The Journal of Immunology

A Novel Targeting Therapy of Malignant Mesothelioma Using
Anti-Podoplanin Antibody
Shinji Abe,* Yuki Morita,† Mika Kato Kaneko,‡ Masaki Hanibuchi,x Yuta Tsujimoto,‡
Hisatsugu Goto,x Soji Kakiuchi,x,{ Yoshinori Aono,x Jun Huang,x Seidai Sato,xMasatoshi Kishuku,† Yuki Taniguchi,† Mami Azuma,* Kazuyoshi Kawazoe,†
Yoshitaka Sekido,‖ Seiji Yano,# Shin-ichi Akiyama,{ Saburo Sone,x,{ Kazuo Minakuchi,†
Yukinari Kato,‡ and Yasuhiko Nishiokax

Malignant pleural mesothelioma (MPM) is an aggressive malignancy arising from the mesothelial cells
lining the pleura (1). Previously, MPM was considered*Central Office for Clinical Pharmacy Training, Institute of Health Biosciences,
University of Tokushima Graduate School, Tokushima 770-8503, Japan; †Department ofPharmacy, Tokushima University Hospital, Tokushima 770-8503, Japan; ‡Regional Innovation Strategy Support Program, Tohoku University Graduate School of Medicine,
Sendai 980-8575, Japan; xDepartment of Respiratory Medicine and Rheumatology,
Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima 770-8503, Japan; {Department of Medical Oncology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima 770-8503, Japan;‖
Division of Molecular Oncology, Aichi Cancer Center Research Institute, Nagoya
464-8681, Japan; and #Division of Medical Oncology, Cancer Research Institute,
Kanazawa University, Kanazawa 920-1192, Japan
Received for publication February 15, 2013. Accepted for publication March 17,
2013.
This work was supported in part by Kakenhi (Grants 24390210, 22390166, 23790185,
23701043, and 23791584), a Grant-in-Aid for Scientific Research (B) (to Y.N. and S.S.)
and Grants-in-Aid for Young Scientists (B) (to S.A., M.K.K., and Y.K.) from the Ministryof Education, Culture, Sports, Science and Technology of Japan, the Mitsubishi
Pharma Research Foundation (to Y.K.), the Children’s Cancer Association of Japan(to Y.K.), the Intelligent Cosmos Academic Foundation (to Y.K.), the Platform for
Drug Discovery, Informatics, and Structural Life Science (to M.K.K. and Y.K.), andthe Regional Innovation Strategy Support Program (to Y.K.).
Address correspondence and reprint requests to Prof. Yasuhiko Nishioka, Departmentof Respiratory Medicine and Rheumatology, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503,
Japan. E-mail address: yasuhiko@clin.med.tokushima-u.ac.jp
Abbreviations used in this article: ADCC, Ab-dependent cellular cytotoxicity; CDC,
complement-dependent cytotoxicity; CHO, Chinese hamster ovary; CRPMI 1640,
RPMI 1640 medium supplemented with 10% FBS; MM, malignant mesothelioma;
MNC, mononuclear cell; MPM, malignant pleural mesothelioma; MSFI, mean specific fluorescence intensity; PDPN, podoplanin; TAA, tumor-associated Ag.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300448

to be rare; however, its incidence is increasing worldwide. According
to various studies, the number of cases is estimated to reach a peak
between 2010 and 2020 in Europe (2) and ∼2030–2040 in Asia(3). Despite the combined therapies including surgery, radiotherapy, and chemotherapy, the prognosis of MPM is very poor: median survival from the time of diagnosis is 4–12 mo, and the 5-ysurvival rate is ,5% (4–6). Therefore, novel therapeutic strategies
for MPM are urgently necessary for improving the prognosis.
On the basis of the current status of MPM, immunotherapy has
also been considered as one of the novel therapeutic approaches.
Recently, tumor-specific immunotherapy using vaccination with
antigenic peptides of tumor-associated Ags (TAAs) with or without
dendritic cells or Abs has been conducted because several TAAs
specific for MPM were reported, although their number is insufficient(7–9). Among them, immunotherapy with specific Abs seems to be
the most promising approach, whereas clinical trials with humanized Abs for mesothelin as well as CD26 are still in phase I (10).
Podoplanin (Aggrus), which is a type I transmembrane sialomucinlike glycoprotein, induces platelet aggregation (11–14). Originally, podoplanin was detected on the surface of podocytes (15).
Although podoplanin is reported to be expressed in limited normal tissues such as endothelium of lymphatic vessels and in type I
alveolar epithelium (16, 17), various tumors such as squamouscell carcinomas, testicular seminomas, malignant brain tumors,fibrosarcomas, and malignant mesotheliomas (MMs) including
MPM are known to show its overexpression (18–22). Furthermore,
it has been reported that podoplanin is associated with cell migration (23, 24), epithelial–mesenchymal transition (25), and tumor
metastasis (26, 27). Moreover, podoplanin serves through extra-

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Podoplanin (Aggrus), which is a type I transmembrane sialomucin-like glycoprotein, is highly expressed in malignant pleural mesothelioma (MPM). We previously reported the generation of a rat anti-human podoplanin Ab, NZ-1, which inhibited podoplanininduced platelet aggregation and hematogenous metastasis. In this study, we examined the antitumor effector functions of NZ-1 andNZ-8, a novel rat-human chimeric Ab generated from NZ-1 including Ab-dependent cellular cytotoxicity (ADCC) and complementdependent cytotoxicity against MPM in vitro and in vivo. Immunostaining with NZ-1 showed the expression of podoplanin in 73%(11 out of 15) of MPM cell lines and 92% (33 out of 36) of malignant mesothelioma tissues. NZ-1 could induce potent ADCC against
podoplanin-positive MPM cells mediated by rat NK (CD161a+) cells, but not murine splenocytes or human mononuclear cells.
Treatment with NZ-1 significantly reduced the growth of s.c. established tumors of MPM cells (ACC-MESO-4 or podoplanintransfected MSTO-211H) in SCID mice, only when NZ-1 was administered with rat NK cells. In in vivo imaging, NZ-1 efficiently
accumulated to xenograft of MPM, and its accumulation continued for 3 wk after systemic administration. Furthermore, NZ-8
preferentially recognized podoplanin expressing in MPM, but not in normal tissues. NZ-8 could induce higher ADCC mediated byhuman NK cells and complement-dependent cytotoxicity as compared with NZ-1. Treatment with NZ-8 and human NK cells
significantly inhibited the growth of MPM cells in vivo. These results strongly suggest that targeting therapy to podoplanin withtherapeutic Abs (i.e., NZ-8) derived from NZ-1 might be useful as a novel immunotherapy against MPM. The Journal of
Immunology, 2013, 190: 6239–6249.

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ANTI-PODOPLANIN Ab AGAINST MALIGNANT MESOTHELIOMA

Materials and Methods
Cell lines
In this study, we used 15 human MPM cell lines. ACC-MESO-1, ACCMESO-4, Y-MESO-8A, Y-MESO-9, Y-MESO-12, and Y-MESO-14 were
established in Aichi Cancer Research Center Institute (35). EHMES-1 wasprovided by Dr. Hironobu Hamada (Ehime University, Matsuyama, Japan)(36). NCI-H290 and NCI-H513 were provided by Dr. Adi F. Gazdar(University of Texas Southwestern Medical Center, Dallas, TX). MSTO211H, NCI-H28, NCI-H226, NCI-H2052, NCI-H2373, NCI-H2452, andChinese hamster ovary (CHO) were purchased from American Type Culture
Collection (Rockville, MD). MPM cell lines are divided into three histological origins, an epithelioid type (ACC-MESO-1, ACC-MESO-4,
Y-MESO-9, Y-MESO-12, NCI-H290, NCI-H513, NCI-H226, and NCIH2452), a sarcomatoid type (NCI-H28, NCI-H2052, and NCI-H2373), anda biphasic type (Y-MESO-8A, Y-MESO-14, EHMES-1, and MSTO-211H)(37–39). These cells were cultured in RPMI 1640 medium supplemented
with 10% FBS (CRPMI 1640; Life Technologies, Grand Island, NY),
100 U/ml penicillin, and 100 mg/ml streptomycin (Meiji Seika Kaisha,Tokyo, Japan). Cell lines were authenticated by DNA fingerprinting.
MSTO-211H cells were transfected with appropriate amounts of plasmids,
pcDNA3/human podoplanin (PDPN) or pcDNA3/mock (11), using Metafectene (Nippon Genetics, Tokyo, Japan) according to the manufacturer’s
instructions. Stable transfectants (MSTO-211H/PDPN and MSTO-211H/mock) were selected by cultivating the transfectants in medium containing
0.5 mg/ml Geneticin (Invitrogen, Carlsbad, CA).

Abs
NZ-1, a rat anti-human podoplanin mAb (IgG2a), was developed as described previously (31). Rat IgG and human IgG were purchased from
Beckman Coulter (Fullerton, CA). For the generation of rat-human chimera anti-human podoplanin (NZ-8), the appropriate VH and VL cDNAs ofa rat NZ-1 Ab and CH and CL of human IgG1 were subcloned into the
pcDNA3.3/Neo or pcDNA3.1/Zeo vectors (Life Technologies), respectively. The cDNAs coding for the VH and VL (k-chain) regions wereconstructed by a PCR-based method. Ab expression vectors were transfected into CHO cells using Lipofectamine 2000 reagent (Life Technologies). Stable transfectants of CHO/NZ-8 were selected by cultivating thetransfectants in medium containing 1 mg/ml Geneticin and 0.5 mg/ml
Zeocin (Life Technologies). CHO/NZ-8 cells were cultivated in CHO-SSFM II medium (Life Technologies). The media containing NZ-8 were
centrifuged, and the obtained supernatant was applied to a column ofprotein G-Sepharose (Thermo Scientific, Rockford, IL). After extensive
washing with PBS, the fusion proteins were eluted using 0.1 M glycine and0.15 M NaCl (pH 2.8) and then neutralized with 1 M Tris (pH 10). The
Abs were dialyzed against PBS. Expression and purity of the proteins wereconfirmed by SDS-PAGE using 5–20% gradient gels (Wako Pure Chemical
Industries, Osaka, Japan). F(ab9)2 fragments of NZ-1 Ab were preparedusing F(ab9)2 Preparation Kits (Thermo Scientific) according to the manufacturer’s instructions. D2-40 (mouse anti-human podoplanin mAb) was
purchased from DakoCytomation (Glostrup, Denmark).

Animals
Male SCID mice, 5 to 6 wk old, and male Wistar rats, 6 wk old, wereobtained from CLEA Japan (Osaka, Japan) and maintained under specificpathogen-free conditions throughout this study. All animals were acclimatized for at least 1 wk before experiments. All experiments were performed in accordance with the guidelines of University of Tokushima,
Committee on Animal Care and Use.

Flow cytometry
Expression of podoplanin was detected by flow cytometry. Cells (5 3 105)
were washed with cold PBS and stained on ice for 30 min with NZ-1
(1 mg/ml), NZ-1 F(ab9)2 fragment (0.675 mg/ml), NZ-8 (1 mg/ml), rat IgG(1 mg/ml), or human IgG (1 mg/ml). After incubation with the primary mAbs,
cells were washed with cold PBS and then incubated on ice for 30 min with
FITC-conjugated goat F(ab9)2 fragment anti-rat IgG (H+L) Ab or FITCconjugated goat F(ab9)2 Fragment anti-human IgG (Fcg) Ab (Beckman
Coulter). The cells were washed again and resuspended in cold PBS. The
analysis was performed on an FACSCalibur flow cytometer with CellQuest
software (BD Biosciences, Franklin Lakes, NJ). The mean specific fluorescence intensity (MSFI) was calculated as the ratio of the mean fluorescenceintensity of NZ-1 to that of control mAb. Positive expression of podoplanin
was defined as an MSFI of .2.0, and high-level expression of podoplanin
was defined as an MSFI of .10.0.

Immunohistochemistry
Tissue arrays of human mesothelioma were obtained from Biomax (Rockville,
MD). Staining was performed using R.T.U. VECTASTAIN Elite ABC
Kit (Vector Laboratories, Burlingame, CA) (40). The tissue arrays weresteamed in sodium citrate buffer for 10 min using a microwave to retrieve
Ag and incubated in 3% H2O2 for 30 min to inhibit endogenous peroxidase. After incubation in blocking serum for 20 min, the slides were incubated overnight with NZ-1, NZ-8, or D2-40 at 4˚C. The slides were
washed and incubated in prediluted biotinylated secondary Ab for 30 min,followed by incubation in ready-to-use streptavidin/peroxidase complex
reagent for 30 min. The tissue arrays were developed with a diaminobenzidine
substrate kit (Vector Laboratories) and counterstained with Mayer’s hematoxylin (Muto Pure Chemicals, Tokyo, Japan).

Western blotting
Cells were rinsed with ice-cold PBS and lysed in M-PER reagent (Pierce,
Rockford, IL) containing phosphatase and protease inhibitor cocktails(Roche, Indianapolis, IN). The concentrations of protein were determinedusing a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA). Cell lysate (15mg/lane) was separated by SDS-PAGE. Immunoblotting was conducted aspreviously described (41).

Preparation of effector cells
Rat splenocytes were harvested from Wistar rat spleens. Spleens wereminced, homogenized in RPMI 1640, and centrifuged after passing throughthe cell strainer (BD Biosciences) (40). The cell pellet was suspended in
RBC lysis buffer (Sigma-Aldrich, St. Louis, MO) on ice for 10 min. After
depletion of RBCs, splenocytes were resuspended in CRPMI 1640 andused as effector cells. The method by which SCID mouse splenocytes were
harvested was the same as that of rat splenocytes. Human PBMCs (MNC)
were obtained from leukocytes in lymphocyte separation medium (Litton
Bionetics, Kensington, MD) as previously described (42). Leukocytes wereseparated from peripheral blood of healthy donors using an RS-6600 rotorof a Kubota KR-400 centrifuge (Kubota, Tokyo, Japan). The human study
was approved by the ethics committee of University of Tokushima, andwritten informed consent was obtained from all of the subjects.

ADCC
ADCC was evaluated using a [51Cr]release assay as described previously(40, 43). Target cells were targeted with 0.1 mCi [51Cr]sodium chromate
at 37˚C for 1 h. 51Cr-labeled target cells were placed in 96-well plates
in triplicate. Effector cells and NZ-1, NZ-1 F(ab9)2 fragment, NZ-8, or
control IgG were added to the plates. After 6 h incubation, 100 ml supernatant was measured in a g counter (PerkinElmer, Waltham, MA).
Percent of cytotoxicity was calculated from the following formula: percent
specific lysis = (E 2 S)/(M 2 S) 3 100, where E is the release in the test
sample, S is the spontaneous release, and M is the maximum release.

Complement-dependent cytotoxicity
CDC was determined by [51Cr]release assay (40). Target cells were incubated with 0.1 mCi [51Cr]sodium chromate at 37˚C for 1 h. After in-

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cellular matrix signaling pathways for cell cycle and cell proliferation (28), and increase of expression of podoplanin is also related
to tumor malignancy and poor clinical outcome (22, 29, 30).
To establish targeted therapy to podoplanin, we previously
generated a rat anti-human podoplanin mAb NZ-1 (31, 32). NZ-1
suppressed podoplanin-induced pulmonary metastasis by inhibiting tumor-induced platelet aggregation (27). Furthermore, we
showed that NZ-1 has not only high specificity and sensitivity but
also high binding affinity against podoplanin, leading to its status
as a candidate for radioimmunotherapy or immunotoxin therapy(33). However, it has not been clarified whether NZ-1 has antitumor effector functions including Ab-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC),
which is the most important mechanism contributing to the clinical efficacy of therapeutic Abs (34).
In the current study, we investigated whether NZ-1 can induce
antitumor effects mediated by ADCC and CDC against MPM
in vitro and in vivo. Furthermore, we also evaluated antitumor activity of NZ-8, a novel rat-human chimeric anti-human podoplanin
Ab, using human mononuclear cells (MNC) as effector cells against
MPM cells.

The Journal of Immunology
cubation, the cells were washed with CRPMI 1640 three times. 51Crlabeled cells were added into 96-well plates and incubated with baby rabbit complement (Cedarlane Laboratories, Ontario, Canada) at a dilution of
1:4 and NZ-1 (1 mg/ml), NZ-8 (1 mg/ml), or control IgG (1 mg/ml) for 6 h.
[51Cr]Release of the supernatant from each well (100 ml) was measured
using a g counter. Percent of cytotoxicity was calculated as above.

Depletion or separation of rat NK cells
Depletion of NK cells from rat splenocytes was performed using the antiasialo GM1 Ab as follows. Harvested rat splenocytes were treated with antiasialo GM1 Ab (Wako, Osaka, Japan) at room temperature for 30 min. After
treatment, splenocytes were incubated with baby rabbit complement at
37˚C for 30 min, then washed with CRPMI 1640 three times, and thenumber of cells for use in experiments was counted. Rat NK cells were separated from Wistar rat splenocytes using a magnetic cell-sorting system.
Splenocytes were incubated with FITC-conjugated anti-CD161a Ab (BDBiosciences) at 4˚C for 30 min and then with anti-FITC mAb-coupled

6241
superparamagnetic microbeads (Miltenyi Biotec, Auburn, CA) at 4˚C for
15 min. CD161a-positive or -negative selection was carried out using an
autoMACS (Miltenyi Biotec) according to the manufacturer’s instructions(44). Isolated CD161a+ and CD161a2 cells that yielded purity $90 and$99.9%, respectively, as determined by flow cytometry were used in experiments. To purify CD14+ cells and CD56+ cells from human MNC,
AutoMACS and CD14 or CD56 microbeads (Miltenyi Biotec) were used.
Human MNC were treated with CD14 or CD56 microbeads at 4˚C for 15
min, and then cells were separated by autoMACS. The purities of CD14+
and CD56+ cells were $90%.

Animal experiments
SCID mice were injected s.c. into the right flank with ACC-MESO-4 (4.0 3106 cells) or MSTO-211H/PDPN (1.0 3 106 cells). Tumor size is expressed
as tumor area (mm2 ): the longest diameter 3 the shortest diameter. The
tumor-bearing mice were divided into several experimental groups before
treatment with Abs was initiated. An i.p. injection of NZ-1 (100 mg), NZ-1

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FIGURE 1. Expression of podoplanin in MM. (A) Expression of podoplanin in MPM cell lines was detected by flow cytometry. MPM cell lines were
stained with NZ-1 (bold line) and control IgG (hatched area). The number in the top right corner in each figure is the MSFI. (B) Immunohistochemistrydemonstrated the expression of podoplanin using the tissue array. Tissues were stained with 1 mg/ml of NZ-1 (original magnification 3200).

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ANTI-PODOPLANIN Ab AGAINST MALIGNANT MESOTHELIOMA

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FIGURE 2. ADCC and CDC of NZ-1 against MPM cells. (A–C) ADCC activity of NZ-1 against various MPM cells was evaluated by 6-h [51Cr]releaseassay in the presence of 1 mg/ml control rat IgG (rIgG; open column) or NZ-1 (filled column) at E:T ratio of 100. Human MNC (A), SCID mousesplenocytes (B), and Wistar rat splenocytes (C) were used as effector cells. (D) CDC activity of NZ-1 was determined using the 6-h [51Cr]release assay inthe presence of 1 mg/ml Ab with baby rabbit complement at a dilution of 1:4. E:T ratio (E) and dose dependence of NZ-1 (F) as well as time course of
ADCC (G) were detected by [51Cr]release assay using rat splenocytes as effector cells. Unchanged experimental conditions were 1 mg/ml Ab, 100 as the
E:T ratio, and duration of 6 h. *p , 0.05 versus control, **p , 0.01 versus control (values are means 6 SE).

The Journal of Immunology
F(ab9)2 fragment (67.5 mg), NZ-8 (100 mg), control rat IgG (100 mg), or
control human IgG (100 mg) was started, on average, 14 d after tumor
inoculation, when the tumor size had reached 15 (6 3 SD) mm2 , andcontinued twice a week for 2 wk. Rat CD161a+ cells (1.0 3 105 cells),
human CD56+ cells (1.0 3 105 cells), or control normal saline injection(s.c.) around the tumors continued weekly for 2 wk.

In vivo fluorescence imaging
In vivo fluorescence imaging was performed using IVIS Spectrum (Caliper
Life Sciences, Hopkinton, MA) (45). ACC-MESO-4 (4.0 3 106 cells) was
injected into the right flank of SCID mice (s.c.). When the tumor grew toa sufficient size (25 mm2), 100 mg NZ-1 or rat IgG labeled with XenoLight
CF770 (Caliper Life Sciences) was injected (i.p.). The fluorescence in eachmouse was determined using IVIS Spectrum (Caliper Life Sciences), withan excitation wavelength of 730–760 nm and an emission wavelengthof 830–850 nm, every day after the labeled Ab injection. Mice wereanesthetized with isoflurane (Abbott Laboratories, Chicago, IL). Images
were acquired and analyzed using Living Image 3.1 software (Caliper
Life Sciences).

Statistical analyses

Results
Detection of podoplanin expression in MPM cell lines andtissues
First, we evaluated the expression of podoplanin on the surface of
MPM cells using NZ-1, which is highly reactive for podoplanin.
Fig. 1A shows that MPM cells expressed various levels of podoplanin as determined by flow cytometry. Expression of podoplanin
was detected on 73% (11 out of 15) of MPM cells, and sarcomatoid type showed a higher expression rate of 100% (3 out of 3)
as compared with 75% of epithelioid type (6 out of 8) and 50% of
biphasic type (2 out of 4), although the number of cell lines is not
sufficient to determine the expression rate. High level of expression of podoplanin, which is defined as a ratio of the mean fluorescence intensity of NZ-1 to that of control IgG of .10, wasfound in 33% (5 out of 15) of all cells, of which four cell lines are
in an epithelioid type.
We next examined podoplanin expression in tissues of mesothelioma by immunohistochemistry with NZ-1. As shown in Fig.
1B, strong intensity of podoplanin staining was observed in tissuesof MM, except benign mesothelioma. Podoplanin expression was
found in 92% (33 out of 36) of all MM tissues.
ADCC and CDC mediated by NZ-1 against MPM cells
Next, we attempted to clarify whether NZ-1 has the potential toinduce ADCC-targeted MPM cells expressing podoplanin. As shownin Fig. 2A and 2B, using human MNC or SCID mouse splenocytes
as effector cells, we could not detect ADCC induced by NZ-1. Bycontrast, significant ADCC against only podoplanin-positive, but
not podoplanin-negative, MPM cells was induced by NZ-1 whensplenocytes of Wistar rat were used as effector cells (Fig. 2C). The
level of ADCC was strongly associated with the level of podoplanin expression (Figs. 1A, 2C). In contrast, CDC induced by
NZ-1 was detected only against ACC-MESO-4 cells (Fig. 2D).
Additionally, treatment with 1 mg/ml NZ-1 alone did not inhibit
proliferation of these MPM cell lines, indicating that NZ-1 has nodirect growth-inhibitory activity on MPM cells in our experimental conditions (data not shown).
To evaluate the kinetics of ADCC activity mediated by NZ-1, we
analyzed the E:T ratio and time course of ADCC and dose dependenceof NZ-1. As shown in Fig. 2E, the level of ADCC was dependent on
E:T ratio. In addition, NZ-1 also effectively induced ADCC against
podoplanin-expressing cells in a dose- and time-dependent manner(Fig. 2F, 2G). The optimal concentration of NZ-1 to induce ADCC
seems to be .0.1 mg/ml. These results suggest that rat antipodoplanin Ab NZ-1 exhibits potent ADCC against MPM cells.
NZ-1 induces ADCC against podoplanin-transfected MPM celllines
To confirm that ADCC of NZ-1 depends on the expression of
podoplanin on target cells, we used MSTO-211H/PDPN as target

FIGURE 3. NZ-1 induces ADCC against MSTO-211H transfected with podoplanin. (A) Expression of podoplanin was detected by FACS analysis. (B)
ADCC activity of NZ-1 using rat splenocytes against MSTO-211H/PDPN was evaluated by 6-h [51Cr]release assay in the presence of 1 mg/ml Ab at the
E:T ratio of 100. (C) E:T ratio–, dose-, and time-dependent effects of ADCC against MSTO-211H/PDPN mediated by NZ-1 with rat splenocytes
were demonstrated by [51Cr]release assay. **p , 0.01 versus control (values are means 6 SE). rIgG, rat IgG.

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The statistical significance of differences in in vitro and in vivo data wereanalyzed by Student t test and one-way ANOVA. In this study, p values,0.05 were considered significant in all experiments.

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ANTI-PODOPLANIN Ab AGAINST MALIGNANT MESOTHELIOMA

FIGURE 4. Rat NK cells are the main
effector cells in ADCC induced by NZ-1.
(A) Effects of depletion of NK cells from rat
splenocytes on ADCC by NZ-1. NK cells in
rat splenocytes were depleted by treatmentof anti-asialo GM1 Ab and baby rabbit complement. (B) Rat NK cells are involved in
ADCC activity of NZ-1. NK (CD161a+) cells
or NK-removed (CD161a2) cells were isolated from rat splenocytes by AutoMACS.
ADCC activity of 1 mg/ml Ab for 6 h was
examined in ACC-MESO-4 as a target
cell. **p , 0.01 versus control (values aremeans 6 SE). rIgG, Rat IgG.

ADCC of NZ-1 is mediated by rat NK cells
For further investigation of the type of effector cells in rat splenocytes, we depleted NK cells using anti-asialo GM1 Ab and baby
rabbit complement. As shown in Fig. 4A, ADCC was not induced
by NK cell–depleted splenocytes. Moreover, we isolated NK(CD161a+) cells and examined ADCC using NZ-1 against ACCMESO-4. Fig. 4B shows that the purified NK (CD161a+) cells
from rat splenocytes showed a significant level of ADCC. In
contrast, CD161a2 splenocytes did not induce ADCC by NZ-1.
Antitumor activity of NZ-1 on the growth of
podoplanin-expressing MPM cells in vivo
To evaluate the antitumor activity of NZ-1 in vivo, we used s.c. xenograft of human MPM (ACC-MESO-4) in SCID mice. NZ-1 orcontrol rat IgG was injected i.p. twice a week, and rat NK(CD161a+) cells or normal saline was injected s.c. weekly for
2 wk because ADCC of NZ-1 was mediated only by rat NK cells
but not mouse splenocytes. Treatment with NZ-1 was started whentumors were established and formed a visible tumor mass (.15mm2) owing to the evaluation of therapeutic antitumor abilityof NZ-1. The antitumor activity of NZ-1 against ACC-MESO-4
in vivo is shown in Fig. 5A. Administration of NZ-1 with rat
NK (CD161a+) cells significantly inhibited the growth of ACCMESO-4 cells. By contrast, the injection of neither NZ-1 nor NK(CD161a+) cells alone showed antitumor effects in our experimental setting. Using an MSTO-211H/PDPN xenograft model,
tumor growth was also significantly inhibited by treatment with
NZ-1 and NK (CD161a+) cells (Fig. 5B).
Critical contribution of Fc portion to antitumor activity of NZ-1
It is known that ADCC activity is mediated by binding of the Fc
portion of Abs to the FcR on NK cells. Therefore, we examined theantitumor activity of NZ-1 F(ab9)2 fragment. Although the NZ-1 F(ab9)2 fragment recognized podoplanin on ACC-MESO-4 in flow
cytometric analysis to a similar extent as intact NZ-1, the F(ab9)2
fragment did not induce ADCC against ACC-MESO-4 (Fig. 6A,

FIGURE 5. Antitumor activity of
NZ-1 against MPM cells in vivo.
SCID mice (n = 5) were injected s.c.
with 4.0 3 106 ACC-MESO-4 cells(A) or 1.0 3 106 MSTO-211H/PDPN cells (B). NZ-1 (100 mg) orcontrol rat IgG (rIgG; 100 mg) injection (i.p.) was continued twicea week for 2 wk. Rat NK (CD161a+)cell (1.0 3 105 cells) injection (s.c.)around the tumors continued weekly
for 2 wk. **p , 0.01 versus control(values are means 6 SE).

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cells. MSTO-211H/PDPN was generated by transfecting pcDNA3/
podoplanin into MSTO-211H, one of the podoplanin-negative
MPM cell lines (Fig. 3A). Although ADCC of NZ-1 was notdetected against MSTO-211H/Mock, NZ-1 exhibited significant
ADCC activity against MSTO-211H/PDPN (Fig. 3B). E:T ratio–,dose-, and time-dependent ADCC activities against MSTO-211H/PDPN were also induced by NZ-1 (Fig. 3C).

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FIGURE 6. Critical contribution of Fc
portion to antitumor activity of NZ-1. (A)Binding ability of NZ-1 F(ab9)2 fragment topodoplanin on the cell surface was compared with that of NZ-1 by FACS analysis.
(B) ADCC activity against ACC-MESO-4
was evaluated by [51Cr]release assay in thepresence of control rat IgG (rIgG), NZ-1, orNZ-1 F(ab9)2 fragment with rat splenocytes.
(C) Antitumor effect of NZ-1 (100 mg), NZ1 F(ab9)2 fragment (67.5 mg), or control rIgG
(100 mg) injection (i.p.) was evaluated using
ACC-MESO-4 s.c. xenograft SCID mousemodel (n = 5). **p , 0.01 versus control(values are means 6 SE).

ACC-MESO-4 after single i.p. injection. Quantification of accumulation to the tumor of NZ-1 or control rat IgG showed a peak of
the accumulation of NZ-1 on day 5, and the accumulation could
be detected over 3 wk (Fig. 7B).

In vivo accumulation of injected NZ-1 to MPM xenograft in
SCID mice

Immunostaining of podoplanin expressing in MPM and normal
tissue with a novel rat-human chimeric anti-human podoplanin
Ab NZ-8

Next, we investigated the accumulation of injected NZ-1 on s.c.
podoplanin-positive MPM tumor in SCID mouse model. As shownin Fig. 7A, Xenolight CF770-labeled NZ-1 accumulated on s.c.

Because it was demonstrated that NZ-1 showed antitumor activity
through ADCC mediated by rat NK cells, to apply targeted therapy

FIGURE 7. In vivo fluorescence imaging of NZ-1 in MPM xenograft model in SCID mice. (A) Fluorescence-labeled NZ-1 (100 mg) or control rat IgG (rIgG;
100 mg) was injected once i.p. to ACC-MESO-4 s.c. xenograft SCID mouse model (n = 3). (B) Accumulation of fluorescence in s.c. tumors was calculated andanalyzed using IVIS Spectrum (Caliper Life Sciences) every day after the labeling Ab injection. **p , 0.01 versus control (values are means 6 SE).

Downloaded from http://www.jimmunol.org/ by guest on June 9, 20196B). Moreover, i.p. injection of NZ-1 F(ab9)2 fragment unalterably
showed no effect on tumor growth of ACC-MESO-4 comparedwith intact NZ-1, even when rat NK cells were simultaneously
administered (Fig. 6C).

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ANTI-PODOPLANIN Ab AGAINST MALIGNANT MESOTHELIOMA

to podoplanin with Ab in a clinic, we generated the chimeric Ab byfusing the VH and VL regions of rat Ab (NZ-1) with the CH and CL
regions of human IgG1, respectively (Fig. 8A). As shown in Fig.
8B and 8C, NZ-8, a novel rat-human chimeric anti-human podoplanin Ab, could recognize the podoplanin expressing in MPM
like NZ-1 by flow cytometry as well as immunohistochemistry.
Moreover, to confirm the recognition pattern of NZ-8 to podoplanin, the lysate of MPM cells were subjected to Western blotting. As shown in Fig. 8D, NZ-8 could detect the ladder bands of
podoplanin. However, the recognizing pattern of NZ-8 was different from that of D2-40. Thinking of clinical application of NZ-

8, the recognition of podoplanin expressing in normal tissue by
NZ-8 is quite important. Therefore, we examined the staining
pattern of podoplanin expressing in normal tissue such as lymphatic vessels, alveolar epithelial cells, and podocytes in thekidney by immunohistochemistry with NZ-8 because these tissues
have been reported to express podoplanin. As shown in Fig. 8E,
NZ-8 preferentially recognized the podoplanin expressing in MPM,
but not in normal tissues including lymphatic vessels, alveolar
epithelial cells, and podocytes. However, D2-40 strongly detected
the podoplanin expressing in lymphatic vessels as compared with
MPM tissues.

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FIGURE 8. Immunostaining of podoplanin expressing in MPM and normal tissue with NZ-8. (A) SDS-PAGE of NZ-1 and NZ-8. SDS-PAGE using 5–
20% gel was performed for purified NZ-1 and NZ-8 under the reducing condition. (B) Expression of podoplanin in MPM cell lines was detected by flow
cytometry. MPM cell lines were stained with NZ-1 or NZ-8 (bold line) and control IgG (hatched area). (C) Immunohistochemistry demonstrated thedetection of podoplanin expression using the tissue array. Tissues were stained with NZ-1 (1 mg/ml) or NZ-8 (1 mg/ml) (original magnification 3200). (D)Podoplanin (PDPN) protein in MPM cell lines was recognized by Western blotting proved with D2-40 (1:500) or NZ-8 (0.1 mg/ml). (E) The expression of
podoplanin was examined in lymphatic vessels (arrowheads) and type I alveolar epithelium (lung) and podocyte (kidney) as well as MM using the tissue
array. The slides of the tissue array were stained with D2-40 (1:500) or NZ-8 (0.1 mg/ml) (original magnification 3200).

The Journal of Immunology
In vitro and in vivo antitumor activity of NZ-8 against MPM
cells

Discussion
In the current study, we have demonstrated that a rat anti-human
podoplanin mAb, NZ-1, possesses potent ADCC mediated by rat

FIGURE 9. Antitumor activity of NZ-8 in vitro andin vivo. (A) ADCC induced by human MNC against
MPM cell lines, ACC-MESO-1, ACC-MESO-4, NCIH226, and MSTO-211H, was determined with 6-h[51Cr]release assay at the E:T ratio of 100 in thepresence of 1 mg/ml NZ-1 or NZ-8. (B) CDC activity
was demonstrated by [51Cr]release assay. The experimental condition was the same as for Fig. 2D. (C)Human NK cells are the main effector cells in ADCC
activity of NZ-8. NK (CD56+) cells or monocytes (CD14+)
were isolated from human MNC by AutoMACS. ADCC
activity of 1 mg/ml NZ-8 mediated by NK (CD56+) cells
or monocytes (CD14+) was evaluated by 6-h [51Cr]release assay at E:T ratio of 10 or 50. SCID mice (n = 5)
were injected s.c. with 4.0 3 106 ACC-MESO-4 cells.
NZ-8 (100 mg) or human IgG (hIgG; 100 mg) (D),and NZ-1 (100 mg), NZ-8 (100 mg), or rat IgG (rIgG;
100 mg), and human IgG (hIgG; 100 mg) (E) injection(i.p.) was continued twice a week for 2 wk. Human NK(CD56+) cell (1.0 3 105 cells) injection (s.c.) around thetumors continued weekly for 2 wk. **p , 0.01 versus
control (values are means 6 SE).

NK cells and CDC activity and mediates in vivo therapeutic antitumor activity against MPM in SCID mice. Moreover, we have also
shown that a novel rat-human chimeric anti-human podoplanin
Ab, NZ-8, effectively induces ADCC mediated by human NK cells
and CDC and mediates in vivo antitumor activity against MPM
cells. Furthermore, NZ-8 preferentially recognized the podoplanin
expressing in MPM than those in normal tissues. These results
strongly suggest the possible clinical application of anti-human
podoplanin Ab against MPM.
In this study, we found that podoplanin was expressed on 73%
of MPM cell lines including 33% of high-expression and 92% of
MM tissues, which is consistent with previous studies (46–48).
Furthermore, NZ-8 could recognize the expression of podoplanin
in MPM like NZ-1. Compared with other mesothelioma-associatedAgs such as calretinin, thrombomodulin, cytokeratin 5, WT1, andmesothelin in previous studies (47–49), the frequency of expression of podoplanin is the same or higher than that of others, suggesting that podoplanin could be a novel immunological target
for MPM therapy. In contrast, although it was reported that podoplanin was expressed in lymphatic vessels and type I alveolar
epithelium and podocyte (15–17), NZ-8 could not detect the expression of podoplanin in lymphatic vessels when it was used at
the lower concentration of 0.1 mg/ml, which could still recognize
the podoplanin in MPM tissues and induce the ADCC and CDC
reactions. This staining pattern of NZ-8 is quite different from that

Downloaded from http://www.jimmunol.org/ by guest on June 9, 2019

We further assessed whether NZ-8 can induce antitumor effectagainst MPM cells mediated by human MNC as effector cells.
As shown in Fig. 9A, ADCC against MPM cell lines was notexhibited by NZ-1 with human MNC, whereas NZ-8 showed theinduction of a significant level of ADCC mediated by human
MNCs against podoplanin-positive MPM cells but not podoplaninnegative MPM cells. CDC was also induced by NZ-8 against ACCMESO-4 and NCI-H226, which was significantly higher than thatof NZ-1 (Fig. 9B). Further effector cell analysis of ADCC clearly
demonstrated that human NK (CD56+) cells, but not human monocytes (CD14+), could induce ADCC by NZ-8 (Fig. 9C). Using the
xenograft model of ACC-MESO-4 in SCID mice, the tumor growth
was also significantly inhibited by treatment with NZ-8 and humanNK (CD56+) cells (Fig. 9D). By contrast, no antitumor effects wereshown by the injection of NZ-8 or human NK (CD56+) cells alone.
Furthermore, administration of NZ-1 did not show any antitumor
effects when human NK (CD56+) cells were used as an effector
cells (Fig. 9E).

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ANTI-PODOPLANIN Ab AGAINST MALIGNANT MESOTHELIOMA
was estimated that NZ-1 has strong binding affinity and specificaccumulation potential against target Ags. These results indicatedthat NZ-1 might have strong antitumor effects against MPM in vivo
by ADCC.
Finally, we have shown that NZ-8, a novel rat-human chimericanti-human podoplanin Ab, generated from NZ-1 has the potency
to induce ADCC against MPM cells mediated by human NK cells
as effector cells. Interestingly, the level of ADCC and CDC of NZ-8
was significantly higher than that of NZ-1. Moreover, NZ-8 has thepromising antitumor effects against MPM cells mediated by humanNK cells in vivo, in contrast to administration of NZ-1 with humanNK cells, which did not induce any antitumor effects. In addition,
NZ-8 preferentially detected the podoplanin expressing in MPM,
but not in normal tissues.
In conclusion, we found that both NZ-1 and NZ-8 possess
potent and therapeutic antitumor effects based on ADCC against
MPM in an SCID mouse model. Importantly, NZ-8 could induce
higher ADCC mediated by human NK cells in addition to CDC as
compared with NZ-1. Based on the ability of NZ-8, which preferentially recognizes the podoplanin expressing in MPM, but not
in normal tissues, the current study suggests that targeting therapy
to podoplanin using NZ-8 might be useful for patients with MPM
as a novel immunotherapy.

Acknowledgments
We thank Tomoko Oka, Imi Saito, Ryo Yanagiya, Shunpei Morita, Kana Nasu,
Hiroko Sasaki, Sachiko Suzuki, and Junko Aita for excellent technicalassistance.

Disclosures
The authors have no financial conflicts of interest.

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ADCC activity of NZ-1 has not been reported because NZ-1 did
not show any ADCC activity mediated by murine splenocytes orhuman MNCs. However, when ADCC of NZ-1 was examined using
rat splenocytes, significant ADCC activity was detected. To use
rat splenocytes as effector cells is unusual to evaluate the ADCC
activity of Ab; however, other studies have also reported that, in
special cases, rat splenocytes are more useful than murine ones fortesting the ADCC activity of rat mAb (51, 52). If the Ab has
potency to induce the ADCC mediated by effector cells of several
species except for humans effectively, it would be possible toprepare chimeric or humanized Ab that induces ADCC mediatedby human effector cells. Therefore, from our study, it is also notedthat rat splenocytes have the same importance to screen the ADCC
activity of Ab as human MNC or mouse splenocytes.
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