Published OnlineFirst January 28, 2016; DOI: 10.1158/2326-6066.CIR-15-0060

CancerImmunology
ResearchResearch Article

CAR T Cells Targeting Podoplanin Reduce
Orthotopic Glioblastomas in Mouse Brains
Satoshi Shiina1, Masasuke Ohno1,2, Fumiharu Ohka1, Shunichiro Kuramitsu1,3,
Akane Yamamichi1, Akira Kato1, Kazuya Motomura1, Kuniaki Tanahashi1,
Takashi Yamamoto1, Reiko Watanabe4, Ichiro Ito4, Takeshi Senga5, Michinari Hamaguchi5,
Toshihiko Wakabayashi1, Mika K. Kaneko6, Yukinari Kato6, Vidyalakshmi Chandramohan7,
Darell D. Bigner7, and Atsushi Natsume1

Abstract
Glioblastoma (GBM) is the most common and lethal primarymalignant brain tumor in adults with a 5-year overall survival
rate of less than 10%. Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, expressed in the lymphatic
endothelium. Several solid tumors overexpress PDPN, including the mesenchymal type of GBM, which has been reported topresent the worst prognosis among GBM subtypes. Chimericantigen receptor (CAR)–transduced T cells can recognize predeﬁned tumor surface antigens independent of MHC restriction, which is often downregulated in gliomas. We constructeda lentiviral vector expressing a third-generation CAR compris-

ing a PDPN-speciﬁc antibody (NZ-1–based single-chain variable fragment) with CD28, 4-1BB, and CD3z intracellulardomains. CAR-transduced peripheral blood monocytes were
immunologically evaluated by calcein-mediated cytotoxic
assay, ELISA, tumor size, and overall survival. The generated
CAR T cells were speciﬁc and effective against PDPN-positive GBM
cells in vitro. Systemic injection of the CAR T cells into an
immunodeﬁcient mouse model inhibited the growth of intracranial glioma xenografts in vivo. CAR T-cell therapy that targets
PDPN would be a promising adoptive immunotherapy to treat
mesenchymal GBM. Cancer Immunol Res; 4(3); 259–68. 2016 AACR.

Introduction

consisted of an extracellular domain derived from a single-chain
variable fragment (scFv) taken from a tumor antigen–speciﬁc
monoclonal antibody (mAb), a transmembrane domain, and acytoplasmic signaling domain from the CD3z chain, a subunit of
the T-cell receptor complex (3). CAR-transduced T cells canrecognize predeﬁned tumor surface antigens independent of the
major histocompatibility complex (MHC) restriction, which isoften downregulated in gliomas (4). CARs can be designed tobind not only to proteins, but also to carbohydrate and glycolipid structures (5). Second-generation CARs, which incorporatea single costimulatory signaling domain such as CD28 (6, 7),
CD137 (4-1BB; refs. 6, 8), or CD134 (OX40; refs. 6, 8), have also
been generated. T cells transduced with a CAR containing theCD28 signaling domain could enhance IL2 production (7),sustain T-cell proliferation (7), and resist immune suppression
mediated by transforming growth factor-b (TGFb) and regulatory
T cells (Treg; ref. 9). The inclusion of domains derived fromthe tumor necrosis factor receptor family members 4-1BB andOX40 into CARs has also been shown to enhance the cytotoxicity
of CAR-transduced T cells (8). Third-generation CARs, combining
two costimulatory domains, such as CD28 and 4-1BB, have been
described and are highly likely to lyse tumor cells (10). Adoptive
transfer of CAR-transduced T cells has been clinically applied in
the treatment of CD19-positive leukemia and lymphoma (11, 12)
as well as GD2-positive neuroblastoma (13). These data provide
a strong rationale for the pursuit of CAR T-cell therapy forother types of cancers, such as GBM. We and other groups havegenerated several CARs against the antigens expressed in GBM,
including epidermal growth factor receptor variant III (EGFRvIII;
ref. 14), human epidermal growth factor receptor 2 (HER2;
ref. 15), IL13 receptor alpha 2 (IL13Ra2; ref. 16), and ephrin

Glioblastoma (GBM) is the most common and lethal primarymalignant brain tumor in adults. After maximal surgical resection,
the current standard of care is concurrent radiotherapy and thealkylating agent temozolomide (TMZ), followed by adjuvant
TMZ (1). Despite the improvement in outcomes with this combined chemoradiotherapy approach, the median survival is 14.6
months and 5-year overall survival (OS) rates are less than 10%(1). Thus, novel therapies are required to improve patient
survival.
In recent years, immunotherapy has emerged as a promisingstrategy for the treatment of GBM (2). The ﬁrst generation ofchimeric antigen receptors (CAR) were recombinant receptors that1
Department of Neurosurgery, Nagoya University School of Medicine,
Nagoya, Japan. 2Division of Neurosurgery, National Hospital Organization Nagoya Medical Center, Nagoya, Japan. 3Division of Neurosurgery, Koujunkai Daido Hospital, Nagoya, Japan. 4Division of Diagnostic Pathology, Shizuoka Cancer Center, Shizuoka, Japan. 5Division ofCancer Biology, Nagoya University School of Medicine, Nagoya,Japan. 6Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan. 7Preston Robert Tisch Brain
Tumor Center at Duke and Department of Pathology, Duke University
Medical Center, Durham, North Carolina.

Note: Supplementary data for this article are available at Cancer Immunology
Research Online (http://cancerimmunolres.aacrjournals.org/).
Corresponding Author: Atsushi Natsume, Nagoya University, 65 Tsurumai-cho,Show-ku, Nagoya 466-8550, Japan. Phone: 81-52-744-2353; Fax: 81-52-7442360; E-mail: anatsume@med.nagoya-u.ac.jp
doi: 10.1158/2326-6066.CIR-15-0060
2016 American Association for Cancer Research.

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Shiina et al.

type A receptor 2 (EphA2; ref. 17). In addition, CAR-transduced
T cells can migrate through the microvascular walls and penetrate
tumors (14, 16). Clinical trials are ongoing using CAR T-celltherapy against GBM targeting EGFRvIII and HER2 (18, 19).
Patient enrollment is completed for clinical trials targeting
IL13Ra2 (20).
Podoplanin (PDPN) is a type I transmembrane mucin-like
glycoprotein expressed in the lymphatic endothelium. It is overexpressed in several solid tumors, such as squamous cell carcinoma (21), malignant mesothelioma (22), Kaposi sarcoma,angiosarcoma (23), testicular seminoma (24), and brain tumors(25). PDPN expression is associated with malignant progression(25–27), epithelial–mesenchymal transition (27), and metastasis(26). In glial tumors, PDPN expression is positively correlatedwith tumor malignancy (25). PDPN is primarily expressed in themesenchymal type of GBM, which presents the worst prognosis
among GBM subtypes (28, 29). We previously produced a highly
reactive mAb to PDPN, called NZ-1 (30), and its recombinantscFv (31).
In the current study, we generated a third generation of CAR
that targeted PDPN, by using the NZ-1-based scFv. We report thegeneration of human CAR T cells that were speciﬁc and effective
against PDPN-positive GBM cells in vitro and that, when infected
systemically, inhibited the growth of intracranial glioma xenografts in vivo.

Materials and Methods
Cell lines
The human GBM cell lines LN319, U87MG, U251MG, T98,
YKG-1, and SK-MG-1 were maintained in Dulbecco's Modiﬁed Eagle Medium (DMEM; Sigma-Aldrich) containing 10%
heat-inactivated fetal bovine serum (FBS; Life Technologies),
100 units/mL of penicillin, and 100 mg/mL of streptomycin (LifeTechnologies) at 37 C in a humidiﬁed atmosphere of 5% CO2.
U251MG, T98, and SK-MG-1 cells were purchased from American
Type Culture Collection in 1995. YKG-1 and LN319 cells were
gifts from Drs. H. Kanno (Yokohama City University, Yokohama,Japan) in 2005 and W. K. Cavenee (Ludwig Institute for Cancer
Research, San Diego, CA) in 2002, respectively. The cell lines
were authenticated by the letters when they were provided.
The GBM-initiating cells GIC0222 were cultured in Neurobasal
Medium (Life Technologies) supplemented with 2 mmol/L of
L-glutamine (Sigma-Aldrich), N-2 and B-27 supplements(Life Technologies), recombinant human FGF basic and EGF(16.7 ng/mL each; R&D Systems), 100 units/mL of penicillin,and 100 mg/mL of streptomycin (Life Technologies) at 37 C in ahumidiﬁed atmosphere of 5% CO2. Primary cultured GBM cells(pcGBM) were from the GBM tissue sample obtained from apatient undergoing surgery at the Nagoya University Hospital,
Japan, after obtaining written informed consent. The study wasapproved by our institutional review board. The dissociation
procedures have been described elsewhere (32). pcGBM cells
were maintained in DMEM at 37 C in a humidiﬁed atmosphereof 5% CO2.
PDPN knockout (KO) LN319 and U87 (PDIS-6 and PDIS-7,
respectively) cells were established using the clustered regularlyinterspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. CRISPR/Cas plasmids, which target thesequence GACACTGAGACTACAGGTTTGG of human PDPN,
were obtained from Sigma-Aldrich. The CRISPR/Cas plasmid was

260 Cancer Immunol Res; 4(3) March 2016transfected into LN319 and U87MG cells using a Gene Pulser Xcell
electroporation system (Bio-Rad Laboratories Inc.). Single-cellcloning was performed by limiting dilution in 96-well plates
using 10% FBS/DMEM medium, and PDPN expression wasassessed by ﬂow cytometry using the NZ-1 mAb. The transfection-cloning cycles were repeated until the complete lack of NZ-1
reactivity was reached. The stable nature of the knockout cells was
conﬁrmed by the lack of NZ-1 reactivity after more than 10
passages.
PDPN expression in public databases
PDPN mRNA expression data from a microarray of normal
tissues were obtained from a public database, BioGPS Dataset
Library (http://biogps.org/dataset/; ref. 33). PDPN expression in
GBM subtypes was obtained from the UCSC Cancer genome
browser (https://genome-cancer.ucsc.edu/; ref. 34).
PDPN immunohistochemical staining
The 10% formalin-ﬁxed, parafﬁn-embedded surgical samples
from 79 patients newly diagnosed with GBM were collected for
immunohistochemical analysis. Immunohistochemical staining was performed as previously described (35). An anti-PDPN
mAb (1:5,000, clone NZ-1.2, rat IgG2a) was used to detect
PDPN. For each immunostained slide, the percentage of positively stained GBM cells on a given slide was evaluated by two
pathologists (R. Watanabe and I. Ito). The tumors in whichstained tumor cells made up more than 50% of the tumor were
graded as positive.
PDPN immunoﬂuorescent staining
GBM and GIC0222 cell lines were plated on a 24-well platecontaining BD BioCoat poly-L-lysine cellware 12-mm roundcoverslips with 10% FBS/DMEM and incubated for 24 hours.
The coverslips were rinsed twice with phosphate-bufferedsaline (PBS; Life Technologies) and placed in 4% paraformaldehyde phosphate buffer solution (PFA; Wako) for 15 minutes.
Blocking was performed in PBS containing 0.1% Triton X-100(Sigma-Aldrich; PBST) with 1.5% goat serum for 1 hour atroom temperature with shaking. The coverslips were incubatedwith the primary antibody NZ-1 diluted to 1 mg/mL in blocking
solution for 1 hour at room temperature with shaking followedby three washes with PBS. The coverslips were stained using asecondary antibody, Alexa Fluor 488 Goat Anti-Rat IgG (HþL;Life Technologies), and DAPI solution (Dojindo) at 1:200
dilution in the blocking solution for 30 minutes in the dark,followed by three washes with PBST. They were then mounted
onto slides. Stained cells were observed under a FV1000 confocal laser scanning biological microscope (Olympus), andpictures were taken.
PDPN expression analysis by quantitative real-time RT-PCR
PDPN expression was examined using quantitative RT-PCR.
Total RNA was prepared from GBM cells using an RNeasy Mini kit(Qiagen). The purity was conﬁrmed with an A260/A280 ratio
greater than 2.0. The ﬁrst-strand complementary DNA (cDNA)
was synthesized using a ReverTra Ace qPCR RT Master Mix with
gDNA Remover (Toyobo). The sequences of the primers used todetect GAPDH mRNA and PDPN mRNA were as follows: GAPDH
forward primer (50 -AGCCACATCGCTCAGACAC-30 ), GAPDH
reverse primer (50 -GCCCAATACGACCAAATCC-30 ), PDPN forward primer (50 -AGAAGGAGCCAGCACAGG-30 ), and PDPN
reverse primer (50 -CGCCTTCCAAACCTGTAGTC-30 ). RT-PCR was

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Chimeric Antigen Receptor Targeting Podoplanin in GBM

performed by using the LightCycler 480 instrument II (Roche
Diagnostics GmbH) and THUNDERBIRD SYBR qPCR Mix(Toyobo). The reaction solution (20 mL) consisted of 2 mL of thetemplate, 10 mL of THUNDERBIRD SYBR qPCR Mix (Toyobo),
1 mL of each primer (10 mmol/L), and 6 mL of distilled water. The
PCR conditions used were denaturation for 5 minutes at 95 C,
followed by 60 PCR cycles of denaturation at 95 C for 15 seconds,
annealing at 60 C for 30 seconds, and extension at 72 C for
1 minute. Respective expression levels of PDPN were normalized
to that of GAPDH in each sample using the DDCT method.
Construction of self-inactivating (SIN) lentiviral vector
The mAb NZ-1 was previously established (30). Its scFv wasthen produced (31). The scFv portion in the pELNS-3C10-CAR(36) was changed to the NZ-1–based scFv to generate pELNSNZ-1-CAR by gene synthesis (Genscript). In this construct, theEF1a promoter drives the CAR fusion protein containing theNZ-1–based scFv targeting PDPN, CD28, 4-1BB, and CD3z
domains. The mock vector was designed to harbor scramble
sequence of the scFv portion that have shown no functional
activity against glioma, breast cancer, colon cancer, and pancreatic cancer cell lines.
Preparation of NZ-1-CAR T and 3C10-CAR T cells
NZ-1-CAR T and 3C10-CAR T (targeting EGFRvIII; ref. 36) cells
were prepared by production and transduction of lentiviral vectors.
HEK293T cells (8  106) were plated on 175-cm2 ﬂask at 37 C in ahumidiﬁed atmosphere of 5% CO2. At 24 hours, the SIN vector,
pMDLg/pRRE, pRSV-Rev, and pMD2.G were cotransfected byusing X-tremeGENE 9 DNA transfection Reagent (Roche Applied
Science). The supernatant was collected at 48 hours, mixed with
PEG-it Virus Precipitation Solution (5; System Biosciences), andincubated for 24 hours at 4 C. The supernatant/PEG-it mixture wasthen centrifuged at 1,500  g for 30 minutes at 4 C. The pellet was
resuspended in 1 of 10 of the original volume using cold, sterile
medium at 4 C and stored at 80 C.
Freshly harvested heparinized peripheral blood mononuclear
cells (PBMC) from healthy donors were separated over a monolayer of Ficoll-Paque PLUS (1,000  g for 20 minutes at 20 C; GE
Healthcare Bio-sciences AB). PBMCs were cultured in 4 mL of
AIM-V Medium (Life Technologies) with 2.5% human serum perwell of a 6-well plate coated with the anti-human CD3 mAb(eBioscience) in the presence of IL2 (50 units/mL; PeproTech) at
37 C in a humidiﬁed atmosphere of 5% CO2 for 24 hours. Next,
the medium (3 mL/well) was gently removed without disturbing
the clustering PBMCs, and the lentiviral pELNS-NZ-1-CAR vector
supernatant (10; 3 mL/well) was added followed by culturing of
the cells for 24 hours. The medium was then replaced by fresh
medium, and the cells were cultured for 48 hours.
Flow cytometric analysis of NZ-1-CAR expression
NZ-1-CAR expression on the cell surface of PBMCs was examined using a FACSCalibur equipped with the CellQuest Pro
software (BD Biosciences). PBMCs were washed twice with PBS
containing 0.5% bovine serum albumin (BSA; Sigma-Aldrich)and 2 mmol/L EDTA (Dojindo). PBMCs were then incubated withbiotin-AfﬁniPure F(ab0 )2 fragment-speciﬁc goat anti-mouse IgG(Jackson Immuno Research Laboratories) at 4 C for 30 minutes.
After washing twice, PBMCs were stained with streptavidin (SA)–phycoerythrin (PE; BD Biosciences) at 4 C for 30 minutes in thedark. After washing twice, PBMCs suspended in 1% PFA werewww.aacrjournals.org

analyzed by FACSCalibur. The data were analyzed using the
WinMDI version 2.9 software (http://en.bio-soft.net/other/
WinMDI.html).
Analysis of IFNg production of effector cells
IFNg production by effector cells was measured by ELISA using
human IFNg ELISA Ready-SET-Go! (eBioscience). Effector cells(1.0  106 NZ-1-CAR–transduced PBMCs or mock-transduced
PBMCs) were cocultured with 1.0  104 target cells (LN319,
U87MG, or T98) in each well of a 96-well plate for 24 hours.
The cell culture supernatants were then harvested and used for
ELISA. PDPN is highly expressed in LN319 cells, but not in T98
cells (30). Therefore, these cells were used as positive and negative
controls, respectively.
Intracellular cytokine staining
Intracellular cytokine staining was performed using the Cytoﬁx/
Cytoperm plus GolgiStop kit (BD Biosciences). Effector cells (2 
105 cells of NZ-1-CAR–transduced PBMCs or mock-transduced
PBMCs) were incubated with 4  105 target cells (LN319 or LN319
PDPN KO) in 200 mL RPMI-1640 (Life Technologies) along with
GolgiStop in a round-bottom, 96-well plate. Following a 4-hour
incubation at 37 C, the cells were incubated with biotin-SP-AfﬁniPure F(ab0 )2 fragment-speciﬁc goat anti-mouse IgG (Jackson
Immuno Research Laboratories) at 4 C for 30 minutes. Afterwashing, cells were stained with SA-PE, allophycocyanin (APC)Cy7–conjugated mAb to human CD8, PerCP-Cy5.5–conjugatedmAb to human CD4, and APC-conjugated mAb to humanCD107a (BD Biosciences) at 4 C for 15 minutes in the dark. After
permeabilization and ﬁxation, the cells were stained intracellularly
with V450-conjugated mAb to human IFNg, PE-Cy7–conjugatedantitumor necrosis factor (TNF) mAb, and ﬂuorescein isothiocyanate (FITC)-conjugated mAb to human IL2 (BD Biosciences) at
4 C for 20 minutes and then washed with BD Perm/Wash solution.
After washing, the labeled cells were suspended in 1% PFA andanalyzed by FACSCanto II (BD Biosciences).
Cytotoxicity assay
Target cells (LN319, PDPN-KO LN319, U87MG, PDPN-KO
U87MG, and pcGBM) were suspended at a ﬁnal concentration of
1.0  106 cells/mL and incubated with 10 mmol/L (100 times)
calcein-AM solution (Dojindo) at 37 C for 30 minutes withoccasional shaking. After washing twice, the cells were adjusted
to 1.0  105 cells/mL and 1.0  104 cells (100 mL) were placed intoa well of a round-bottom, 96-well plate. Effector cells (NZ-1-CAR–
transduced PBMCs, 3C10-CAR–transduced PBMCs, or mocktransduced PBMCs) were incubated for 48 hours. The cells werethen harvested and added to each well at an appropriate effector:
target ratio (50:1, 25:1, 12.5:1, 6:1, and 3:1) and incubatedat 37 C for 5 hours. After centrifugation at 300  g for 2 minutes,
75 mL of supernatant were aspirated carefully and loaded into a
96-well white/clear ﬂat-bottom plate (BD Biosciences). The absorbance was then measured to evaluate the tumor-killing efﬁcacy.
Target cells in the medium with 3 mL of 10% sodium dodecyl
sulfate (SDS; Wako) were used to determine the maximum
release, and target cells alone were used to measure spontaneous
release. The percentage of speciﬁc lysis was calculated as follows:100(experimental releasespontaneous release)/(maximum releasespontaneous release)

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Intracranial glioma xenograft model
Immunoﬂuorescence and RT-PCR analyses indicated that
PDPN is highly expressed in LN319 cells. However, this cellline is not tumorigenic in vivo (37). Thus, we utilized LN319
cells as a positive control only in in vitro experiments. U87MG
cells that are tumorigenic presented the second highest level of
PDPN expression, whereas other cell lines such as GIC0222
cells grew slowly and expressed relatively little PDPN. Thus,
U87MG cells were used in animal experiments. All animal
experiments were approved by the Nagoya University ethics
committee and performed according to our institutional animal care and use guidelines. Five- to 6-week-old NOD/Shi-scid,
IL2Rr KO Jic (NOG) female mice (Central institute for Experimental Animals, Kawasaki, Japan) were used for the experiments. Mice were anesthetized intraperitoneally (i.p) with 25
mg/kg body weight (BW) pentobarbital sodium (somnopentyl,
Kyoritsu). After ﬁxing the head and incising the scalp, a burrhole was made using an 18-gauge needle in the right side of theskull, 2 mm lateral to the midline and 3 mm posterior to thelateral angle of the eye. Using a 26-gauge Hamilton syringeA

GBM#33B(Hamilton), 5.0  104 U87MG cells/mouse suspended in 2-mL
PBS were stereotactically injected 3.5 mm below the dura
matter through the burr hole by using a stereotactic apparatus.
Seven days after tumor inoculation, NZ-1-CAR–transduced
PBMCs or mock-transduced PBMCs (2  106) were suspended
in 200 mL PBS and infused intravenously (i.v.) via the tail vein.
The nontreated mice were infused with PBS alone. Survival wasmonitored following the tumor inoculation.
Tumor imaging
The growth of intracranial tumors was measured by 3T
magnetic resonance imaging (MRI; MRS 3000; MR Solutions)
every other week. Each mouse was placed on an animal
holder and anesthetized with 1% to 2% isoﬂurane at a rate of
1.5 L/minute of air. During the examination, the respiratory
rate was monitored using a respiratory sensor connected to amonitoring and gating system and kept at 80 to 100 breaths per
minute to the extent that was possible. Each mouse received
i.p. injection of 0.05 mmol/kg BW gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA; Magnevist, Bayer) for

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Figure 1.
The expression of PDPN in GBMs and cell lines. Out of 79 newly diagnosed GBM cases, 22 cases (27.8%) showed robust staining. A, 4 representative cases(2 positive and 2 negative) are shown. B, PDPN expression was the highest in GBM mesenchymal subtypes from UCSC Cancer Genome Browser(https://genome-cancer.ucsc.edu). C, PDPN expression in U87MG and GIC0222 was approximately 10% of that of LN319, which was used as a positive control
for PDPN, as determined by quantitative RT-PCR. T98 was almost negative. D, immunoﬂuorescence for PDPN (green) is consistent with the results ofquantitative RT-PCR.

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Chimeric Antigen Receptor Targeting Podoplanin in GBM

LeaderA
RSV/HIV-1
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38.4%

0

Figure 2.
Construction of NZ-1-CAR T cells. A,
a lentiviral vector construct with the
EF1a promoter followed by theleader sequence, NZ-1–based scFv,
CD28, 4-1BB, and CD3z. The
transduction efﬁciency was
examined by ﬂow cytometry withmouse-derived F(ab0 )2 recognizing
biotin antibody. B, the efﬁciency was
reproducibly 35% to 40%.

EF1αLinker

100

101

102

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contrast-enhanced MRI. Tumor volume was estimated using theABC/2 method as follows: An MRI slice with the largest area oftumor was identiﬁed in the contrast-enhanced MRI. The largest
diameter (A) of the tumor on this slice was measured. Next, thewidth perpendicular to (A) on the same slice was measured (B).
Finally, the approximate number of slices with tumor multiplied by the slice thickness (1 mm) was calculated (C). Then, A,
B, and C were multiplied and the product divided by 2, whichyielded the tumor volume in cubic millimeters.
Immunohistochemical staining in vivo
The U87MG-bearing mice treated with NZ-1-CAR or mocktransduced PBMCs were euthanized on day 12, 22, or 38 after the
PBMC injection. Brain tissues were harvested and embedded in
optimum cutting temperature (OCTTM) compound (Sakura Fine
Technical) and frozen in liquid nitrogen. Six-micrometer-thickfrozen sections were prepared with a cryostat (CM3050S, Leica).
After drying, the sections were ﬁxed with 4% formaldehyde. The
sections were then blocked with 1.5% normal goat serum (Vector
Laboratories) in PBS containing 0.05% Tween 20 at room temperature for 1 hour, and were stained with rabbit anti-human CD3
antibody (1:100, Thermo Lab Vision) diluted to 1 mg/mL. The
secondary labeled polymer from EnVision HRP kit (Dako) wasapplied, and sections were incubated for 30 minutes. The substrate-chromogen solution from the EnVision HRP kit (Dako) wasapplied for 10 minutes. After washing, sections were counterstained with hematoxylin and mounted in Multi Mount (Matsunami Glass Ind).
Statistical analysis
The statistical signiﬁcance of differences between two groups
was determined using the Student t test. A two-tailed P value of<0.05 was considered statistically signiﬁcant. In mouse experiments, survival curves were obtained by using the Kaplan–Meiermethod and compared by using the log-rank test.

Results
PDPN expression in human GBM specimens and cell lines
PDPN is expressed in lymphatic endothelial cells and basal cells
in normal tissues (Supplementary Fig. S1A). The analysis of publicwww.aacrjournals.org

databases indicates that PDPN expression is the highest in theplacenta with a 4-fold difference with that in the brain (Supplementary Fig. S1B). However, PDPN is overexpressed in several
solid tumors (21–25). In particular, PDPN expression in GBMs is
16-fold higher than that in the brain (Supplementary Fig. S1C).
First, we evaluated PDPN expression in human GBMs by immunohistochemistry with the PDPN mAb, NZ-1.2. Out of 79 newly
diagnosed GBM cases, 22 cases (27.8%) showed robust, butheterogeneous, staining. Four representative cases (2 positive and2 negative) are presented (Fig. 1A). Although most GBM specimens were not positive for PDPN, specimens presenting themesenchymal type of GBMs predominantly expressed PDPN (Fig.
1B). The mesenchymal type has been reported to present the worst
prognostic among GBM subtypes (28, 29). Likewise, PDPN was
not highly expressed in all human glioma cell lines used in thisstudy. However, quantitative RT-PCR indicated that PDPN expression in U87MG and GIC0222 cells was approximately 10% of thatin LN319 cells, which was used as a positive control (Fig. 1C). The
immunoﬂuorescence analysis using the PDPN mAb conﬁrmedthat PDPN was expressed on the cellular membrane of LN319 and
U87MG cells, whereas T98 cells were almost negative for PDPN
(Fig. 1D).
Construction of NZ-1-CAR T cells
We constructed a lentiviral vector tandem linked with the EF1apromoter followed by the leader sequence (so that the productwas able to protrude extracellularly), and NZ-1–based scFv,
CD28, 4-1BB, and CD3z (Fig. 2A, the third generation). The
lentiviral vector was used to infect human PBMCs. The transduction efﬁciency was examined by ﬂow cytometry with a mousederived F(ab0 )2–biotin antibody. The percentage of transduced
PBMCs with NZ-1 CAR was 35% to 40% (Fig. 2B). These results
were reproducible.
Functional assay of NZ-1-CAR T cells in vitro
The calcein-based nonradioisotope cytotoxic assay indicatedthat PDPN-positive LN319 cells were signiﬁcantly lysed by NZ1-CAR–transduced PBMCs in an effector:target (E:T) ratio–
dependent manner. Cytotoxic effects of NZ-1-CAR–transduced
PBMCs on PDPN-positive U87MG cells and primary cultured

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GBM cells (pcGBM) were not as drastic as that on LN319 cells,
but a signiﬁcant speciﬁc lysis was still observed. In contrast,
speciﬁc lysis was not observed against PDPN-KO LN319 and
PDPN-KO U87MG cells (Fig. 3A). EGFRvIII-targeting (3C10)CAR PBMCs did not lyse LN319 cells, suggesting that cells were
not lysed nonspeciﬁcally (Supplementary Fig. S2A). These
results suggest that NZ-1-CAR T cells present PDPN-speciﬁccytotoxicity.
Coculture of LN319 or U87MG cells with NZ-1-CAR–transduced PBMCs resulted in the production of approximately 350
to 400 pg/mL of IFNg, whereas mock-transduced PBMCs
released signiﬁcantly less IFNg (Fig. 3B). Thus, we successfully
generated functional active NZ-1-CAR T cells that recognize
PDPN. CD4þ, and CD8þ NZ-1-CAR T cells may secrete different
sets of cytokines. T-cell functionality (production of IFNg,
TNFa, IL2, and CD107a) was separately analyzed for CD4þ andCD8þ CAR T cells by FACS (Fig. 3C). IFNg, IL2, and CD107awere predominantly produced by CD4þ CAR T cells cocultured
with LN319 and U87MG cells, whereas CD8þ CAR T cells

60

60

PDPN-KO LN319

40

40

40

PDPN-KO U87MG
60

60
40

40

20
20

20

20

0

0
3

3

−20
50 25 12.5 6
E/T ratio

E/T ratio3

50 25 12.5 6

3050 25 12.5 6

E/T ratio

3

E/T ratio

U87MG

CD8

lFNγ

lgG-Fab

CD8

CD4

CD4

CD107

IL2

CD107

IL2

CD4

CD8

CD4

CD4

CD107

CD4

CD4

lFNγ

lgG-Fab

CD4

CD8

CD8

CD107

LN319
U87MG
NZ-1-CAR
Mock-CAR

CD8

CD8

IL2

0

TNFα

100

CD8

CD8

IL2

CD8
lgG-Fab

200

TNFα

LN319

300

lFNγ

lgG-Fab

400

TNFα

C
500

TNFα

B

0
50 25 12.5 6

20

0

lFNγ

% Specific lysis

60

50 25 12.5 6
NZ-1-CAR E/T ratio
Mock-CARIFNγ (pg/mL)

Distribution and antitumor effect of NZ-1-CAR T cells on
human glioma in the mouse brain
After conﬁrming the presence of intracranial tumors by MRI,
mock-transduced PBMCs or NZ-1-CAR–transduced PBMCs
were injected in the tail vein of mice. Brain tissues were
harvested and embedded in OCT compound, and the sections
were stained with rabbit antibody to human CD3 on days 12,
22, and 38 after injection (Fig. 4A). Whereas human CD3þ cells
were not found in the tumors obtained from the mock-treatedmice on day 12, CD3þ cells were still observed on day 38 in the
pcGBM

U87MG

LN319

A

mainly produced TNFa. The sorted CAR T cells were subjected
to RT-PCR (Supplementary Fig. S2B). TNFa was signiﬁcantly
produced in NZ-1-CAR T cells that were stimulated by LN319
cells (P < 0.01), but IL2 and CD107a were expressed even in
mock-CAR T cells, which suggested that the secretion of IL2 andCD107a was not speciﬁc to the PDPN antigen. The mock-CAR T
cells did not produce IFNg, TNFa, IL2, or CD107a (Supplementary Fig. S3).

CD4

CD4

Figure 3.
Functional assay of NZ-1-CAR T cells in vitro. A, PDPN-positive glioma cells are lysed signiﬁcantly by NZ-1-CAR–transduced PBMCs compared with mocktransduced PBMCs in an E:T ratio–dependent manner; however, speciﬁc lysis was not observed in PDPN-knockout glioma cells.  , P < 0.05. B, the cocultureof LN319 or U87MG with NZ-1-CAR–transduced PBMCs produced approximately 350 to 400 pg/mL of IFNg. The IFNg levels were signiﬁcantly higher than those fromþþ
mock-transduced PBMCs.  , P < 0.05. Cytokine secretion proﬁles (IFNg, TNFa, and IL2) and those of CD107a were separately analyzed for CD4 and CD8þ
CAR T cells by an intracellular cytokine assay using FACS. C, IFNg, IL2, and CD107a were secreted predominantly by CD4 CAR T cells cocultured with LN319
þand U87MG cells, whereas CD8 CAR T cells mainly produced TNFa.

264 Cancer Immunol Res; 4(3) March 2016

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Published OnlineFirst January 28, 2016; DOI: 10.1158/2326-6066.CIR-15-0060

Chimeric Antigen Receptor Targeting Podoplanin in GBM

A

Confirmation of in vivo tumor
i.v. infusion of 2 × 106 PBMC/mouse

Histology

Day 0

NZ-1-CAR (Day 38)

NZ-1-CAR (Day 22)HE

Anti-human CD3

200 μm200 μm500 μm500 μm

100 μm

100 μmHE

Mock-CAR (Day 12)

B

Day 12, 22, 38

Anti-human CD3500 μm500 μm200 μm200 μmFigure 4.
Distribution of NZ-1-CAR T cells on human glioma in mouse brain. A, after the presence of intracranial tumors was conﬁrmed by MRI, mock-transduced PBMCs, orNZ-1-CAR–transduced PBMCs were injected in the tail vein of mice. Brain tissues were harvested and embedded in an OCT compound, and the sections wereþ
stained with rabbit anti-human CD3 antibody on days 12, 22, and 38 after injection. B, while human CD3 cells were not found in the tumors obtained from theþ
mock-treated mice on day 12, CD3 cells were still observed on day 38 in the tumors of NZ-1-CAR–treated mice. Dotted lines indicate the tumors. The higher
magniﬁcation of images in the rectangular insets is also shown below.

tumors of NZ-1-CAR–treated mice (Fig. 4B). Thus, NZ-1-CAR T
cells spread to the brain tumor and persisted for at least 38 days
after injection.
The treatment design is summarized in Fig. 5A. On day 7 after
intracranial implantation of tumor cells, PBS, mock-transduced
PBMCs, or NZ-1-CAR–transduced PBMCs were injected in the tailvein of the mice. The volume of gadolinium-enhanced tumors
was evaluated sequentially. In approximately 60% of the micetreated with NZ-1-CAR PBMCs, the tumor grew markedly more
slowly than that in the other two groups (Fig. 5B and C). Median
OS durations of the three groups were 59, 56.5, and 79 days,
respectively. In addition, a log-rank test indicated that mice fromthe NZ-1-CAR group survived signiﬁcantly longer (Fig. 5D). The
survival curve of approximately 40% of the NZ-1-CAR miceoverlapped with those of the two control groups. We repeatedthe animal experiment twice, and the results were reproducible.
The tumor growth and effective CAR T-cell proliferation may bealmost equivalent; thus, the adoptive transferred CAR T cells
started to elicit an effect around 60 days. We speculate that a

www.aacrjournals.org

certain amount of time is required for effective T-cell proliferation,
so that the number of cells is sufﬁcient to eradicate an intracranialtumor through the blood–brain barrier (BBB).

Discussion
In this study, we report the construction of a third-generation
CAR that targets PDPN and its successful lentivirus-mediatedexpression on human T cells. We showed that the generated T
cells were speciﬁc and effective against PDPN-positive GBM cells
in vitro, and systemic injection of the T cells signiﬁcantly increased
survival time in vivo (P ¼ 0.035).
PDPN is especially expressed in the mesenchymal type of GBM,
which presents the worst prognosis among GBM subtypes(Fig. 1B; refs. 28, 29). This study and previous reports showedthat the expression of PDPN in GBM was observed in 27.8% (thisstudy), 47% (25), and 31% of patients with GBM (28). Considering that PDPN is one of the factors associated with poor
prognosis, PDPN-targeted CAR T-cell therapy would be useful inCancer Immunol Res; 4(3) March 2016

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265

Published OnlineFirst January 28, 2016; DOI: 10.1158/2326-6066.CIR-15-0060

Shiina et al.

Ai.c. implantation of U87MG
5 × 104/mousei.v. infusion of 2 × 106 PBMC/mouse

MRI

Day 7

Day 8, 15, 23, 35,
43, 49, 56

OS
Day 0

B

C
Tumor volume (mm3)

PBS

400

Mock

PBS
Mock-CAR
NZ-1-CAR

300

200

100

0
Day 8 Day
15

NZ-1-CAR

Day 23

Day 35

Day 43

Day 49Day 56

Days after implantation

Survival rate (%)

D

Day
23

Day
35

Day
43

Day Day
49
56

Day
64

100

PBS
Mcok-CAR
NZ-1-CAR

80
60
40

Log-rank
P = 0.035

20
0
0

20

40

60

80

100

120

Days after implantation
Figure 5.
Antitumor effect of NZ-1-CAR T cells on human glioma in mouse brain. A, the experimental design. B and C, in approximately 60% of the mice treated with NZ-1-CAR–
transduced PBMCs, the tumor grew markedly more slowly than those from the other two groups. D, median OS durations of the three groups were 59, 56.5,
and 79 days, respectively. P < 0.05 by a log-rank test i.c., intracranial.

the treatment of patients with relapsed/resistant tumors followingﬁrst-line chemotherapy.
Recent studies focused on immune checkpoint targeting tocytotoxic T lymphocyte–associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1). CTLA-4 is expressed on T
cells in which it regulates the amplitude of early stages of T-cell
activation by counteracting the activity of the T-cell costimulatory
receptor CD28 (38). Moreover, it enhances Treg immunosuppressive activity (39). In contrast, PD-1 limits the activity of T cells
in peripheral tissues to avoid autoimmunity (38). The samemechanism is observed in tumor immune evasion (40). The
major PD-1 ligand, PD-1 ligand 1 (PD-L1; known as B7-H1 andCD274), is overexpressed in various solid tumors, including
malignant melanoma, ovarian cancer, lung cancer, and GBM(41). PD-L1 signaling inhibits T-cell proliferation and IFNg secretion (42). One of the fully humanized antibodies to CTLA-4,
ipilimumab, was clinically tested (43). John and colleagues
demonstrated for the ﬁrst time that the administration of an
antibody to PD-1 can signiﬁcantly enhance the therapeutic efﬁcacy of CAR T cells (44). Combination of NZ-1-CAR T cells and

266 Cancer Immunol Res; 4(3) March 2016

ipilimumab (anti–CTLA-4) or nivolumab (anti–PD-1) would be
another potential strategy. Such strategies may lead to an increasein effector T-cell proliferation in vivo, so that there are enough cells
to penetrate the BBB.
One of the concerns with PDPN-targeted CAR therapy is that
PDPN is expressed in normal tissues, including the lymphatic
endothelium, lung type I alveolar cells, kidney glomerular podocytes, and mesothelium (21, 23). In the central nervous system,
PDPN is expressed in the choroid plexus, ependyma, meninges(21), and Purkinje cells (45). We established a cancer-speciﬁc
mAb (CasMab) to human PDPN (46). The newly establishedmAb clone LpMab-2 recognizes the cancer-type PDPN, which isaberrantly glycosylated, and was puriﬁed from a human PDPNtransfected GBM cell line. LpMab-2 can react with PDPN-expressing cancer cells, but not with normal cells such as lymphatic cells
and type I alveolar cells. It may be useful to produce a new CAR
using LpMab-2 for CAR therapy targeting PDPN.
Although there may be a number of issues to be addressed
for clinical application, such as off-targets, slow responses, andshort effectiveness, overall we successfully established CAR T cells

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Published OnlineFirst January 28, 2016; DOI: 10.1158/2326-6066.CIR-15-0060

Chimeric Antigen Receptor Targeting Podoplanin in GBM

against a promising tumor antigen, PDPN, and provide new
insights toward therapies targeting solid tumors that have failed
other treatments.

Other (contributed to histological and morphological evaluation):
R. Watanabe
Other (contributed to microscopical or morphological appraisal): I. Ito
Other (provided the scFv targeting the podoplanin antigen): V. Chandramohan

Disclosure of Potential Conﬂicts of Interest
No potential conﬂicts of interest were disclosed.

Authors' Contributions
Conception and design: T. Wakabayashi, A. Natsume
Development of methodology: M. Ohno, T. Wakabayashi, Y. Kato, A. Natsume
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): S. Shiina, M. Ohno, F. Ohka, A. Kato, K. Motomura,T. Yamamot, M. Hamaguchi, T. Wakabayashi, D.D. Bigner
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): F. Ohka, A. Yamamichi, K. Motomura, T. Wakabayashi,
M. Ohno, A. Natsume
Writing, review, and/or revision of the manuscript: M. Ohno, F. Ohka,
T. Senga, D.D. Bigner, A. Natsume
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): M. Ohno, A. Yamamichi, A. Kato, K. Tanahashi,
T. Wakabayashi, M.K. Kaneko, D.D. Bigner
Study supervision: M. Ohno, T. Wakabayashi, A. Natsume

Grant Support
This work was supported by Grant-in Aid for Scientiﬁc Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science andTechnology (MEXT; A. Natsume; #23107010) and Grant-in-Aid for Young
Scientists B (M. Ohno; #25861270). This work was also supported in part by
the Platform for Drug Discovery, Informatics, by Structural Life Science (PDIS)
from Japan Agency for Medical Research and development, AMED (Y. Kato), by
the Basic Science and Platform Technology Program for Innovative Biological
Medicine from AMED (Y. Kato), and by the Regional Innovation Strategy
Support Program from MEXT (Y. Kato).
The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received March 3, 2015; revised October 27, 2015; accepted November 30,
2015; published OnlineFirst January 28, 2016.

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Published OnlineFirst January 28, 2016; DOI: 10.1158/2326-6066.CIR-15-0060

CAR T Cells Targeting Podoplanin Reduce Orthotopic
Glioblastomas in Mouse Brains
Satoshi Shiina, Masasuke Ohno, Fumiharu Ohka, et al.
Cancer Immunol Res 2016;4:259-268. Published OnlineFirst January 28, 2016.

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