Antitumor effect of novel anti-podoplanin antibody
NZ-12 against malignant pleural mesothelioma in an
orthotopic xenograft model
Shinji Abe,1,2 Mika Kato Kaneko,3 Yuki Tsuchihashi,4 Toshihiro Izumi,1 Satoshi Ogasawara,3 Naoto Okada,1Chiemi Sato,1 Makoto Tobiume,2 Kenji Otsuka,2 Licht Miyamoto,4 Koichiro Tsuchiya,4 Kazuyoshi Kawazoe,1Yukinari Kato3 and Yasuhiko Nishioka2
Departments of 1Clinical Pharmacy Practice Pedagogy; 2Respiratory Medicine and Rheumatology, Institute of Biomedical Sciences, Tokushima University
Graduate School, Tokushima; 3Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai; 4Department of Medical
Pharmacology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan

Key words
antibody-dependent cellular cytotoxicity, mesothelioma,
NZ-12, orthotopic xenograft model, podoplanin
Correspondence
Yasuhiko Nishioka, Department of Respiratory Medicine
and Rheumatology, Institute of Biomedical Sciences,
Tokushima University Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
Tel: +81-88-633-7127; Fax: +81-88-633-2134;
E-mail: yasuhiko@tokushima-u.ac.jp
Funding Information
Ministry of Education, Culture, Sports, Science and Technology of Japan KAKENHI (24390210, 25460189,
26440019, and 25462242); Tokushima University; Japan
Agency for Medical Research and Development.
Received April 2, 2016; Revised June 7, 2016; Accepted
June 10, 2016
Cancer Sci 107 (2016) 1198–1205
doi: 10.1111/cas.12985

Podoplanin (aggrus) is highly expressed in several types of cancers, including
malignant pleural mesothelioma (MPM). Previously, we developed a rat antihuman podoplanin mAb, NZ-1, and a rat–human chimeric anti-human podoplaninantibody, NZ-8, derived from NZ-1, which induced antibody-dependent cellularcytotoxicity (ADCC) and complement-dependent cytotoxicity against podoplaninpositive MPM cell lines. In this study, we showed the antitumor effect of NZ-1, NZ8, and NZ-12, a novel rat–human chimeric anti-human podoplanin antibody
derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment
with NZ-1 and rat NK (CD161a+) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft
mouse models. NZ-8 and human natural killer (NK) (CD56+) cells also inhibitedtumor growth and pleural effusion in MPM orthotopic xenograft mice. Furthermore, NZ-12 induced potent ADCC mediated by human MNC, compared with either
NZ-1 or NZ-8. Antitumor effects were observed following treatment with NZ-12
and human NK (CD56+) cells in MPM orthotopic xenograft mice. In addition, combined immunotherapy using the ADCC activity of NZ-12 mediated by human NK(CD56+) cells with pemetrexed, led to enhanced antitumor effects in MPM orthotopic xenograft mice. These results strongly suggest that combination therapy
with podoplanin-targeting immunotherapy using both NZ-12 and pemetrexed
might provide an efficacious therapeutic strategy for the treatment of MPM.

E

xpression of podoplanin (aggrus), a transmembrane sialomucin-like glycoprotein, has been detected in various normal tissues, including kidney podocytes, endothelium oflymphatic vessels, and type I alveolar epithelium,(1–3) as wellas many types of cancers, including malignant brain tumor,
oral cancers, esophageal cancers, squamous carcinoma, testicular seminomas, bladder cancers, fibrosarcomas, and malignant
pleural mesothelioma (MPM).(4–9) Podoplanin, which binds tothe platelet aggregation-stimulating domain of PDPN–C-type
lectin-like receptor 2 in platelets, induces platelet aggregation,
resulting in cancer metastasis.(4,10) Furthermore, high expression of podoplanin in cancer-associated fibroblasts is associated with severe malignancy and poor prognosis in cancer
patients.(11–14) Therefore, it is expected that podoplanin will
become a target for cancer diagnosis and therapies.
Malignant pleural mesothelioma, which is mainly caused byexposure to asbestos, develops in the pleural cavity with a highlikelihood of malignancy.(15) It is expected that the number of
MPM patients will increase from 2030 to 2040 in Asia, andfrom 2010 to 2020 in Europe.(16,17) The standard therapy for
MPM involves a combination of surgical operations, radiation
therapy, and systemic chemotherapy. However, the prognosis

of MPM is very poor as MPM is one of the most progressive
cancers and frequently resists treatment.(18,19) Treatment withpemetrexed, which is the only validated chemotherapy drugfor MPM, combined with cisplatin, is the standard chemotherapy used in MPM patients; however, this combination therapy
often only prolongs progression-free survival by approximately
2.8 months, compared with treatment without pemetrexed.(20)Thus, development of novel therapies for MPM is warranted
in order to improve the prognosis.
Immunotherapy using therapeutic antibodies against tumorassociated antigens or antigenic peptides derived from pemetrexed is a novel therapy for the treatment of various cancers,
including MPM.(21,22) Several therapeutic antibodies, including trastuzumab and rituximab, are already being used inclinical practice. Antibody-dependent cellular cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC) are
critical mechanisms by which therapeutic antibodies provide
their antitumor effects.(23) Previously, we generated a rat
anti-human podoplanin mAb, NZ-1,(24–26) and a rat–humanchimeric anti-human podoplanin antibody, NZ-8, derived
from NZ-1.(9,27) These anti-podoplanin antibodies induce
potent ADCC and CDC activity against podoplanin-positive

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MPM cell lines in vitro, and had significant antitumor effects
in an MPM s.c. transplantation SCID mouse model. However,whether the therapeutic effects of anti-podoplanin antibodies
in MPM orthotopic xenograft mice are similar to clinical presentation of MPM is still unknown. In the present study, weinvestigated whether anti-human podoplanin antibodies NZ-1,
NZ-8, and NZ-12, a novel rat–human chimeric anti-human
podoplanin antibody derived from NZ-1, can induce antitumor effects in an MPM orthotopic xenograft SCID mousemodel. Furthermore, we evaluated the antitumor effects ofcombined treatment using anti-human podoplanin antibodybased immunotherapy, and pemetrexed.
Materials and Methods
Cell lines. In this study, we used four human MPM cell lines.
NCI-H290, ACC-MESO-1, and ACC-MESO-4 were provided
by Dr. Yoshitaka Sekido (Division of Molecular Oncology,
Aichi Cancer Center Research Institute, Nagoya, Japan).(28)
NCI-H226 and Chinese hamster ovary (CHO) were purchasedfrom ATCC (Rockville, MD, USA). NCI-H290 cells weretransfected with appropriate amounts of pcDNA3/human podoplanin or pcDNA3/mock plasmids, using Metafectene (Nippon
Genetics, Tokyo, Japan), according to the manufacturer’s
instructions. Stable transfectants (NCI-H290/PDPN and NCIH290/mock) were selected by culture in medium containing
0.5 mg/mL Geneticin (Invitrogen, Carlsbad, CA, USA). Thesecells were maintained in RPMI-1640 medium supplemented
with 10% FBS (CRPMI-1640; Gibco, Grand Island, NY,
USA), 100 U/mL penicillin, and 100 lg/mL streptomycin(Meiji Seika Kaisha, Tokyo, Japan) in 5% CO2 at 37°C.
Antibodies and reagents. Rat anti-human podoplanin mAb,
NZ-1, and rat–human chimeric anti-human podoplanin antibody, NZ-8, were developed as described previously.(24,27) For
the generation of rat–human chimera anti-human podoplanin(NZ-12), the appropriate VH of a rat NZ-1 antibody and CH
of human IgG1 were subcloned into the pCAG-Neo (Wako
Pure Chemical Industries, Osaka, Japan), and VL of a rat NZ1 antibody and CL of human lambda chain were subcloned
into pCAG-Ble vectors (Wako Pure Chemical Industries).
Antibody expression vectors were transfected into CHO cells
using a Lipofectamine LTX kit (Life Technologies, Carlsbad,
CA, USA). Stable transfectants of CHO/NZ-12 were selectedby cultivating the transfectants in medium containing 1 mg/
mL Geneticin and 0.5 mg/mL Zeocin (Life Technologies).
The CHO/NZ-12 cells were cultivated in CHO-S-SFM II medium (Life Technologies). The media containing NZ-12 were
centrifuged, and the obtained supernatant was applied to a column of protein G-Sepharose (Thermo Fisher Scientific, Rockford, IL, USA). After extensive washing with PBS, the fusion
proteins were eluted using 0.1 M glycine and 0.15 M NaCl
(pH 2.8), and then neutralized with 1 M Tris (pH 10.0). The
antibodies were dialyzed against PBS. Expression and purityof the proteins were confirmed by SDS-PAGE, using 5–20%
gradient gels (Wako Pure Chemical Industries). Rat IgG(rIgG) was purchased from Southern Biotechnology (Birmingham, AL, USA). Human IgG (hIgG) was purchased from Cappel (Cochranville, PA, USA). Pemetrexed was obtained from
Eli Lilly Japan (Kobe, Japan).
Animals. Five- to 6-week-old male SCID mice and 6-weekold male Wistar rats were obtained from CLEA Japan (Osaka,
Japan) and maintained under specific pathogen-free conditions
throughout experiments. All animals were acclimatized for atleast 1 week before experiments. All experiments were carried
Cancer Sci | September 2016 | vol. 107 | no. 9 | 1199

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Abe et al.

out in accordance with the guidelines of the Committee on Animal Care and Use of Tokushima University (Tokushima, Japan).
Flow cytometry. Expression of podoplanin was detected byflow cytometry, as described previously.(9) Cells (5 9 105)
were washed with PBS and stained with NZ-1 (1 lg/mL) orrIgG (1 lg/mL). After 30 min of incubation, cells were
washed with PBS and incubated for 30 min with FITC-conjugated goat F(ab’)2 fragment anti-rat IgG (H+L) antibody(Beckman Coulter, Fullerton, CA, USA). Cells were washedagain and resuspended in PBS. A FACSCalibur flow cytometerwith CellQuest software (BD Biosciences, Franklin Lakes, NJ,
USA) was used for the analysis.
Preparation of effector cells. Effector cells were prepared aspreviously described.(9,29,30) Rat splenocytes were harvested
from Wistar rat spleens. Spleens were homogenized in RPMI1640 and centrifuged. To deplete red blood cells, the cellpellet was suspended in red blood cell lysis buffer (SigmaAldrich, St. Louis, MO, USA). After washing and resuspension
in CRPMI-1640, splenocytes were used as effector cells. To
separate rat natural killer (NK) cells from rat splenocytes, amagnetic cell-sorting system was used. Splenocytes wereincubated with FITC-conjugated anti-CD161a antibody (BDBiosciences), followed by anti-FITC mAb-coupled super-paramagnetic microbeads (Miltenyi Biotec, Auburn, CA, USA).
CD161a+ selection was carried out using an autoMACS (Miltenyi Biotec). Isolated CD161a+ cells yielding purity ≥90%,
determined by flow cytometry, were used in experiments.
Human peripheral blood mononuclear cells (MNCs) wereobtained from leukocytes in lymphocyte separation medium(Litton Bionetics, Kensington, MD, USA). Leukocytes wereseparated from peripheral blood of healthy donors using a
Kubota KR-400 centrifuge with an RS-6600 rotor (Kubota,
Tokyo, Japan). CD56+ cells were purified from human MNCs,
which were treated with CD56 microbeads (Miltenyi Biotec),and then cells were separated by autoMACS. The purity of
CD56+ cells was ≥90%. The human study was approved by
the ethics committee of University of Tokushima, and written
informed consent was obtained from all donors.
Antibody-dependent cellular cytotoxicity. Antibody-dependent
cellular cytotoxicity was determined using 51Cr releaseassays.(9,29,31) Target cells were incubated with 51Cr-sodium
chromate (3.7 MBq) at 37°C for 1 h. After washing withCRPMI-1640 three times, 51Cr-labeled target cells were placedin triplicate in 96-well plates. Effector cells and anti-human
podoplanin antibody or control IgG were added to the plates.
After 6 h of incubation, 51Cr release of the supernatant fromeach well (100 lL) was measured using a gamma counter(PerkinElmer, Waltham, MA, USA). Percent of cytotoxicity
was calculated using the following formula: % specificlysis = (ES) / (MS) 9 100, where E is the release in thetest sample, S is the spontaneous release, and M is the maximum
release.
Complement-dependent cytotoxicity. Complement-dependent
cytotoxicity was evaluated by 51Cr release assay, as described
previously.(9,32) Target cells were incubated with 51Cr-sodium
chromate (3.7 MBq) for 1 h at 37°C. Following this, cells were
washed in CRPMI-1640. The 51Cr-labeled cells were incubatedwith baby rabbit complement (dilution of 1:4) (Cedarlane,
Burlington, VT, Canada) and NZ-12 (1 lg/mL) or control hIgG
(1 lg/mL) for 6 h in 96-well plates. After incubation, the supernatant, including 51Cr, was measured using a gamma counter.
Percent cytotoxicity was calculated as described above.
Animal experiments. SCID mice were injected into the thoracic cavity with NCI-H290/PDPN (1.0 9 106 cells) or NCI© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Original Article
Anti-podoplanin antibody against MPM

H226 (1.0 9 106 cells) on day 0. Intrathoracic administration
or i.p. injection of anti-human podoplanin antibody or control
IgG began on day 0, and continued twice a week for 2–
3 weeks. Rat CD161a+ cells (1.0 9 106 cells), human CD56+
cells (1.0 9 106 cells), or control normal saline were injected
into the thoracic cavity from day 3, and continued weekly for
2–3 weeks. SCID mice of the pemetrexed combination groupwere treated with pemetrexed (100 mg/kg, i.p.) on days 4, 5,
6, 11, 12, and 13, as described previously.(33) Three weeks(NCI-H290/PDPN) or 9 weeks (NCI-H226) after tumor cell
inoculation, the mice were killed, thoracic tumors were
weighed, and the volume of pleural effusion was measured
using a 1-mL syringe.
Statistical analyses. The statistical significance of differences
in in vitro and in vivo data was analyzed using standard Student’s t-test and one-way ANOVA. P-values < 0.05 were considered significant in all experiments.
Results
Rat anti-human podoplanin antibody NZ-1 induces ADCC
against podoplanin-transfected MPM cell lines. First, we con-firmed that NZ-1 induces ADCC against NCI-H290/PDPN in
target cells. NCI-H290, one of the podoplanin-negative MPM
cell lines, develops thoracic tumors and pleural effusion in an
orthotropic xenograft SCID mouse model.(34) We generated
NCI-H290/PDPN to transfect pcDNA3/podoplanin into NCIH290. As shown in Fig. 1(a), podoplanin expression in
NCI-H290/PDPN was determined by flow cytometry. Using
NCI-H290/PDPN as target cells, we could detect ADCC
induced by NZ-1 with rat splenocytes (Fig. 1b). Figure 1(c)shows that effector/target cell-dependent and dose-dependent
ADCC activities against NCI-H290/PDPN were also induced
by NZ-1. Moreover, NZ-1 showed significant ADCC activity
against NCI-H290/PDPN and NCI-H226 when rat NK cells
were used as target cells (Fig. 1d).
Antitumor activity of NZ-1 in MPM orthotopic xenograftmodel. Previously, we reported that injection of both NZ-1 and

rat NK cells inhibited the growth of podoplanin-positive MPM

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cells inoculated s.c. in SCID mice.(9) To determine whether
NZ-1 also induced an antitumor effect in an orthotopic xenograft model, we first evaluated the effects of local administration of NZ-1 in NCI-H290/PDPN orthotopic xenograft mice.
For 2 weeks, NZ-1 or control IgG was injected into the thoracic cavity twice a week, and rat NK (CD161a+) cells or PBS
was injected into the thoracic cavity once a week. Three weeks
after tumor cell inoculation, tumor growth and production of
pleural effusion was almost entirely inhibited in both the NZ-1
and rat NK cells treatment groups (Fig. 2). In contrast, treatment with NZ-1 alone did not produce an antitumor effect inour model. Next, we investigated whether systemic treatment
of NZ-1 induced antitumor effects in the orthotopic xenograft
SCID mouse model. For 3 weeks, NZ-1 or rIgG was injected
i.p. twice a week, and rat NK cells or PBS were injected oncea week into the thoracic cavity. In both NCI-H290/PDPN- and
NCI-H226-inoculated mice, systemic administration of NZ-1
with rat NK cells significantly inhibited growth of intrathoracic
tumors and production of pleural effusion, compared witheither NZ-1 or rat NK cell treatment alone (Fig. 3).
Antitumor activity of NZ-8 in MPM orthotopic xenograft model.

We previously reported that NZ-8 induced ADCC activity
mediated by human NK cells.(9) To evaluate the antitumor
effects of NZ-8 combined with human NK cells in an MPM
orthotopic xenograft model, we used the NCI-H290/PDPN
orthotopic xenograft mouse model. As shown in Figure 4(a),ADCC activity against NCI-H290/PDPN was observed following treatment with NZ-8 and human MNC. Injection of NZ-8
(i.p.) twice a week and human NK (CD56+) cells injected into
the thoracic cavity weekly for 2 weeks significantly inhibitedtumor weight and pleural effusion production, compared with
NZ-8 or human NK cells alone (Fig. 4b).
In vitro and in vivo antitumor effects of NZ-12. Given that
NZ-1 and NZ-8 induced antitumor effects in MPM in an orthotopic xenograft model, we generated a novel rat–human chimeric anti-human podoplanin antibody, NZ-12, derived from
NZ-1, in order to establish a more potent target therapy forpodoplanin. As shown in Figure 5(a), NZ-12 induced a significant level of ADCC, mediated by human MNC, against

Fig. 1. Antibody-dependent cellular cytotoxicity(ADCC) activity of rat anti–human podoplaninantibody NZ-1 against NCI-H290/PDPN in vitro. (a)Expression of podoplanin was detected by FACS
analysis. (b) ADCC activity of NZ-1 using rat
splenocytes against NCI-H290/PDPN or NCI-H290/
Mock was evaluated by 6-h 51Cr release assay in thepresence of antibody (1 lg/mL; effector/target [E/T]
ratio 100). (c) E/T ratio-dependent and antibody
dose-dependent effects of ADCC against NCI-H290/
PDPN mediated by NZ-1 with rat splenocytes areshown by 51Cr release assay. (d) Rat natural killer(NK) (CD161a+) cells were isolated from rat
splenocytes. ADCC activity of NZ-1 (1 lg/mL)
mediated by NK (CD161a+) cells was evaluated by 6h 51Cr release assay at an E/T ratio of 10. **P < 0.01
versus control (values represent mean  SE). rIgG,
rat IgG.
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Abe et al.

Fig. 2. Antitumor effects of NZ-1 injected into thethoracic cavity in an NCI-H290/PDPN malignant
pleural mesothelioma orthotopic xenograft model.
SCID mice (n = 5) were injected into the thoracic
cavity with 1.0 9 106 NCI-H290/PDPN cells. NZ-1
(100 lg) or rat IgG (rIgG; 100 lg) intrathoracic
injection began on day 0, and continued twice aweek for 2 weeks. Rat NK (CD161a+) cells (1.0 9 106
cells) or control normal saline intrathoracic
injections continued weekly for 2 weeks. Three
weeks after tumor cell inoculation, the mice were
killed, and the weight of thoracic tumors (whitearrows) and volume of pleural effusion were
measured. **P < 0.01 (values represent mean  SE).

podoplanin-positive MPM cells. The ADCC activity induced
by NZ-12 was significantly higher than that of NZ1 or NZ-8.
NZ-12 also induced CDC activity against podoplanin-positive
MPM cells (Fig. 5b). Moreover, ADCC activity of NZ-12 wasmediated by human NK (CD56+) cells (Fig. 5c). In the NCIH290/PDPN orthotopic xenograft SCID mouse model, tumor
weight and production of pleural effusion was significantly
inhibited by 2 weeks of injections of NZ-12 (i.p.), given twicea week, co-administered with injections of human NK(CD56+) cells into the thoracic cavity, given once a week
(Fig. 5d,e). By contrast, treatment with NZ-12 alone did notinhibit tumor growth.
Effect of combination treatment of NZ-12 and pemetrexed. Our result suggested that combined treatment with NZ-

12 and human NK cells inhibited tumor growth in an MPM
orthotropic model. Additionally, we further revealed the efficacy of NZ-12 against MPM using combined treatment of NZ12 and pemetrexed in an MPM SCID mouse model. As shownin Figure 6(a), expression of podoplanin in NCI-H290/PDPN
was unchanged following 72 h of incubation with pemetrexed(0.1 lM). In contrast, the same treatment condition of pemetrexed inhibited the proliferation of NCI-H290/PDPN in vitro(data not shown). Furthermore, treatment with pemetrexed intarget cells did not inhibit ADCC activity induced by NZ-12
with human MNC (Fig. 6b). Using the orthotopic xenograft
SCID mouse model, injection of NZ-12 (i.p.), human NK(CD56+) cells injected into thoracic cavity, and pemetrexed(i.p.) significantly reduced intrathoracic tumor growth and production of pleural effusion, compared with the immunotherapy
of NZ-12 (NZ-12 with human NK cells) or pemetrexed alone(Fig. 6c).
Discussion

In the present study, we have shown that anti-human podoplanin antibodies NZ-1, NZ-8, and NZ-12 possess therapeutic
antitumor effects in an MPM orthotopic xenograft SCID
mouse model. Furthermore, NZ-12 induced more ADCC
Cancer Sci | September 2016 | vol. 107 | no. 9 | 1201

activity than NZ-1 or NZ-8. In addition, we have shown thatcombination treatment of NZ-12 and pemetrexed produced significantly greater antitumor activity than each single therapy.
These findings suggested that NZ-12 is an effective antibody
against MPM.
We previously established a human MPM orthotopic xenograft SCID mouse model, in which MPM cells were inoculated
into the thoracic cavity.(33,35) Several weeks after inoculation,
the mice died due to increased size of thoracic tumors and production of bloody pleural effusion inside the thoracic cavity.
As this disease state is similar to that of human MPM patients,
this model could be used to evaluate the efficacy of therapeutic
treatments of MPM.(34,36) Although our previous study only
showed the antitumor effects of an anti-human podoplaninantibody given with NK cells in an MPM s.c. xenograftmodel,(9) in this study, administration of anti-human podoplanin antibodies NZ-1, NZ-8, and NZ-12 with NK cells reduced
growth of thoracic tumors and production of pleural effusion
in an MPM orthotopic xenograft model. By contrast, no
antitumor effects were observed following injection of theanti-human podoplanin antibody alone in this experimental
condition. Treatment of NK cells with control IgG partly
inhibited the tumor growth because of antitumor activity of
NK cells alone. Production of pleural effusion closely depends
on the tumor size above a certain level in MPM orthotopic
xenograft model. Therefore, pleural effusion was completely
inhibited in Figure 2. However, anti-human podoplanin antibody with NK cells significantly inhibited tumor weight compared with NK cells alone. These results indicate that the
antitumor effects of anti-human podoplanin antibodies in the
MPM orthotopic xenograft mouse model are mediated by NK
cell-mediated ADCC. Furthermore, cancer progression was
reduced not only by intrathoracic injection of the anti-human
podoplanin antibody, but also by i.p. injection. As the drug
injection into the thoracic cavity produced several side-effects,
including pneumothorax, inflammation, and infection, systemic
administration is preferable in clinical practice. Therefore, inthe current study, it appears that systemic treatment with anti© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Original Article
Anti-podoplanin antibody against MPM

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Fig. 3. Antitumor effects of NZ-1 i.p. injection in amalignantpleuralmesothelioma
orthotopic
xenograft model. SCID mice (n = 5) were injected
into the thoracic cavity with NCI-H290/PDPN (a) or
NCI-H226 (b) (1.0 9 106 cells). Intraperitoneal
injection of NZ-1 (100 lg) or rat IgG (rIgG; 100 lg)
began on day 0, and continued twice a week for
3 weeks. Intrathoracic injection of rat CD161a+ cells(1.0 9 106 cells) or control normal saline began on
day 3, and continued weekly for 3 weeks. Three
weeks (NCI-H290/PDPN) or 9 weeks (NCI-H226) after
tumor cell inoculation, mice were killed. *P < 0.05,**P < 0.01 (values represent mean  SE).

Fig. 4. Antitumor activity of rat–human chimericanti-human podoplanin antibody NZ-8 in an NCIH290/PDPN
malignantpleuralmesothelioma
orthotopic xenograft model. (a) Antibodydependent cellular cytotoxic activity against NCIH290/PDPN was determined with 6-h 51Cr releaseassay (effector/target ratio 100) in the presence ofhuman IgG (hIgG; 1 lg/mL), NZ-1 (1 lg/mL), or NZ-8
(1 lg/mL),
with
human
peripheralbloodmononuclear cells (MNC). (b) NCI-H290/PDPN
6(1.0 9 10 cells) was inoculated into the thoracic
cavity on day 0 (n = 5). NZ-8 (100 lg) or hIgG
(100 lg) injection (i.p.) also began on day 0, andcontinued twice a week for 2 weeks. Humannatural killer (NK) (CD56+) cells (1.0 9 106 cells) orcontrol normal saline were injected into thethoracic cavity from day 3, and continued weekly
for 2 weeks. Mice were killed 3 weeks after tumor
cell inoculation. *P < 0.05, **P < 0.01 (values
represent mean  SE).

human podoplanin antibodies would be beneficial in podoplanin-positive MPM patients.
We have shown that NZ-12, a novel rat–human chimericanti-human podoplanin antibody generated from NZ-1, withhuman NK cells induced antitumor effects against MPM
in vitro and in vivo. The ADCC activity of NZ-12 mediated byhuman NK cells was much higher than that of NZ-1 or NZ-8.
In our previous study, NZ-1 could only induce ADCC activity
by rat NK cells because NZ-1 was a rat anti-human podoplaninantibody.(9) However, NZ-8, another rat-human chimeric antihuman podoplanin antibody generated from NZ-1, could

induce ADCC activity by human NK cells as effector cells.
The reason why ADCC activity of NZ-12 was significantly
higher than that of NZ-8 is still unknown. However, it may berelated to differences in the development method of NZ-12
and NZ-8. In development of the human chimeric antibody,the kappa chain was generally used as the antibody light chain,
and NZ-8 was also established using the kappa chain. In contrast, because NZ-1 has a lambda chain of the rat antibody,
NZ-12 was also generated using the human lambda chain.
Therefore, it might be considered that the potent ADCC activity of NZ-12, compared with NZ-8, was caused by differences© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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Original Article
Abe et al.

Fig. 5. Antitumor effects of novel rat–humanchimeric anti-human podoplanin antibody NZ-12
in vitro and in vivo. (a) Antibody-dependent
cellular cytotoxicity induced by human peripheral
blood mononuclear cells (MNC) against malignant
pleural mesothelioma cell lines ACC-MESO-1, ACCMESO-4, NCI-H226, and NCI-H290/PDPN, wasevaluated by 6-h 51Cr release assay (effector/target
ratio 100) in the presence of human IgG (hIgG;
1 lg/mL), NZ-1 (1 lg/mL), NZ-8 (1 lg/mL), and NZ-12(1 lg/mL), with human MNC. (b) Complementdependent cytotoxic activity was indicated by 6-h51
Cr release assay in the presence of NZ-12 (1 lg/mL) or hIgG with baby rabbit complement (1:4
dilution). (c) Human natural killer (NK) (CD56+) cells
were isolated from human MNC. Antibodydependent cellular cytotoxic activity of NZ-12 (1 lg/mL) mediated by human NK (CD56+) cells wasevaluated by 6-h 51Cr release assay at an effector/
target ratio of 5, 10, and 25. (d, e) SCID mice(n = 5)were
injected
with
NCI-H290/PDPN(1.0 9 106 cells) into the thoracic cavity. NZ-12(100 lg) or hIgG (100 lg) injection (i.p.) was
continued twice a week for 2 weeks. Human NK(CD56+) cells (1.0 9 105 cells) were injected into thethoracic cavity weekly for 2 weeks. Three weeks
after tumor cell inoculation, the mice were killed
and the weight of thoracic tumors (white arrows)
and volume of pleural effusion were measured.
*P < 0.05, **P < 0.01 (values represent mean  SE).

Fig. 6. Combinatory effects of treatment for
malignant pleural mesothelioma with NZ-12-based
immunotherapy and pemetrexed in vivo. (a) NCIH290/PDPN was incubated with pemetrexed(0.1 lM). After 72 h of incubation, expression of
podoplanin was evaluated by FACS analysis. (b)Antibody-dependent cellular cytotoxic activity of
NZ-12 (1 lg/mL) against NCI-H290/PDPN mediatedby human peripheral blood mononuclear cells(MNC) was evaluated by 6-h 51Cr release assay(effector/target ratio 100) in the presence orabsence of pemetrexed (0.1 lM). NCI-H290/PDPN
treated with pemetrexed (0.1 lM) for 72 h was also
used in target cells. (c) SCID mice (n = 5) were
injected with NCI-H290/PDPN (1.0 9 106 cells) into
the thoracic cavity. NZ-12 (100 lg) or human IgG(hIgG; 100 lg) was injected i.p. twice a week for
2 weeks. Human natural killer (NK) (CD56+) cells(1.0 9 105 cells) or normal saline was injected into
the thoracic cavity weekly for 2 weeks. Pemetrexed(100 mg/kg, i.p.) was given on days 4, 5, 6, 11, 12,
and 13. *P < 0.05, **P < 0.01 (values represent
mean  SE). N.S., not significant.
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© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Original Article
Anti-podoplanin antibody against MPM

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in the binding affinity of NZ-12 against podoplanin due to thedevelopment method. In addition, CDC activity in ACCMESO-1 and ADCC activity in ACC-MESO-4 were relatively
lower compared to other cell lines. We previously showed that
ACC-MESO-1, ACC-MESO-4, and NCI-H226 express highlevels of podoplanin.(9) Therefore, the expression level of
podoplanin was probably not related. We speculated that thesensitivity of several cell lines against the cytotoxic mechanisms induced by NK cells or complements were related todifferences in ADCC and CDC activity.
Pemetrexed combined with cisplatin is the standard
chemotherapy used in the treatment of MPM patients. However, this combined treatment often only prolongs survival inpatients by approximately 2.8 months, compared with cisplatintreatment alone.(20) Therefore, development of novel therapeutic strategies in combination with pemetrexed are needed to
improve therapeutic treatments for MPM. In this study, prolonged treatment with pemetrexed did not change the expression level of podoplanin in MPM cells, suggesting that ADCC
activity of NZ-12 against MPM was not inhibited by pemetrexed in vitro. In addition, the combined immunotherapy of
NZ-12, mediated by human NK cells, with pemetrexed injection enhanced antitumor effects by both induction of ADCC
activity and antifolate action in an MPM orthotopic xenograftmodel. Although many anticancer chemotherapy drugs suppress immune function due to myelosuppression, several
chemotherapy drugs, including gemcitabine, combine with theeffects of immunotherapy to modulate immune function.(37,38)
Pemetrexed, which is an antifolate antimetabolite agent, islikely to induce myelosuppression. Nevertheless, it is reported
that antifolate agents enhanced the production of interleukin-2
and interleukin-12.(39) The detailed mechanism is still unclear,but our results suggest that treatment with both NZ-12mediated immunotherapy and pemetrexed induces combined

effects against intrathoracic MPM. These are likely additiveeffects, as pemetrexed had no effect on the expression of
podoplanin or ADCC activity of NZ-12 mediated by humanNK cells.
In conclusion, we found that anti-human podoplanin antibodies possess ADCC activity-induced antitumor effects in an
MPM orthotopic xenograft model. Furthermore, combined
treatment of NZ-12, which induced potent ADCC activity
mediated by human NK cells, and pemetrexed produced
enhanced antitumor effects in an MPM orthotopic xenograftmodel. These findings suggest that combination therapy using
both NZ-12 podoplanin-targeting immunotherapy and pemetrexed could provide a promising therapeutic strategy in thetreatment of MPM.

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Acknowledgments
We thank Tomoko Oka for excellent technical assistance. This work
was supported in part by: KAKENHI (24390210, 25460189, 26440019,
and 25462242) for Scientific Research (C) (to Y.N., S.A., M.K.K., andY.K.) from the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) of Japan; a research program for development ofintelligent Tokushima artificial exosome (iTEX) from Tokushima
University (to S.A.); the Platform for Drug Discovery, Informatics, andStructural Life Science from the Japan Agency for Medical Researchand Development (AMED) (to Y.K.); the Basic Science and Platform
Technology Program for Innovative Biological Medicine from AMED
(to Y.K.); and by the Regional Innovation Strategy Support Programfrom MEXT, Japan (to Y.K.).

Disclosure Statement
Yasuhiko Nishioka received research funding from Eli Lilly Japan.
The other authors have no conflicts of interest.

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