukocytes, which were separated from peripheral blood of healthy
donors as described previously.(31) The human study wasapproved by the ethics committee of the University of Tokushima, and written informed consent was obtained from all
subjects.
Antibody-dependent cellular cytotoxicity (ADCC). The ADCC
was evaluated using a 51Cr release assay as described
previously.(30) Target cells were targeted with 0.1 lCi of
51
Cr-sodium chromate at 37°C for 1 h. 51Cr-labeled target cells
were placed in 96-well plates in triplicate. Effector cells andantibodies (NZ-1, NZ-8, control rat IgG, control human IgG)

2

were added to the plates. After a 6-h incubation, 100 lL
supernatant was measured in a gamma counter (PerkinElmer,
Waltham, MA, USA). Percent of cytotoxicity was calculated
from the following formula: %Specific lysis = (E – S)/(M – S) 9 100, where E is the release in the test sample, S is
the spontaneous release, and M is the maximum release.
Complement-dependent cytotoxicity (CDC). The CDC wasdetermined by 51Cr release assay.(30) Target cells were incubated with 0.1 lCi of 51Cr-sodium chromate at 37°C for 1 h.
After incubation, the cells were washed three times with medium. 51Cr-labeled cells were added into 96-well plates, andincubated with baby rabbit complement and NZ-1, NZ-8, control rat IgG, or control human IgG for 6 h. 51Cr release of thesupernatant from each well (100 lL) was measured using a
gamma counter. Percent of cytotoxicity was calculated asabove.
Antitumor activity of anti-podoplanin antibodies. CHO/
hPDPN was suspended after trypsin treatment, washed with
PBS, suspended in Hanks’ balanced salt solution without calcium or magnesium, adjusted to a density of 3.0 9 107 cells/mL,
and s.c. implanted into BALB/c nude mice at a dose of
100 lL/animal. NZ-1 or rat IgG was loaded in an Alzet miniosmotic pump (model 2002; Durect, Cupertino, CA, USA),implanted into the peritoneal cavity of mice, and given at
2.5 lg/h for 2 weeks sustainably (NZ-1 experiments: control
group, n = 10; NZ-1 group, n = 9). One hundred microgramsof NZ-8 or normal human IgG was given i.p. once a week,
four times in total (NZ-8 experiments: n = 10/group).
Platelet aggregation assay. Platelet aggregation was monitored by measuring electric impedance using a whole-blood
aggregometer Model 560 (Chrono-Log, Havertown, PA,
USA).(9) Heparinized blood was drawn from BALB/c mice bycardiac puncture, and 500 lL whole blood was diluted with
500 lL normal saline. The specimen was placed in a plastic
cuvette containing a magnetic stir bar, and incubated at 37°C
for 10 min before testing. The platelet aggregation was theninitiated by the addition of CHO/hPDPN (2 9 106 cells) andmonitored for up to 20 min.
Experimental lung metastasis. CHO/hPDPN was harvested,washed, and resuspended in PBS (5 9 106 cells/mL). Next,
the cells were incubated with NZ-1, NZ-8, or human IgG, andinoculated i.v. (5 9 105 cells/mouse) into the lateral tail veinof female BALB/c-nu/nu mice. After 19–20 days, the mice
were killed, and the surface lung metastatic foci were counted
and measured.
Statistical analysis. All data are shown as the mean ± SEM.
Student’s t-test, one-way ANOVA followed by Tukey–Kramer
multiple comparisons, or two-way ANOVA were carried out,
where appropriate. P-values <0.05 were considered statisticallysignificant. All statistical tests were two-sided.
Results
Production and characterization of chimeric anti-podoplaninantibody (NZ-8). We first generated the rat–human chimericantibody (NZ-8) by fusing the VH and VL regions of rat antibody (NZ-1) with CH and CL regions of human IgG1, respectively. As depicted in Figure 1(A), NZ-8 reacted with bothhuman glioblastoma cell lines LN319 and D397, and human
mesothelioma cell line H226 in flow cytometry, although the
NZ-8 reaction seems to be weaker than that of NZ-1. We
previously produced several podoplanin-transfected glycandeficient CHO cell lines, such as Lec1, Lec2, and Lec8,
to investigate the involvement of O-glycan or N-glycan to
podoplanin-induced platelet aggregation.(27) As shown in
Figure 1(B), NZ-8 reacted with all podoplanin-transfected
glycan-deficient CHO cell lines in the same way as podoplanin-transfected CHO/hPDPN. Furthermore, NZ-8 reacted
doi: 10.1111/j.1349-7006.2012.02385.x© 2012 Japanese Cancer Association

(A)

(B)

Fig. 1. Characterization of chimeric anti-podoplanin antibody, NZ-8. (A) LN319 and D397 human glioblastoma cells and H226 human malignant
mesothelioma cells were treated with NZ-1 or NZ-8 (1 lg/mL). (B) The membrane was incubated with NZ-1 or NZ-8 (1 lg/mL). Lane 1, CHO cells
transfected with human podoplanin (CHO/hPDPN); lane 2, Lec1/hPDPN; lane 3, Lec2/hPDPN; lane 4, Lec8/hPDPN; lane 5, CHO; lane 6, LN319; lane
7, lymphatic endothelial cells; lane 8, H226. WB, Western blot.

(A)

(B)

(C)

(D)

Fig. 2. Determination of binding-affinity using flow cytometry and ELISA. LN319 human glioblastoma cells (2 9 105) were resuspended at
100 lL serially diluted NZ-1 (A) or NZ-8 (B) (0.01–20 lg/mL). The plates were incubated with serially diluted NZ-1 (C) or NZ-8 (D) (150 pg/mL–
2.5 lg/mL). KD, dissociation constant.

Kaneko et al.

Cancer Sci | 2012 | 3© 2012 Japanese Cancer Association

(A)

(A)

(B)
(B)

(C)

Fig. 3. Antibody-dependent cellular cytotoxicity activities induced by
rat splenocytes against CHO cells transfected with human podoplanin(CHO/hPDPN) (A), CHO (B), and glioblastoma cell line LN319 (C), were
determined with 6-h 51Cr release assays at the effector/target (E/T)
ratio of 100 and 250 in the presence of 1 lg/mL of NZ-1. **P < 0.01
versus control (values are means ± SE).

with glioblastoma cell line LN319, mesothelioma cell line
H226, and human lymphatic endothelial cells. Because thebinding-affinity of antibodies is critical for antibody-based
cancer therapy, dissociation constant (KD) was next determinedusing flow cytometric analysis or ELISA.(28) As shown in
Figure 2(A,C), KD of NZ-1 was determined to be
7.2 9 10 9 M by flow cytometry or 4.1 9 10 10 M by
ELISA. Using the same methods, KD of NZ-8 was determined
to be 7.6 9 10 8 M by flow cytometry or 1.8 9 10 9 M by
ELISA (Fig. 2B,D).
Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) mediated by anti-podoplaninantibodies. To apply targeted therapy to podoplanin, we further

assessed whether anti-podoplanin antibodies can induce ADCC
against podoplanin-expressing cell lines mediated by rat
splenocytes or human MNC as effector cells. Antibody-dependent cellular cytotoxicity by NZ-1 was observed against CHO/
hPDPN cells, when rat splenocytes were used as effector cells(Fig. 3A), whereas it was not observed against CHO cells
4Fig. 4. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities of anti-podoplanin antibodies. (A) ADCC activities induced by human mononuculear cells
against glioblastoma cell lines LN319 and D397 were determined with
6-h 51Cr release assay at the effector/target ratio of 100 in the presence of 1 lg/mL NZ-1, NZ-8, rat IgG, and human IgG. (B) CDC activities
against glioblastoma cell lines LN319 and D397 were shown by 51Cr
release assay. **P < 0.01 versus control (values are means ± SE).

(Fig. 3B). Antibody-dependent cellular cytotoxicity by NZ-1
was also observed against LN319 glioblastoma cells (Fig. 3C).
We next compared the ADCC and CDC activities between
NZ-1 and NZ-8 using LN319 and D397. Antibody-dependent
cellular cytotoxicity against glioblastoma cell lines was notshown by NZ-1 using human MNC, whereas NZ-8 showed theinduction of significant levels of ADCC mediated by human
MNCs against LN319 and D397 glioblastoma cells (Fig. 4A).
Furthermore, CDC was induced by NZ-8 against LN319 andD397, which was significantly higher than that of NZ-1
(Fig. 4B).
Antitumor activity of anti-podoplanin antibodies on thegrowth of podoplanin-expressing cells in vivo. We first investi-

gated the antitumor activity of NZ-1 in vivo (Fig. 5). The subcutaneous tumor formation rate was 9/10 (90%) for the control
group and 6/9 (66.6%) for the group given NZ-1 antibody.
Subcutaneous tumors were significantly smaller in the group
given NZ-1 antibody after 50 days of cell implantation, as
compared to the control group (Fig. 5A), indicating that inoculation with NZ-1 significantly inhibited the growth of CHO/
hPDPN cells in vivo. Spontaneous metastasis of CHO/hPDPN
was also observed; the lung nodules of CHO/hPDPN of NZ-1
groups were less than those of rat IgG groups (Fig. 5B,
P < 0.05). Staining with H&E revealed that several metastatic
foci were detected in control lungs, whereas much fewer metastatic foci were observed in lung NZ-1 groups (Fig. 5C). We
next investigated the antitumor activity of NZ-8 in vivo.
Tumor formation incidence was 10/10 (100%) for the control
group and 4/10 (40%) for the NZ-8 group. Subcutaneous
doi: 10.1111/j.1349-7006.2012.02385.x© 2012 Japanese Cancer Association

(A)

(A)

(B)
(B)

(C)

Fig. 5. Antitumor activity of NZ-1 on the growth of podoplaninexpressing cells in vivo. (A) CHO cells transfected with human podoplanin (CHO/hPDPN; 3.0 9 107 cells/mL) were s.c. implanted into BALB/cnude mice at a dose of 100 lL/animal. After 1 day, NZ-1 (5 mg/mL) or
rat IgG (5 mg/mL) was given at 0.5 lL/h for 2 weeks sustainably (control group, n = 10; NZ-1 group, n = 9). Twenty days after cell implantation, tumor diameter was measured at intervals of 3 days. *P < 0.05;**P < 0.01; ***P < 0.001 by two-way ANOVA. (B,C) Spontaneous metastasis of CHO/hPDPN was observed. (B) number of lung metastatic foci.
*P < 0.05 by t-test. (C) H&E staining.

tumors were significantly smaller in the group given NZ-8
antibody compared to the control group (Fig. 6), indicatingthat inoculation with NZ-8 also significantly inhibited thegrowth of CHO/hPDPN cells in vivo.
Suppression of tumor metastasis by anti-podoplanin antibodies. We then investigated the inhibitory effect of anti-podoplaninantibodies against podoplanin-induced platelet aggregation.
We previously clarified that NZ-1 recognizes the platelet
aggregation-stimulating domain, and neutralizes podoplanininduced platelet aggregation. Results showed that NZ-8 inhibited
platelet aggregation by CHO/hPDPN in a dose-dependent manner (Fig. 7A), although NZ-1 inhibited CHO/hPDPN-induced
platelet aggregation more effectively than NZ-8 at a dose of
1 mg/mL.
We next investigated whether anti-podoplanin antibodies
could suppress the podoplanin-induced pulmonary metastasis
in an experimental metastasis model. Injection of CHO/hPDPN
cells led to development of multiple lung metastatic foci. The
number of metastatic lung nodules in mice injected with CHO/
Kaneko et al.

Fig. 6. Antitumor activity of NZ-8 on the growth of podoplaninexpressing cells in vivo. (A) Podoplanin-expressing CHO cells transfected with human podoplanin (CHO/hPDPN; 3.0 9 106 cells) were s.c.
implanted into BALB/c nude mice. NZ-8 or normal human IgG (100 lg/
mouse) was given every week (n = 10/group). (B) Comparison oftumor size (day 40).*P < 0.05; **P < 0.01; ***P < 0.001 by two-way
ANOVA.

hPDPN + NZ-1 was significantly lower than that in
CHO/hPDPN + control PBS (Fig. 7B, P < 0.05). We did notobserve, macroscopically, metastatic foci in the liver, kidney,spleen, colon, or ovary in any of the mice (data not shown).
Furthermore, we investigated whether NZ-8 could also suppress podoplanin-induced pulmonary metastasis in the samemodel. As shown in Figure 7(B), NZ-8 also suppressed pulmonary metastasis. The number of metastatic lung nodules inmice injected with CHO/hPDPN + NZ-8 was significantly
lower than that in CHO/hPDPN + control PBS in the same
way with NZ-1 (Fig. 7B, P < 0.05). Staining with H&E
revealed that many metastatic foci were detected in control
lung, whereas much fewer metastatic foci were observed inlung in NZ-1 and NZ-8 groups (Fig. 7C). Furthermore, dosedependency of the antimetastatic effects by NZ-8 was examined. As shown in Figure 7(D,E), CHO/hPDPN-induced lungmetastasis was reduced significantly by both 10 lg (P < 0.05)
and 50 lg (P < 0.001) NZ-8 in a dose-dependent manner.
These results indicate that both NZ-1 and NZ-8 inhibited significantly pulmonary metastasis as compared to control, suggesting that anti-podoplanin antibodies may be effective in
podoplanin-induced tumor metastasis.
Discussion

In this study, we produced a human–rat chimeric anti-podoplanin mAb, NZ-8, which showed high reactivity with podoplanin-expressing cell lines in flow cytometry and Western
blot analyses (Fig. 1). Although NZ-8 possesses lower affinity
compared with NZ-1 (Fig. 2), NZ-8 showed the induction ofsignificant levels of ADCC and CDC against glioblastoma
cells compared with NZ-1 (Fig. 4), indicating that targeting
podoplanin-expressing tumors with NZ-8 seems to be usefulfor antibody-based immunotherapy. Generally, the CDC activity of antibody is known to be dependent on the IgG subclass;Cancer Sci | 2012 | 5© 2012 Japanese Cancer Association

(B)

(A)

(C)

(D)

(E)

Fig. 7. Suppression of tumor metastasis by anti-podoplanin antibodies. (A) Platelet aggregation was initiated by the addition of CHO cells
transfected with human podoplanin (CHO/hPDPN; 5 9 106) and monitored for 20 min. (B,C) CHO/hPDPN cells (5 9 106 cells/mL) were incubatedwith 100 lg NZ-1 or NZ-8, and inoculated i.v. (5 9 105 cells/mouse) into BALB/c-nu/nu mice. (B) Number of lung metastatic foci. *P < 0.05 by ANO6
VA with Tukey–Kramer multiple comparisons. (C) H&E staining. (D,E) CHO/hPDPN cells (5 9 10 cells/mL) were incubated with 50 lg human IgG,
10 lg NZ-8, or 50 lg NZ-8, and inoculated i.v. (5 9 105 cells/mouse) into BALB/c-nu/nu mice. (D) Lung metastatic foci. (E) Number of lung metastatic foci. *P < 0.05; ***P < 0.001 by ANOVA with Tukey–Kramer multiple comparisons.

human IgG1 and IgG3 possess higher CDC activity, and rat
IgG2b subclass has higher CDC activity compared with othersubclasses. Therefore, NZ-8 of human IgG1 might show muchhigher CDC activity compared with NZ-1 of rat IgG2a.
It has been shown that NZ-1 suppresses podoplanin-induced
pulmonary metastasis through inhibiting platelet aggregation.(12) In this study, NZ-1 also inhibited platelet aggregation
using another whole blood aggregometer system (Fig. 7A).
NZ-8 inhibited platelet aggregation in a dose-dependent manner; however, the inhibition seems to be weaker than that of
NZ-1, probably because of the low binding-affinity against
podoplanin-expressing cells. To date, the suppression of tumor
metastasis by podoplanin has been reported to be dependent onplatelet aggregation, because not only whole IgGs of NZ-1 but
also F(ab′)2 fragments of NZ-1 almost completely suppressedpulmonary metastasis of CHO/hPDPN cells.(12) However, othereffector functions, such as ADCC and CDC, of NZ-1 remain
to be investigated. In the present study, NZ-1 and NZ-8 clearly
showed ADCC and CDC activities (Figs 3,4). NZ-1 and NZ-8
also inhibited the growth of CHO/hPDPN cells in vivo
(Figs 5,6). Although the neutralizing activity of NZ-8 seems to
be weaker than that of NZ-1 (Fig. 7A), effective suppressionof pulmonary metastasis by NZ-8 was observed at the samelevel as NZ-1 (Fig. 7B–E), indicating that suppression of pulmonary metastasis may be dependent on both neutralizing
activity and ADCC/CDC activities. We carried out additional
experiments of 51Cr release assay to show that NZ-1 and NZ-8
possess ADCC activities against tumor cells, using mouse6

splenocytes. However, neither NZ-1 nor NZ-8 could clearly
induce ADCC activity using mouse splenocytes (data notshown). In contrast, both NZ-1 and NZ-8 showed high CDC
activities (Fig. 4B); therefore, we speculate that the antitumor
effects of NZ-1 and NZ-8 against tumor cells in vivo may be
induced mainly by their CDC activity, especially in a mousemodel. Importantly, podoplanin has been reported to be
expressed in lymphatic endothelial cells, podocytes, and type I
alveolar cells, suggesting that there are possibilities to induceside-effects for human cancer treatment using NZ-8. Therefore,
the toxicity test should be carried out using monkeys in thenear future.(32) We have shown that podoplanin expression innormal tissue is much lower than that of tumors;(1) therefore,
the side-effects of anti-podoplanin antibodies could be reduced
by decreasing the dosage of NZ-8 antibody.
In conclusion, we clarified that anti-podoplanin antibodies
possess potent and therapeutic antitumor effects based on
ADCC and CDC against podoplanin-expressing cells. These
results suggest that targeting therapy to podoplanin with therapeutic antibodies might be useful as a novel immunotherapy
against podoplanin-expressing tumors such as malignant brain
tumors, mesotheliomas, and squamous cell carcinoma.
Acknowledgments
We thank Shunpei Morita, Ryo Yanagiya, Junko Aita, Kimiko Takeshita,
and Kei Sakuma for their excellent technical assistance. This work was
supported by KAKENHI (22390345, 23659884, 18590855, 23790185,

doi: 10.1111/j.1349-7006.2012.02385.x© 2012 Japanese Cancer Association

23701043, and 23791584), a Grant-in-Aid for Scientific Research (B)(Y.S.), a Grant-in-Aid for Exploratory Research (Y.S.), a Grant-in-Aid
for Scientific Research (C) (Y.N.), and a Grant-in-Aid for Young Scientists (B) (M.K.K., Y.K., S.A.), respectively, from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by SENSHIN
Medical Research Foundation (Y.K., M.K.K.), by the Children’s Cancer
Association of Japan (Y.K.), by the Intelligent Cosmos Academic Foun-dation (Y.K.), and by the Office for Gender Equality of Yamagata University (M.K.K.).

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Kaneko et al.

Disclosure Statement
The authors have no conflicts of interest.

Cancer Sci | 2012 | 7© 2012 Japanese Cancer Association