IJC
International Journal of Cancer

Recombinant anti-podoplanin (NZ-1) immunotoxin for thetreatment of malignant brain tumors
Vidyalakshmi Chandramohan1, Xuhui Bao1,2, Mika Kato Kaneko3, Yukinari Kato3, Stephen T. Keir1, Scott E. Szafranski1,
Chien-Tsun Kuan1, Ira H. Pastan4 and Darell D. Bigner1
1

Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, NC
Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China
3
Molecular Tumor Marker Research Team, The Oncology Research Center, Advanced Molecular Epidemiology Research Institute,
Yamagata University Faculty of Medicine, Yamagata, Japan
4
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD

Our study demonstrates the glioma tumor antigen podoplanin to be present at very high levels (>90%) in both glioblastoma(D2159MG, D08-0308MG and D08-0493MG) and medulloblastoma (D283MED, D425MED and DAOY) xenografts and cell line.
We constructed a novel recombinant single-chain antibody variable region fragment (scFv), NZ-1, specific for podoplanin fromthe NZ-1 hybridoma. NZ-1-scFv was then fused to Pseudomonas exotoxin A, carrying a C-terminal KDEL peptide(NZ-1-PE38KDEL). The immunotoxin (IT) was further stabilized by a disulfide (ds) bond between the heavy-chain andlight-chain variable regions as the construct NZ-1-(scdsFv)-PE38KDEL. NZ-1-(scdsFv)-PE38KDEL exhibited significant reactivity
to glioblastoma and medulloblastoma cells. The affinity of NZ-1-(scdsFv), NZ-1-(scdsFv)-PE38KDEL and NZ-1 antibody forpodoplanin peptide was 2.1 3 1028 M, 8.0 3 1028 M and 3.9 3 10210 M, respectively. In a protein stability assay,
NZ-1-(scdsFv)-PE38KDEL retained 33–98% of its activity, whereas that of NZ-1-PE38KDEL declined to 13% of its initial levels
after incubation at 37C for 3 days. In vitro cytotoxicity of the NZ-1-(scdsFv)-PE38KDEL was measured in cells isolated fromglioblastoma xenografts, D2159MG, D08-0308MG and D08-0493MG, and in the medulloblastoma D283MED, D425MED and
DOAY xenografts and cell line. The NZ-1-(scdsFv)-PE38KDEL IT was highly cytotoxic, with an 50% inhibitory concentration in
the range of 1.6–29 ng/ml. Significantly, NZ-1-(scdsFv)-PE38KDEL demonstrated tumor growth delay, averaging 24 days
(p < 0.001) and 21 days (p < 0.001) in D2159MG and D283MED in vivo tumor models, respectively. Crucially, in the
D425MED intracranial tumor model, NZ-1-(scdsFv)-PE38KDEL caused a 41% increase in survival (p  0.001). In preclinicalstudies, NZ-1-(scdsFv)-PE38KDEL exhibited significant potential as a targeting agent for malignant brain tumors.

Glioblastoma is the most frequent and most malignant
primary brain tumor in adults and accounts for 17% of all
primary brain tumors, whereas medulloblastoma, an invasive,
malignant tumor, accounts for about 13% of all childhood
brain tumors in children of ages 0–14 years.1 Despite
aggressive multimodality therapy, the median survival of glioblastoma patients until the late 1990s was less than a year.2
Current clinical trials incorporating temozolomide or the

monoclonal antibody (mAb) bevacizumab have been shown
to be effective. However, they produced only a modest
increase in glioblastoma patient survival, with less than 5% of
patients surviving 5 years postdiagnosis.1,3,4 Similarly, recent
advances in surgery, radiotherapy and chemotherapy have led
to an increase in 5-year survival rate of medulloblastoma
patients, from 30% to upward of 60–70%.5 However, theaggressive nature of these therapies results in long-term

Key words: podoplanin, glioblastoma multiforme, medulloblastoma, recombinant immunotoxin, single-chain disulﬁde Fv
Abbreviations: BSA: bovine serum albumin; EDTA: ethylenediaminetetraacetic acid; FACS: ﬂuorescence-activated cell sorter; FR: framework;
HC: heavy chain; HSA: human serum albumin; i.c.: intracranial; IC50: 50% inhibitory concentration; i.t.: intratumoral; IT: immunotoxin;
KDEL: lysine-aspartic acid-glutamic acid-leucine; LC: light chain; mAb: monoclonal antibody; MED: medulloblastoma; MG: malignant glioma;
QFACS: quantitative FACS; s.c.: subcutaneous; scFv: single-chain antibody fragment
Additional Supporting Information may be found in the online version of this article.
Grant sponsor: The National Cancer Institute; Grant numbers: NINDS 5P50 NS20023-29, NCI Merit Award 5R37 CA011898-42; Grant
sponsors: Southeastern Brain Tumor Foundation, Silvian Foundation, Intramural Research Program of the NIH, National Cancer Institute,
Center for Cancer Research
DOI: 10.1002/ijc.27919
History: Received 8 Jan 2012; Accepted 5 Oct 2012; Online 1 Nov 2012
Correspondence to: Darell D. Bigner, MD, PhD, Department of Pathology, Duke University Medical Center, Box 3156, 177 MSRB1,
Research Drive, Durham, NC 27710, USA, Tel.: þ1-919-684-5018, Fax: þ1-919-684-6458, E-mail: bigne001@mc.duke.edu

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Int. J. Cancer: 000, 000–000 (2012) V

Tumor Immunology22Anti-podoplanin immunotoxin for brain tumor therapy

Tumor Immunology

What’s new?
Podoplanin is a glycoprotein that is overexpressed in several types of malignant tumor, including glioblastoma,
medulloblastoma, mesothelioma, and germ-cell tumors. In this study, the authors constructed a novel immunotoxin to target
podoplanin, by fusing a monoclonal antibody fragment to exotoxin A from Pseudomonas. In preclinical studies, thisimmunotoxin, called NZ-1-(scdsFv)-PE38KDEL, increased survival by 41%. Its specificity and high binding affinity allow for
specific targeting of tumor cells while avoiding adjacent normal tissue, thereby having the potential to improve survival ofpatients with brain tumors.

adverse effects, including cerebellar mutism, cognitive andendocrine impairments, hearing loss, infertility and neuropathies.6 To overcome these difﬁculties, there is a need for thedevelopment of novel and effective therapies speciﬁcallytargeting the tumor cells while preserving the surrounding
normal central nervous system (CNS).
Current advances in the understanding of brain tumor
biology have identiﬁed unique molecular abnormalities thatare speciﬁcally associated with brain tumors but which areabsent in normal brain cells.7 Molecular characterization ofbrain tumors has led to the identiﬁcation of tumor-speciﬁc
proteins that are being exploited for the development oftargeted therapies for brain tumor treatment.8 Several mAbs
against brain tumor antigens have been generated.9 Over the
past 2 decades, recombinant single-chain variable-region antibody fragment (scFv)-based immunotoxins (ITs), composedof the heavy-chain and light-chain variable regions (VH andVL) of a mAb-targeting tumor antigen fused to a toxin molecule, have been widely investigated for the treatment ofseveral cancers.10 The smaller size of a scFv-IT fusion proteinshould provide greater tumor penetration and therefore
enhanced therapeutic efﬁcacy.
Podoplanin is a 162-amino acid type I transmembranesialomucin-like glycoprotein consisting of a serine- andthreonine-rich extracellular domain, a single transmembrane domain and a short cytoplasmic domain containingcAMP and protein kinase C phosphorylation sites.11 It ishighly expressed by cultured human lymphatic endothelial
cells, and podoplanin/ mice die at birth because of
impaired lymphatic vessel pattern formation and respiratory failure.12 Podoplanin expression has been described inseveral tumors, including brain tumors,11,13,14 and is associated with malignant progression involving epithelial–mesenchymal transition, metastasis and invasion.15–18 Among the
CNS tumors, podoplanin expression is observed in 83%
and 27% of glioblastoma and medulloblastoma cases,
respectively,14 making it an attractive target for brain tumor therapy. Several anti-podoplanin antibodies (NZ-1,
D2-40, AB3 and 18H5) have been developed and wellcharacterized.19,20 A functional immunotherapeutic agent
should be able to bind to the target tumor cells with highafﬁnity and be internalized. The radiolabeled NZ-1 mAb
has been previously demonstrated to bind avidly by
D397MG cells and rapidly internalized by D2159MG xenograft cells.21 Further, the [131I]SGMIB-NZ-1 mAb exhibited
signiﬁcant tumor accumulation in D2159MG tumor-bearing

mice, which thereby established the potential utility of NZ1 as an immunotherapeutic agent against malignant brain
tumors.21
Herein, we demonstrate the expression of the tumor antigen podoplanin in several glioblastoma and medulloblastoma
xenograft cells. Further, we report the construction of arecombinant Pseudomonas exotoxin-based IT, NZ-1-(scdsFv)PE38KDEL, from the NZ-1 mAb. The NZ-1-(scdsFv)-PE38KDEL IT inhibited protein synthesis in brain tumor xenograft
cells in vitro and exhibited signiﬁcant delay in tumor growthof brain tumor xenografts in vivo, establishing this IT as apromising candidate for the therapy of brain tumors.

Material and Methods
Cell line

The medulloblastoma cell line DAOY (ATCC Number: HTB186) was cultured in complete zinc option (ZO)–10% fetal bovine serum [Improved Modiﬁed Eagle Medium ZO (Richter’s
Modiﬁcation, Cat. No. 10373-017) liquid; Invitrogen, San
Diego, CA] and passed at conﬂuence with 0.05% Trypsin–
Ethylenediaminetetraacetic acid (EDTA) (Invitrogen).
Dissociation of xenografts

Human-biopsy-derived xenograft tissue from malignant
glioma lines, D2159MG, D08-0308MG, D08-0493MG, D080695MG and D08-0537MG, and medulloblastoma lines,
D283MED and D425MED, obtained under sterile conditions
from the Duke animal facility, was prepared for cell culture
in a laminar ﬂow hood with a sterile technique. Tumor material was ﬁnely minced and digested with 100 lg Liberase(Roche, Indianapolis, IN). This mixture was stirred at 37 C
for 10 min, and a cell-rich supernatant was obtained. The
cells were washed with complete ZO medium, further treatedwith Ficoll-Hypaque to remove any red blood cells and thenwashed once in ZO medium.
Cloning of variable heavy and variable light domainsof the NZ-1 and P588 mAb

P588 is a mouse mAb (IgG1j) puriﬁed from the mouse
myeloma cell line P3X63Ag8, which is a subclone of the
myeloma cell line MOPC-21.22 The antigen speciﬁcity of
MOPC-21 is unknown and is widely used as an isotype control antibody (Cell Signaling Technology, Danvers, MA, Cat.
No. 4878). Total cellular mRNA from 106 NZ-1 andC 2012 UICC
Int. J. Cancer: 000, 000–000 (2012) VP3X63Ag8 hybridoma cells was isolated by using a Dynabeads, mRNA direct kit (Invitrogen). A SMART RACE
cDNA ampliﬁcation kit (Clontech, Palo Alto, CA) was used
to obtain VH and VL cDNAs of the NZ-1 and P588 mAb.
The VH domain was fused to the VL domain by a 15-aminoacid peptide (Gly4Ser)3 linker by PCR. The NZ-1 and P588
scFv fragment was cloned into pRB199 vector using a T4
DNA ligase kit (Roche) to obtain NZ-1-(scFv)-PE38KDEL
and P588-(scFv)-PE38KDEL. The NZ-1-(scdsFv)-PE38KDEL
and P588-(scdsFv)-PE38KDEL constructs were obtained bymutating residues 44 of VH and 103 of VL, and 44 of VH
and 100 of VL, respectively, by site-directed mutagenesis
using a QuickChange Multi-Site Directed Mutagenesis Kit(Stratagene, La Jolla, CA), and the sequence was veriﬁed.

1,000 ng/ml in phosphate-buffered saline containing 0.2%
bovine serum albumin (BSA; 0.2% BSA/PBS), and 10 ll of
diluted toxin was added to each well. Plates were incubatedfor 20 hr at 37 C and then pulsed with 1 lCi per well of
3
L-[4,5- H]leucine (PerkinElmer, Shelton, CT) in 25 ll of
0.2% BSA/PBS for 3 hr at 37 C. Radiolabeled cells were captured on ﬁlter-mats and counted in a MicroBeta scintillation
counter (PerkinElmer). The cytotoxic activity of NZ-1(scdsFv)-PE38KDEL was deﬁned by 50% inhibitory concentration (IC50), which was the toxin concentration that inhibited radioactivity incorporation by 50% when compared to
the radioactivity measured in untreated cells. All the assays
were performed in triplicate for each cell line. Results arepresented as mean 6 standard deviation where n  3.

Preparation of recombinant ITs

In vivo xenograft model

The different NZ-1 and P588 ITs were generated by fusing
the speciﬁc scFv or scdsFv with the sequences for domains II
and III of Pseudomonas exotoxin A (PE38KDEL; KDEL:
lysine-aspartic acid-glutamic acid-leucine) according to apreviously described protocol.23 NZ-1-(scdsFv), NZ-1-(scFv)PE38KDEL, NZ-1-(scdsFv)-PE38KDEL, P588-(scFv)-PE38KDEL and P588-(scdsFv)-PE38KDEL were expressed undercontrol of the T7 promoter in Escherichia coli BL21 (k DE3)
(Stratagene). All recombinant proteins were accumulated in
the inclusion bodies. The inclusion bodies were puriﬁed andsolubilized in 8 M guanidine (Thermo Scientiﬁc, Rockford, IL)
and reduced with 0.3 M Dithioerythritol (Sigma, St. Louis,MO) for 5 hr at room temperature, as described previously.23
The reduced protein was rapidly diluted 100-fold in the refolding buffer consisting of 0.1 M Tris, pH 8.0 (Invitrogen), 0.5 M
L-arginine (Sigma), 9 mM L-Glutathione oxidized (Sigma) and2 mM EDTA (Invitrogen). Protein samples in refolding buffer
at a concentration of 100 lg/ml were incubated at 10 C for 72
hr. After renaturation the samples were dialyzed against 20
mM Tris, pH 7.5 (Invitrogen) and 100 mM urea (MP Biomedicals, Solon, OH) overnight at 4 C. The dialyzed protein sample was ﬁltered and loaded on to a Q-Sepharose fast ﬂow column (GE Healthcare, Pittsburgh, PA), and the IT was eluted
over a linear salt gradient with 1 M Sodium chloride (Sigma)
in 20 mM Tris, pH 7.5. The IT was further puriﬁed as a monomer (64 kDa) by size exclusion chromatography on a TSKgel
SuperSW3000 column (TOSOH Bioscience, King of Prussia,
PA) to greater than 95% purity.

Female athymic nude mice (20 g; 4–6 weeks) were injectedsubcutaneously (s.c.) in the right ﬂank with 3  106 D2159MG
or D283MED cells suspended in 50 ll of PBS, pH 7.4. Whenthe implanted tumors reached 150–300 mm3, ten mice per arm
were randomly selected for inoculation. The test mice weretreated by intratumoral (i.t.) injection every other day with a
total of three doses of 0.3 mg/kg of NZ-1-(scdsFv)-PE38KDEL
diluted in 200 ll of PBS containing 0.2% human serum albumin (PBS-HSA). The control mice were handled in the samemanner, but treated with either 0.2% PBS-HSA or 0.3 mg/kg of
the negative control IT, P588-(scFv)-PE38KDEL. Tumors were
measured twice weekly with a handheld vernier caliper, and thetumor volumes were calculated in cubic millimeters by using
the formula: ([length]  [width2])/2. When tumor volume met
both of the following criteria: (i) >1,000 mm3 and (ii) ﬁve times
its original treatment size, animals were tested out of the study.

In vitro cytotoxicity assay

Intracranial implantation of tumor cells

The cytotoxicity of the NZ-1-(scdsFv)-PE38KDEL on cells
isolated from D2159MG, D08-0308MG, D08-0493MG, D080695MG, D08-0537MG, D283MED and D425MED xenografts and DAOY cells was assayed by inhibition of protein
synthesis as described previously.24 P588-(scdsFv)-PE38KDEL
was used as negative control. Cells were seeded in 96-wellplates at a density of 2  104 cells per well in 200 ll of complete ZO medium 24 hr before the assay. Immunotoxins
were serially diluted to achieve a ﬁnal concentration of 0.01–

Male NOD scid gamma (NSG) mice (20–30 g; 12 weeks)
were anesthetized by an intraperitoneal injection of a solutionof ketamine 50 mg/kg (Fort Dodge Animal Health, Overland
Park, Kansas) and xylazine 10 mg/kg (Akorn, Decatur, IL).
The anterior cranial region was shaved under sterile conditions and an incision 1 cm in length was made in the skinover the skull, and a small burr hole was drilled at coordinates 2.5 mm left lateral of the sagittal and 1 mm anterior to
the bregma. A 25-gauge 10-ll Hamilton needle was inserted

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Assessment of response

The s.c. xenograft response was assessed by delay in tumor
growth in test mice (T) versus control mice (C). The growth
delay (T  C) was the difference between the median times
required for tumors in treated and control mice to reach avolume that was ﬁve times the size at the initiation of therapy and >1,000 mm3. Tumor regression was the decrease intumor volume over two successive measurements.
Statistical analysis was performed as described previously,25 using the Wilcoxon rank-sum test for growth delay
and Fisher’s exact test for tumor regressions.

Tumor Immunology

3

Chandramohan et al.

4vertically to a depth of 2.5 mm from the dura mater. All
mice were injected with 105 D425MED cells in 5 ll of 1
PBS into the cortex.
Intracranial convection-enhanced delivery

Four days post-tumor implantation, a 3-day Alzet pump
(Durect Corporation, Cupertino, CA) containing 0.3 lg of
the P588-(scdsFv)-PE38KDEL or NZ-1-(scdsFv)-PE38KDEL
IT diluted in 100 ll of 0.2% PBS-HSA was implanted subcutaneously on the back of the anesthetized mice slightly posterior to the scapulas. The pump was connected to the cannula
through a catheter passing through a tunnel created under
the skin of mice, and the IT was infused through the cannula
into the tumor site over a 3-day period. The control animals
were handled in the same manner, but were either left
untreated (sham) or treated with 0.2% PBS-HSA alone. The
mice were followed to assess tumor development and death.
Evaluation of intracranial xenograft response to treatment

Tumor Immunology

The response of intracranial (ic) xenografts to treatment wasassessed by percentage increase in time to a speciﬁc neurologic
endpoint (seizure activity, repetitive circling or other subtle
changes such as grooming or decrease in appetite) or to death.
Statistical analysis was performed using the Wilcoxon rankorder test as described previously.26,27 Animals were observed
twice daily for signs of distress or development of neurologic
symptoms, at which time, the mice were euthanized.

Results
Expression of podoplanin on glioblastoma andmedulloblastoma xenografts and cell line

Before the construction of a recombinant IT from a mAb, it isessential to demonstrate the reactivity of the mAb to brain tumor cells. Hence, the expression of podoplanin on brain tumor
xenograft cells was analyzed by ﬂow cytometry with NZ-1
mAb (Fig. 1, left panel). Fluorescence-activated cell sorter(FACS) data revealed signiﬁcant levels of podoplanin to bepresent on both glioblastoma (D2159MG, D08-0308MG and
D08-0493MG) and medulloblastoma (D283MED, D425MED
and DAOY) tumors (Fig. 1, left panel). The speciﬁcity of NZ-1
mAb was demonstrated by testing its reactivity on two additional glioblastoma xenograft cells (D08-0695MG and D080537MG), exhibiting almost complete absence of podoplanin(Supporting Information Fig. S1). The levels of podoplanin receptor on both podoplanin-positive and -negative brain tumor
cells were quantiﬁed using NZ-1 mAb by quantitative FACS(QFACS), and the data are presented in Table 3.

Anti-podoplanin immunotoxin for brain tumor therapy

The NZ-1 (scFv) was then fused with the sequences fordomains II and III of Pseudomonas exotoxin A, PE38KDEL,
carrying a modiﬁed C-terminus, which increases its intracellular retention, in turn enhancing its cytotoxicity. The stability ofan IT can be enhanced by mutating a single, key residue in the
VH and VL domains to cysteine, which forms a stabilizing disulﬁde bond.28 Thus, we constructed a scdsFv containing botha disulﬁde bond, generated by cysteine residues that replace
Gly44 in the framework region 2 (FR2) of VH and Gly103 in
the FR4 of VL, and a peptide linker. The NZ-1 (scdsFv) PCR
fragment was then fused to PE38KDEL. The NZ-1-(scFv)PE38KDEL and NZ-1-(scdsFv)-PE38KDEL IT constructs wereexpressed in E. coli under the control of T7 promoter and collected as inclusion bodies. Both NZ-1-(scFv)-PE38KDEL and
NZ-1-(scdsFv)-PE38KDEL were refolded and puriﬁed as
described in ‘‘Materials and Methods’’ section to >95% purity.
Size exclusion chromatography fraction 68 of both NZ-1(scFv)-PE38KDEL and NZ-1-(scdsFv)-PE38KDEL was concentrated (Supporting Information Fig. S3). The protein concentration was determined, and 2.0 lg of the puriﬁed proteins
R 4–12% Bis-Tris Gels (Invitrogen)was run in a NuPAGEV(Supporting Information Fig. S3). The ﬁnal yield of the puriﬁedprotein was 2 mg/l of bacterial culture.
Antigen-binding characteristics of NZ-1-(scdsFv)-PE38KDEL

To determine whether the NZ-1-(scdsFv)-PE38KDEL IT
binds to podoplanin expressed on the cell surface, indirect
ﬂow cytometric analysis was performed (Fig. 1, right panel).
Similar to the NZ-1 mAb, FACS analysis revealed signiﬁcantNZ-1-(scdsFv)-PE38KDEL binding to both glioblastoma(D2159MG, D08-0308MG and D08-0493MG) and medulloblastoma (D283MED, D425MED and DAOY) tumors (Fig. 1,
right panel).
The antigen-binding kinetics of NZ-1-(scdsFv), NZ-1(scdsFv)-PE38KDEL and NZ-1 mAb was assessed by surface
plasmon resonance (Fig. 2). The puriﬁed NZ-1-(scdsFv), NZ1-(scdsFv)-PE38KDEL and NZ-1 mAb were applied to sensor
chips coated with a biotinylated podoplanin peptide (hpp3851, corresponding to amino acids 38–51 of human podoplanin), which was used as an immunogen for the generation of
NZ-1 mAb.19,20 All three proteins bound to the podoplanin
peptide-coated chips, as expected NZ-1 mAb (KD ¼ 3.9 
1010 M) displayed a much higher afﬁnity as opposed toboth NZ-1-(scdsFv) (KD ¼ 2.1  108 M) and NZ-1(scdsFv)-PE38KDEL (KD ¼ 8.0  108 M) (Fig. 2). These
results demonstrate that the NZ-1-(scdsFv)-PE38KDEL IT
binds to both podoplanin peptide and to native protein molecules expressed on brain tumor cells.

Cloning of the variable domains of NZ-1 scFv andgeneration of NZ-1-(scdsFv)-PE38KDEL IT

Stability of NZ-1 ITs

Once the cell surface reactivity of NZ-1 was established, theVH and VL cDNAs were cloned and sequenced. The cloned
VH and VL fragments were 351 and 342 bp, respectively.
The NZ-1 VH and VL domain amino acid sequences areshown in Supporting Information Figure S2.

The stability of an IT plays a signiﬁcant role in determining
its therapeutic efﬁcacy. Earlier studies indicated scdsFvs
might be more stable than scFvs.28 Hence, the stability of
NZ-1-(scFv)-PE38KDEL was compared to that of NZ-1(scdsFv)-PE38KDEL over a 6- or 7-day period at 37 C by
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Chandramohan et al.

Figure 1. Flow cytometric analysis of brain tumor xenografts to determine reactivity of the NZ-1 mAb and NZ-1-(scdsFv)-PE38KDEL. Cells isolated
from brain tumor xenografts (a) D2159MG, (b) D08-0308MG, (c) D08-0493MG, (d) D283MED, (e) D425MED and (f) DAOY cell line were stainedwith NZ-1 mAb/ NZ-1-(scdsFv)-PE38KDEL (gray open peaks) or an isotype control IgG2a antibody/P588-(scdsFv)-PE38KDEL (filled black peaks).

measuring its cytotoxic activity against D2159MG xenograft
cells (Table 1) and DAOY cells (Table 2). Although the IC50
value of NZ-1-(scdsFv)-PE38KDEL increased eightfold fromday 0 to day 7, the IC50 value of NZ-1-(scFv)-PE38KDEL
increased 33.3-fold over the same time period on D2159MG
xenograft cells (Table 1). Similarly, on the medulloblastoma
cells DAOY, the IC50 value of NZ-1-(scdsFv)-PE38KDEL
increased 1.3-fold from day 0 to day 6; however, there was amuch higher increase in the IC50 value of NZ-1-(scFv)PE38KDEL, 21.4-fold over the same time period (Table 2),
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demonstrating that NZ-1-(scdsFv)-PE38KDEL was more stable than NZ-1-(scFv)-PE38KDEL.
Cytotoxicity of NZ-1-(scdsFv)-PE38KDEL toward
podoplanin-expressing brain tumor cells

The cytotoxic activity of NZ-1-(scdsFv)-PE38KDEL wasassessed on three podoplanin-positive (D2159MG, D080308MG and D08-0493MG) and two podoplanin-negative
glioblastoma xenograft cells (D08-0695MG and D08-0537MG),
and two medulloblastoma xenograft cells (D283MED andTumor Immunology

6Anti-podoplanin immunotoxin for brain tumor therapy

Figure 2. Surface plasmon resonance analysis of NZ-1-(scdsFv), NZ-1-(scdsFv)-PE38KDEL and NZ-1 mAb. Binding kinetics and affinity
constants of NZ-1-(scdsFv) (a), NZ-1-(scdsFv)-PE38KDEL (b) and NZ-1 mAb (c) were determined by surface plasmon resonance against
biotinylated podoplanin peptide (hpp38-51). The association and dissociation rates from the sensogram were KA ¼ 4.8  107 M1 and KD¼ 2.1  108 M for NZ-1-(scdsFv) (a), KA ¼ 1.2  107 M1 and KD ¼ 8  108 M for NZ-1-(scdsFv)-PE38KDEL (b) and KA ¼ 2.6  109 M1
and KD ¼ 3.9  1010 M for NZ-1 mAb (c).
Table 1. Stability of NZ-1-(scFv)-PE38KDEL vs. NZ-1-(scdsFv)PE38KDEL1 on D2159MG

Table 2. Stability of NZ-1-(scFv)-PE38KDEL vs. NZ-1-(scdsFv)PE38KDEL1 on DAOY

IC50 (ng/ml)

IC50 (ng/ml)

Sample

0

2

3

4

5

6

7 (days)

Sample0

NZ-1-(scFv)-PE38KDEL

0.6

3

4.5

5.5

6

7

20

NZ-1-(scFv)-PE38KDEL

28 81 220 220 380 400 600

1

NZ-1-(scdsFv)-PE38KDEL

0.5

1

1.5

1.5

3.5

2.5

4

NZ-1-(scdsFv)-PE38KDEL 40 42

2

41

3

41

4

48

5

6 (days)

48

51

1

1

NZ-1-(scFv)-PE38KDEL and NZ-1-(scdsFv)-PE38KDEL were incubatedwith 0.2% BSA/PBS at 7.5 lg/ml over a 7-day period at 37 C and thenassayed for cytotoxic activity on D2159MG xenograft cells. Cytotoxicity
data are given as an IC50 value, the concentration of immunotoxin thatcauses a 50% inhibition of protein synthesis after a 20-hr incubation
with immunotoxin.

NZ-1-(scFv)-PE38KDEL and NZ-1-(scdsFv)-PE38KDEL were incubatedwith 0.2% BSA/PBS at 7.5 lg/ml over a 6-day period at 37 C and thenassayed for cytotoxic activity on DAOY medulloblastoma cell line.
Cytotoxicity data are given as an IC50 value, the concentration of
immunotoxin that causes a 50% inhibition of protein synthesis after a
20-hr incubation with immunotoxin.

D425MED) and a medulloblastoma cell line (DAOY) (Table 3
and Supporting Information Fig. S4). Inhibition of protein synthesis by NZ-1-(scdsFv)-PE38KDEL was used as a measure ofits cytotoxic effect. The NZ-1-(scdsFv)-PE38KDEL was highly
cytotoxic, with IC50 in the range of 1.6–3.3 and 9.7-29 ng/ml on
the glioblastoma and medulloblastoma cells, respectively (Table
3 and Supporting Information Fig. S4). An IgG1j isotype con-trol-derived IT P588-(scdsFv)-PE38KDEL exhibited a nonspeciﬁc cytotoxic effect only at the highest concentration tested(1,000 ng/ml) on some of the xenograft cells (D2159MG, D080308MG, D08-0493MG and D283MED) (Supporting
Information Fig. S4). Both NZ-1-(scdsFv)-PE38KDEL andP588-(scdsFv)-PE38KDEL had a nonspeciﬁc cytotoxic effectat the highest concentration tested (1,000 ng/ml) on the
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Int. J. Cancer: 000, 000–000 (2012) V7Chandramohan et al.

Xenograft

NZ-1-(scdsFv)PE38KDEL,
IC50 (ng/ml)

Number of
podoplaninmolecules
per cell

Percent
internalized

D08-0695MG>1,0001.0  103

ND

D08-0537MG>1,0002

6.9  10ND

D2159MG

1.6 6 1.2

3.4  10680%

D08-0308MG

3.3 6 2.05

1.1  1087%

D08-0493MG

2.7 6 1.7

6.1  107

42%

D283MED9.7 6 4.0

7.3  103

ND

D425MED
DAOY

26.7 6 4.0
29.0 6 14.4

4

6.5  10

93%

6

21%

1.3  101

Cytotoxicity data are given as an IC50 value, the concentration of
immunotoxin that causes a 50% inhibition of protein synthesis after a
20-hr incubation with immunotoxin.
Abbreviation: ND: not determined.

podoplanin-negative glioblastoma xenograft cells (D080695MG) and no cytotoxic effect on the D08-0537MG xenograft cells (Supporting Information Figs. S4a and S4b).
Determination of podoplanin receptor number on brain
tumor cells using NZ-1 mAb

Under conditions assuming 1:1 stoichiometry between antibody and antigen, Alexa Fluor 488 (AF488)-labeled NZ-1
mAb was used to estimate the number of podoplanin molecules expressed on each brain tumor cell surface by QFACS.29
The results of QFACS analysis of glioblastoma and medulloblastoma cells with NZ-1-AF488 are summarized in Table 3.
The levels of surface podoplanin receptors by QFACS on glioblastoma and medulloblastoma xenograft cells varied widelywith 3.4  106 on D2159MG, 1.1  105 on D08-0308MG,
6.1  107 on D08-0493MG, 7.3  103 on D283MED, 6.5 
104 on D425MED and 1.3  106 on DAOY. The control glioblastoma xenografts, D08-0695MG (1.0  103) and D080537MG (6.9  102), expressed comparatively lower levels of
surface podoplanin molecules. QFACS analysis revealed thatthe number of podoplanin receptors expressed on glioblastoma cells (D2159MG, D08-0308MG and D08-0493MG) tobe signiﬁcantly higher compared to the levels on medulloblastoma cells (D283MED, D425MED and DAOY).
NZ-1-(scdsFv)-PE38KDEL internalization rates
on brain tumor cells

Upon binding to its cognate receptor, NZ-1-(scdsFv)-PE38KDEL is internalized by endocytosis and kills the target cells.
Internalization rates of NZ-1-(scdsFv)-PE38KDEL-AF488 on
different brain tumor cells were measured at various time
points (1–6 hr) and were compared to the total amount of IT
bound to the cell surface in the absence of internalization. Thisvalue was set as 100%. As shown in Table 3, the percent of internal NZ-1-(scdsFv)-PE38KDEL-AF488 in D2159MG (80%)
C 2012 UICC
Int. J. Cancer: 000, 000–000 (2012) V

and D08-0308MG (87%) was twofold greater than that of D080493MG (42%). On the D425MED xenograft cells, NZ-1(scdsFv)-PE38KDEL-AF488 was internalized at a much fasterrate. At a concentration of 10 lg/ml, 93% of NZ-1-(scdsFv)PE38KDEL-AF488 on D425MED cells was internalized,
whereas only 21% of the IT was internalized on DAOY cells(Table 3). Hence, different brain tumor cells show variation in
their rates of NZ-1-(scdsFv)-PE38KDEL internalization.
Antitumor activity of NZ-1-(scdsFv)-PE38KDEL
in subcutaneous tumor model

The antitumor activity of NZ-1-(scdsFv)-PE38KDEL wasinvestigated in female athymic nude mice bearing D2159MG
or D283MED subcutaneous tumors. D2159MG or D283MED
tumors were treated intratumorally with three doses of theNZ-1-(scdsFv)-PE38KDEL at a 0.3 mg/kg concentration. The
control groups were treated with either 0.2% PBS-HSA or 0.3
mg/kg of the nonspeciﬁc control IT, P588-(scFv)-PE38KDEL.
The D2159MG xenograft tumors regressed in eight of ten
mice treated with NZ-1-(scdsFv)-PE38KDEL. Compared tothe control IT-treated animals, a growth delay of 19 days,
with a p-value of <0.001, was observed in NZ-1-(scdsFv)PE38KDEL-treated group (Fig. 3a). Similarly, with the
D283MED model, a tumor growth delay of 10 days, with ap-value of <0.001, along with two tumor regressions wasobserved in the NZ-1-(scdsFv)-PE38KDEL group as opposedto the control group (Fig. 3b). No toxicity-related deaths oradverse effects were observed in any of the treated animals.
Intracranial efficacy of NZ-1-(scdsFv)-PE38KDEL
on D425MED tumor model

To determine the time course of D425MED intracranialtumor growth in the xenograft model, 1  105 cells/5 ll was
injected into male NSG mice (11 mice) and a survival curve
was plotted (Supporting Information Fig. S5). The D425MED
survival curve demonstrated that 100% death occurred at day
14 post-tumor implantation; hence, post-tumor implantation
day 4 was chosen as the optimal day to study the efﬁcacy of
NZ-1-(scdsFv)-PE38KDEL. Further, to determine whether
NZ-1-(scdsFv)-PE38KDEL would be tolerated intracranially,
NSG mice were given different concentrations of the NZ-1(scdsFv)-PE38KDEL IT (0.1–3.0 lg/100 ll) through an Alzet
pump over a 3-day period (ﬁve mice per group). Supporting
Information Figure S6 depicts that both 0.1 and 0.3 lg of
NZ-1-(scdsFv)-PE38KDEL had no toxicity-associated mortality. Therefore, 0.3 lg of NZ-1-(scdsFv)-PE38KDEL wasselected to be the ideal concentration for intracranial therapy.
On the basis of the survival and toxicity studies, the efﬁcacy study was performed on day 4 post-D425MED tumor
implantation with 0.3 lg dose of the NZ-1-(scdsFv)-PE38KDEL. The control groups were either left untreated (sham) or
treated with 0.2% PBS-HSA or 0.3 lg of the nonspeciﬁc
control IT, P588-(scdsFv)-PE38KDEL. Treatment of the tumor-bearing mice with NZ-1-(scdsFv)-PE38KDEL increased
survival by 41% (Fig. 4) and the Kaplan–Meier survival curve

Tumor Immunology

Table 3. Cytotoxicity, receptor number and internalization of
NZ-1-(scdsFv)-PE38KDEL on brain tumor cells18Anti-podoplanin immunotoxin for brain tumor therapy

Tumor Immunology

Figure 3. Effect of NZ-1-(scdsFv)-PE38KDEL on D2159MG and D283MED subcutaneous tumors in athymic nude mice. Female athymic nude
mice (20 g; 4–6 weeks) bearing D2159MG tumors (a) or D283MED tumors (b) were randomized into three groups: 0.2% PBS-HSA (n),P588-(scFv)-PE38KDEL (~) and NZ-1-(scdsFv)-PE38KDEL (l). Once the tumors reached an average volume of 150–300 mm3, the mice weretreated by i.t. injections every other day with a total of three 0.3 mg/kg doses of P588-(scFv)-PE38KDEL or NZ-1-(scdsFv)-PE38KDEL diluted
in 0.2% PBS-HSA or with 0.2% PBS-HSA alone.

Figure 4. Effect of NZ-1-(scdsFv)-PE38KDEL on D425MED intracranial tumors in NSG mice. Male NSG mice (20–30 g; 12 weeks) bearing
D425MED tumors were randomized into four groups: sham (l), 0.2% PBS-HSA (n), P588-(scdsFv)-PE38KDEL (D) and NZ-1-(scdsFv)PE38KDEL (^). Four days post-tumor implantation, the mice were either left untreated (sham) or treated with 0.3 lg of P588-(scdsFv)PE38KDEL or NZ-1-(scdsFv)-PE38KDEL diluted in 100 ll of 0.2% PBS-HSA or with 0.2% PBS-HSA alone, delivered intracranially through an
Alzet pump over a 3-day period.

C 2012 UICC
Int. J. Cancer: 000, 000–000 (2012) Vdemonstrated a statistically signiﬁcant (p  0.001) increase insurvival time in the 0.3 lg NZ-1-(scdsFv)-PE38KDEL-treatedmice when compared to the controls (Fig. 4).

Discussion
The clinical success of mAbs and recombinant IT-basedtargeted therapies against solid intracranial tumors has led
investigators to identify novel brain-tumor-speciﬁc antigen
targets.30 Podoplanin is one such antigen that is highly
expressed on several CNS tumors.13,14 NZ-1 is a mAb exhibiting high reactivity to podoplanin (KD ¼ 1.2  1010 M)
and when bound by glioblastoma cells expressing podoplanin,
NZ-1 mAb is efﬁciently internalized,21 thereby demonstrating
its potential as an important immunotherapeutic agent. In
our study, we demonstrate the expression of podoplanin on
both glioblastoma and medulloblastoma xenografts by ﬂow
cytometry using NZ-1 mAb. Further, we constructed arecombinant IT, NZ-1-(scdsFv)-PE38KDEL, by fusing the
scFv segment of the NZ-1 mAb with the PE38KDEL
molecule. Similar to the NZ-1 mAb, the NZ-1-(scdsFv)PE38KDEL IT exhibited considerable binding to both glioblastoma and medulloblastoma cells by ﬂow cytometry. The
afﬁnity of NZ-1-(scdsFv)-PE38KDEL was determined to be
KD ¼ 8.0  108 M by Biacore. NZ-1-(scdsFv)-PE38KDEL
displayed considerable stability when incubated at 37 C forlong time periods. NZ-1-(scdsFv)-PE38KDEL demonstrated
signiﬁcant cytotoxicity against both glioblastoma and medulloblastoma cells. Further, in subcutaneous and intracranial
animal models, with glioblastoma or medulloblastoma xenografts, NZ-1-(scdsFv)-PE38KDEL demonstrated signiﬁcanttumor growth delays (p < 0.001), which makes it a noveland efﬁcacious immunotherapeutic agent against malignant
brain tumors.
Consistent with earlier studies, we demonstrate podoplanin to be present in both glioblastoma and medulloblastoma
xenografts.14 Flow cytometry analysis with cells isolated fromglioblastoma (D2159MG, D08-0308MG and D08-0493MG)
and medulloblastoma xenografts (D283MED and D425MED)
and a medulloblastoma cell line (DAOY) demonstrates different levels of reactivity to both the NZ-1 mAb and NZ-1(scdsFv)-PE38KDEL, which directly correlates with podoplanin expression (Fig. 1). Further, the QFACS data demonstratea higher level of podoplanin to be present on glioblastoma
xenografts than on the medulloblastoma xenografts. This difference in cell surface levels of podoplanin could contributeto the lower cytotoxicity values obtained with NZ-1-(scdsFv)PE38KDEL against glioblastoma xenografts as opposed tomedulloblastoma cells (Table 3). Moreover, despite having
similar cytotoxicity values within themselves, the levels of
podoplanin expression and the rate of podoplanin receptor
internalization varied considerably among both glioblastoma
and medulloblastoma xenografts (Table 3). Thus, boththe cell surface levels of podoplanin and the internalization
rate determine the cytotoxic activity of NZ-1-(scdsFv)PE38KDEL.
C 2012 UICC
Int. J. Cancer: 000, 000–000 (2012) V

9

The afﬁnity of NZ-1-(scdsFv)-PE38KDEL was determinedby surface plasmon resonance with podoplanin peptide(hpp38-51). The association rate, ka ¼ 4.9  104 (Ms)1, andthe dissociation rate, kd ¼ 3.9  103 s1, of NZ-1-(scdsFv)PE38KDEL were 2.6-fold lower and 80-fold higher than thatof the NZ-1 mAb, respectively. This difference in the association and dissociation rates observed between the NZ-1(scdsFv)-PE38KDEL and NZ-1 mAb could be explained by
the monovalent and bivalent nature of the IT and mAb,
respectively. Despite having lower afﬁnity, the cytotoxic activity (IC50 values) of NZ-1-(scdsFv)-PE38KDEL was comparable to the activity obtained with a mutant epidermal growth
factor receptor-speciﬁc IT currently being evaluated in Phase
I clinical trial for glioblastoma patients.24,31
Additionally, an IT has to be signiﬁcantly stable over long
periods of time to demonstrate therapeutic efﬁcacy whenadministered to brain tumor patients. The VH and VL
domains of NZ-1 scFv are held together by a 15-amino acidpeptide linker, and it is well established that the noncovalent,interdomain interactions provided by the peptide linker are
not strong enough to keep them together.32 The linker israther ﬂexible, and this could promote the interaction
between the VH and VL domains of NZ-1-(scFv)-PE38KDEL
and a nearby IT molecule, which could thereby affect the stability of the IT. We hypothesized that this problem could beovercome by introducing an interchain disulﬁde bond
between the structurally conserved framework regions of theNZ-1 VH and VL domains.28 The new disulﬁde-stabilized IT
is termed NZ-1-(scdsFv)-PE38KDEL. As hypothesized, theincorporation of a disulﬁde bond, in addition to the peptide
linker, offered higher stability to NZ-1-(scdsFv)-PE38KDEL,
which was evident from its lower IC50 values compared to
NZ-1-(scFv)-PE38KDEL on both D2159MG xenograft cells
and DOAY cells (Tables 1 and 2).
The in vivo results were compatible with the in vitro ﬂow
cytometry and cytotoxic activity data. Our in vitro results
demonstrated higher reactivity and cytotoxicity of NZ-1(scdsFv)-PE38KDEL against glioblastoma xenografts. Accordingly, NZ-1-(scdsFv)-PE38KDEL caused greater tumor
regression (eight of ten mice) and a signiﬁcant tumor growth
delay (p-value of <0.001) against the glioblastoma xenograft
D2159MG. Moreover, it was also effective in producing a tumor growth delay of 10 days (p-value of <0.001) against the
medulloblastoma model D283MED. In this regard, NZ-1(scdsFv)-PE38KDEL is a truly novel IT that can target two
different CNS malignancies effectively. In our in vivo animalmodels, the NZ-1-(scdsFv)-PE38KDEL IT was administeredby intratumoral injections. In the treatment of malignant
brain tumors, the NZ-1-(scdsFv)-PE38KDEL IT will beadministered directly into the tumor site and thus bypass the
blood–brain barrier by convection-enhanced delivery(CED).31 To replicate an actual brain tumor model, theD425MED xenograft cells were implanted intracranially andwere treated by CED with NZ-1-(scdsFv)-PE38KDEL. Similar
to the subcutaneous brain tumor models, NZ-1-(scdsFv)-

Tumor Immunology

Chandramohan et al.

10Anti-podoplanin immunotoxin for brain tumor therapy

PE38KDEL was extremely efﬁcacious in the intracranialmodel and increased the survival of tumor-bearing mice by
41% (p-value of 0.001).
In conclusion, our results clearly demonstrate thatthe disulﬁde-stabilized recombinant IT NZ-1-(scdsFv)PE38KDEL is highly efﬁcacious against CNS tumors expressing podoplanin in a preclinical setting and hence is asuitable candidate for targeted therapy of malignant brain
tumors.

Acknowledgements
This work was supported by The National Cancer Institute [Grant numbers:
NINDS 5P50 NS20023-29, and NCI Merit Award 5R37 CA011898-42 (D.D.
Bigner)] and also by Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research (I.H. Pastan).

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