Available online at www.sciencedirect.com

Nuclear Medicine and Biology 37 (2010) 785 – 794
www.elsevier.com/locate/nucmedbio

Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting
malignant gliomas☆
Yukinari Katoa,b,⁎, Ganesan Vaidyanathanc , Mika Kato Kanekoa,b , Kazuhiko Mishimad ,Nidhi Srivastavaa , Vidyalakshmi Chandramohana , Charles Pegrama , Stephen T. Keire ,Chien-Tsun Kuana , Darell D. Bignera , Michael R. ZalutskycaDepartment of Pathology, Duke University Medical Center, Durham, NC 27710, USA
The Oncology Research Center, Advanced Molecular Epidemiology Research Institute, Yamagata University Faculty of Medicine,
Yamagata 990-9585, Japanc
Department of Radiology, Duke University Medical Center, Durham, NC 27710, USAd
Saitama Medical University International Medical Center 1397-1 Yamane Hidaka-shi, Saitama 350-1298, Japane
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA
Received 30 December 2009; received in revised form 4 March 2010; accepted 25 March 2010b

Abstract
Introduction: Podoplanin/aggrus is a mucin-like sialoglycoprotein that is highly expressed in malignant gliomas. Podoplanin has beenreported to be a novel marker to enrich tumor-initiating cells, which are thought to resist conventional therapies and to be responsible forcancer relapse. The purpose of this study was to determine whether an anti-podoplanin antibody is suitable to target radionuclides tomalignant gliomas.
Methods: The binding affinity of an anti-podoplanin antibody, NZ-1 (rat IgG2a), was determined by surface plasmon resonance andScatchard analysis. NZ-1 was radioiodinated with 125I using Iodogen [125I-NZ-1(Iodogen)] or N-succinimidyl 4-guanidinomethyl 3-[131I]
iodobenzoate ([131I]SGMIB-NZ-1), and paired-label internalization assays of NZ-1 were performed. The tissue distribution of 125I-NZ-1(Iodogen) and that of [131I]SGMIB-NZ-1 were then compared in athymic mice bearing glioblastoma xenografts.
Results: The dissociation constant (KD) of NZ-1 was determined to be 1.2×10−10 M by surface plasmon resonance and 9.8×10−10 M for
D397MG glioblastoma cells by Scatchard analysis. Paired-label internalization assays in LN319 glioblastoma cells indicated that [131I]
SGMIB-NZ-1 resulted in higher intracellular retention of radioactivity (26.3±0.8% of initially bound radioactivity at 8 h) compared to thatfrom the 125I-NZ-1(Iodogen) (10.0±0.1% of initially bound radioactivity at 8 h). Likewise, tumor uptake of [131I]SGMIB-NZ-1 (39.9±8.8 %ID/g at 24 h) in athymic mice bearing D2159MG xenografts in vivo was significantly higher than that of 125I-NZ-1(Iodogen) (29.7±6.1 %ID/g
at 24 h).
Conclusions: The overall results suggest that an anti-podoplanin antibody NZ-1 warrants further evaluation for antibody-based therapy
against glioblastoma.
© 2010 Elsevier Inc. All rights reserved.
Keywords: Podoplanin; Monoclonal antibody; Malignant gliomas; SGMIB; Internalization; Biodistribution

1. Introduction☆

This study was supported by NIH grants CA42324, 5P50 CA108786,
5P50 NS20023 and R37 CA011898. Y.K. was supported by the Global COE
Program for Medical Sciences, Japan Society for the Promotion of Science.
⁎ Corresponding author. Department of Pathology, Duke University
Medical Center, Durham, NC 27710, USA. Tel.: +1 919 613 6154; fax: +1
919 684 6791.
E-mail address: yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
0969-8051/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2010.03.010

Gliomas are the most common primary brain tumors,and glioblastoma multiforme is the most frequent andmalignant type of gliomas [1]. Despite advances in surgical
techniques, radiation therapy and adjuvant chemotherapy,
their prognoses remain poor. Many antigens, such as
epidermal growth factor receptor (EGFRwt), its gliomaassociated deletion variant EGFRvIII, lacto-series ganglio-

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Y. Kato et al. / Nuclear Medicine and Biology 37 (2010) 785–794

sides, tenascin and chondroitin sulfate proteoglycans, havebeen found in gliomas, and up-regulation of thosemolecules has been observed in brain tumor cells [2–7].
Although these molecules are under investigation as
therapeutic targets, multiple entities ultimately may haveto be targeted for optimal therapy in order to compensate
for tumor heterogeneity.
Podoplanin is a platelet aggregation-inducing factor[8–11] and its expression has been reported in many tumors
including malignant brain tumors [12–23]. Importantly,
recent investigations have suggested that expression of
podoplanin is associated with tumor metastasis [24,25],
malignant progression [15,22] and epithelial-mesenchymaltransition [26]. Podoplanin expression has also been reported
to be associated with clinical outcome [14,16–18,27,28]. In
solid tumors such as brain tumors, only a small andphenotypically distinct subset of cells could be responsible
for generating and sustaining tumors and thus be considered
cancer stem cells or tumor- initiating cells (TICs) [29].
Because TICs are thought to be resistant to conventional
therapies and are responsible for relapse, targeting TICs
could be a promising approach to cancer therapy [30,31].
Podoplanin has been reported to be a TIC marker; therefore,
immunotherapy using specific antibodies reactive to podoplanin may eradicate TICs in cancers [32].
We previously produced an anti-podoplanin antibody,
NZ-1 [23,33]. NZ-1 should have not only high specificity
and sensitivity but also high binding affinity against
podoplanin to be applied for radioimmunotherapy or
immunotoxin therapy. Furthermore, particularly for its use
as an immunotoxin, NZ-1 should be internalized into tumor
cells and also well accumulated into tumors in vivo. The
object of this study was to determine the affinity of NZ-1 andto investigate whether NZ-1 is a suitable candidate fortherapy against malignant gliomas by performing internalization assays in vitro using several glioblastoma cell lines
and biodistribution experiments in vivo using glioblastoma
xenograft models.

Committee and National Institutes of Health guidelines. The
LN319 glioblastoma cell line was donated by Dr. Webster K.
Cavenee (Ludwig Institute for Cancer Research, San Diego,
CA, USA). D397MG and D245MG glioblastoma cell lines
and the D2159MG xenograft were established at Duke
University. D2159MG xenograft cells were dissociated with
Liberase (Roche, Indianapolis, IN, USA) at a 100 μg/ml
concentration. LN319 and D2159MG were cultured at 37°C
in a humidified atmosphere of 5% CO2 and 95% air in
Dulbecco's Modified Eagle's Medium (Invitrogen Corp.,
Carlsbad, CA, USA), including 2 mM L-glutamine and 1%of a penicillin–streptomycin solution, and D397MG and
D245MG were cultured in Zinc Option medium (Invitrogen)supplemented with 10% heat-inactivated fetal bovine serum(Sigma-Aldrich).
2.3. Anti-podoplanin monoclonal antibody NZ-1
The development of the anti-podoplanin mAb NZ-1 wasdescribed previously [23]. Briefly, Sprague-Dawley rats
were immunized by neck subcutaneous injections of thesynthetic peptide EGGVAMPGAEDDVV (hpp3851),
corresponding to amino acids 38–51 of human podoplaninplus C-terminus cysteine conjugated with KLH with
Complete Freund's Adjuvant (Difco Laboratories, Detroit,
MI, USA). One week later, secondary intraperitoneal
immunization was performed. The booster injection wasgiven intraperitoneally 2 days before spleen cells were
harvested. The spleen cells were fused with mouse myeloma
P3U1 cells by using polyethylene glycol (Mr 4000); thehybridomas were grown in RPMI medium with hypoxanthine, aminopterin and thymidine selection medium supplement (Sigma-Aldrich). The culture supernatants werescreened by ELISA for binding to the synthetic peptide.
The characterization of NZ-1 was performed as in a previous
study [23,33]. NZ-1 was produced in ascites of athymic miceand purified with a Protein-G column (Thermo Scientific,
Inc., Rockford, IL, USA).
2.4. Flow cytometry

2. Materials and methods
2.1. General
All reagents were purchased from Sigma-Aldrich (St.
Louis, MO, USA) except where noted. Sodium 125 I-iodide(2200 Ci/mmol) and sodium 131I-iodide (1200 Ci/mmol) in
0.1N NaOH were supplied by Perkin-Elmer Life andAnalytical Sciences (Boston, MA, USA).
2.2. Animals, cell lines and the xenograft model
Female athymic mice (nu/nu genotype, BALB/c background, 6 weeks or older) were used for all antitumor studies.
Animals were maintained in Thoren filter-top cages (Thoren
Caging Systems, Hazleton, PA, USA). All animal procedures conformed to Institutional Animal Care and Use

Glioblastomas cells were collected by trypsin-EDTA
treatment and were incubated with NZ-1 (1 μg/ml) or isotype
control (rat IgG2a) for 30 min at 4°C. Then the cells wereincubated with an Oregon green-conjugated anti-rat antibody(1:200 diluted; Invitrogen) for 30 min. Flow cytometry was
performed using a FACSCalibur instrument (Becton Dickinson, Franklin Lakes, NJ, USA).
2.5. Affinity determination by surface plasmon resonanceTo determine the affinity, biotinylated podoplanin peptide(hpp3851) was immobilized on the surface of streptavidin
chips for analysis by using the BIAcore 3000 system(BIAcore, Piscataway, NJ, USA). The running buffer was 10mM HEPES, 150 mM NaCl and 3.4 mM EDTA (pH 7.4).
The NZ-1 was passed over the biosensor chip, and affinity
rate constants (association rate constant, kassoc, and disasso-

Y. Kato et al. / Nuclear Medicine and Biology 37 (2010) 785–794

ciation rate constant, kdiss) were determined by nonlinear
curve fitting using the Langmuir one-site binding modelof the BIAevaluation software (BIAcore). The affinity
constant (KA) at equilibrium was calculated as KA=kassoc/
kdiss, and the dissociation constant (KD) was determined
as 1/KA.
2.6. Radiolabeling of NZ-1 with
I-NZ-1(Iodogen)125

I using Iodogen:125

A solution of NZ-1 (250–300 μg; 5 mg/ml) in phosphatebuffer (pH 7.4) was added to a 2-dram vial coatedwith Iodogen (10 μg) followed by a solution of sodium[125 I]iodide (∼1 mCi; 2–3 μl). The mixture was incubated atroom temperature for 10 min with occasional shaking, andthe labeled protein was isolated by gel filtration on a PD-10
column (GE Healthcare, Piscataway, NJ, USA) using PBS as
running buffer.
2.7. Radiolabeling of NZ-1 with SGMIB
The radioiodinated prosthetic group was synthesizedfrom the corresponding trialkylstannyl precursor andpurified as previously reported [34]. For labeling with[125 I]SGMIB or [131I]SGMIB, NZ-1 in borate buffer (pH8.5; 50 μl of 5 mg/ml) was incubated with 0.4–1.6 mCi(0.33–1.33 nmol) of the tracer at room temperature for
15–20 min, and the labeled NZ-1 ([125I]SGMIB-NZ-1 or[131 I]SGMIB-NZ-1) was purified by gel filtration. The
integrity of labeled NZ-1 was determined by gel filtration
HPLC using a TSK2000SW column.
2.8. Determination of immunoreactive fraction
The immunoreactivity of radiolabeled NZ-1 was evaluated by a magnetic bead assay [35] using streptavidin-coated
MPG magnetic beads (Controlled Pore Glass, Lincoln Park,
NJ, USA) coated with the biotin-labeled podoplanin peptide(hpp3851) or, to control for nonspecific binding, biotinlabeled BSA. Each labeled NZ-1 solution (5 ng in 50 μl PBS)
in triplicate was added to increasing volumes (25, 50 and 100μl) of both positive and control beads in 115 mM phosphatebuffer (pH 7.4) with 0.05% BSA and 0.05% Brij 35, and thepercent of total radioactivity that bound to the beads wasdetermined. The immunoreactive fractions were calculatedby the method of Lindmo et al. [36].
2.9. Scatchard analysis
A modified Scatchard analysis was performed to measure
the binding affinity of 125I-NZ-1(Iodogen) against LN319
and D397MG glioblastoma cells. Cells were plated in 24well plates at a density of 5×104 per well and incubatedovernight at 37°C in a 5% CO2 humidified atmosphere.
125
I-NZ-1(Iodogen) was serially diluted from 8 μg/ml andwas incubated with podoplanin-positive glioblastoma celllines LN319 and D397MG at 4°C for 4 h. The podoplaninnegative cell line D245MG was used as a negative control.
The cell-bound radioactivity was measured as a proportion787

of input activity, and nonlinear regression analysis was
performed to calculate the dissociation constant (KD) with
GraphPad Prism software (GraphPad Prism Software, San
Diego, CA, USA).
2.10. Internalization assay
The podoplanin-expressing cell lines or xenograftderived cells used in these studies were LN319, D397MG
and D2159MG. Cells were plated in six-well plates at adensity of 5×105 per well and incubated overnight at 37°C in
a 5% CO humidified atmosphere. On the day of the assay,
the cells were incubated at 4°C for 30 min. The assays were
performed in a single- or dual-label format. Both labeled(125I and 131 I; about 1 μg each) NZ-1 mAbs were added to
the wells, creating conditions of mAb excess. The cells wereincubated at 4°C for 1 h, washed with fresh medium toremove unbound radioactivity and brought to 37°C. Cells
were then processed at 0, 1, 2, 4, 8, 16 and 24 h as follows:
acidic medium (zinc option, pH 2.0; 1 ml) was added to eachwell, and the cells were incubated for 10 min at 4°C. The
acid wash was transferred to a counting vial, and the cells
were further washed with 1 ml of the acidic medium andpooled with the previous wash. The cells were solubilized byincubation at room temperature with 0.5 ml of 0.5N NaOH
overnight. Cell culture supernatant aliquots, acid washes andsolubilized cell fractions were then counted in the gamma
counter using a dual-label program.
2.11. Biodistribution
Subcutaneous tumor transplantation was done in theright flank of the animals with an inoculation volume of 50
μl using tumor homogenate from xenografts as previously
described [37]. Groups of five mice were used for each time
point of the studies. In the first experiment, performed in apaired-label format, mice were injected with 4.0 μCi each of
125
I-NZ-1(Iodogen) and [131I]SGMIB-NZ-1 via the tailvein, and biodistribution of the radiolabeled mAb wasdetermined at 6, 24, 48, 72, 144 and 192 h. A second
experiment was conducted to determine the specificity of
uptake. For this experiment, the control mice were injectedwith 4 μCi of [125I]SGMIB-NZ-1 with or without coinjection of a 100-fold excess of unlabeled NZ-1, andbiodistribution was examined at 6, 24 and 48 h. At eachtime point, mice were killed with an overdose ofisofluorane, and tumor and other organs of interest were
harvested, blot-dried, weighed and counted in an automatedgamma counter for 125 I and 131I activity along with
injection standards. The results are expressed as percent
injected dose per gram of tissue (%ID/g). The statisticalsignificance of the difference in uptake between the two
tracers was calculated by a paired or unpaired Student's t
test using the Microsoft Excel program statistical function.
The differences were considered to be significant if the Pvalues were less than .05.

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3. Results
3.1. Radiolabeling
NZ-1 was radiolabeled using Iodogen in almost quantitative radiochemical yields and with a specific activity of 3.9
mCi/mg. When SGMIB was used for radiolabeling, theconjugation yield was 66.6±14.1% (n=2) with a specificactivity of 2.6±0.4 mCi/mg. Size-exclusion HPLC indicatedthat more than 95% of the radioactivity was associated with apeak corresponding to the retention time of the intact mAb
with little or no aggregation. Immunoreactive fractions foreach batch of the labeled NZ-1 are given in appropriate
subsections below.
3.2. Kinetics of NZ-1 binding to podoplaninanalysis, NZ-1 was labeled with 125 I using Iodogen, with
an immunoreactive fraction of 76%. 125 I-NZ-1(Iodogen)exhibited KD values of 2.58×10−9 and 9.79×10−10 (mol/L)against LN319 and D397MG, respectively, indicating that
NZ-1 also has high affinity against podoplanin-expressing
glioblastoma cells.
3.3. Internalization of 125I-NZ-1(Iodogen) by D397MG and
LN319 glioblastoma cells
An internalization assay was performed in a single-label
format using 125 I-NZ-1(Iodogen) and the podoplaninpositive glioblastoma cells, D397MG and LN319. The
immunoreactive fraction of 125 I-NZ-1(Iodogen) was 76%.
Cell surface-bound counts and culture supernatant counts in
D397MG showed little change over time (Fig. 2A), whereas

To determine the affinity of NZ-1, we performed a kinetic
analysis of the interaction of NZ-1 with a podoplanin peptide(hpp3851) by surface plasmon resonance (BIAcore).
Determination of the association and dissociation rates
from the sensorgrams revealed a kassoc of 3.2×105 (mol/L s)−1
and a kdiss of 4.3×105 s−1. The KA at binding equilibrium,
calculate as KA=kassoc/kdiss, was 7.4×109 (mol/L)−1, KD=1/
KA=1.2×10−10 (mol/L), indicating that NZ-1 has highaffinity binding to a podoplanin peptide. NZ-1 was also
analyzed by conventional Scatchard analysis using podoplanin-positive glioblastoma cell lines LN319 and D397MG
and a podoplanin-negative glioblastoma cell line, D245MG.
Podoplanin expression in the positive cell lines was
confirmed by flow cytometry (Fig. 1). For Scatchard

Fig. 1. NZ-1 recognition of podoplanin on the cell surface of glioblastoma
cells by flow cytometry. D397MG, LN319, D2159MG and D245MG
glioblastoma cells were collected by trypsin-EDTA treatment, and cells wereincubated with NZ-1 or an isotype-matched control antibody (rat IgG2a). The
cells were next incubated with an Oregon green-conjugated anti-rat
antibody. Flow cytometry was performed using a FACSCalibur instrument.

Fig. 2. Internalization of NZ-1 into glioblastoma cells. D397MG (A) and
LN319 (B) cells were incubated with 125I-NZ-1(Iodogen) at 4°C for 1 h andthen washed with fresh medium to remove unbound radioactivity. Cells
were cultured at 0, 1, 2, 4, 8, 16 and 24 h at 37°C and processed at various
time periods.

Y. Kato et al. / Nuclear Medicine and Biology 37 (2010) 785–794

cell surface-bound counts in LN319 gradually decreased,accompanied by a slow increase in counts in the cell culture
supernatant (Fig. 2B). The intracellularly trapped radioactivity of 125I-NZ-1(Iodogen) in D397MG cells gradually
increased to a maximum of 21.6±0.3% at the 4-h time point(Fig. 2A). A similar trend was seen in LN319 cells with22.4±0.5% of initially bound radioactivity within the cells at
4 h (Fig. 2B).
3.4. Paired-label internalization of NZ-1 by LN319 cells:
Iodogen vs. SGMIB
The internalization of 125 I-NZ-1(Iodogen) and[ I]SGMIB-NZ-1 by LN319 cells was compared inpaired-label format after binding during 1-h incubation at
4°C. The immunoreactive fraction was 62% and 55% for 125I131

789

131

NZ-1(Iodogen) and [ I]SGMIB-NZ-1, respectively. At
every time point studied, the percentage of the radioiodine
activity initially bound to the cells for [131I]SGMIB-NZ-1(Fig. 3B) that was present in the intracellular compartment
was significantly higher (Pb.01) than that for 125 I-NZ-1(Iodogen) (Fig. 3A). The percent of radioactivity retained
intracellularly for 125 I-NZ-1(Iodogen) peaked at 1 h (13.8±0.6%) and declined thereafter (Fig. 3A). In contrast,
internalized radioactivity peaked at 8 h (26.3±0.8%) with[131 I]SGMIB-NZ-1 (Fig. 3B). At this time point, theintracellularly trapped 131I activity was more than 2.5 times
higher than the 125I activity (9.95±0.15%). To confirm thisresult, internalization assay using the D2159MG xenograft
cells was also performed. The immunoreactive fraction of[125I]SGMIB-NZ-1 was 75%. As shown in Fig. 4, cellsurface-bound counts for D2159MG gradually decreased,accompanied by a slow increase in counts in the cell culture
supernatant. The internalization of [125I]SGMIB-NZ-1 into
D2159MG cells gradually increased to a maximum of 30.7±
2.0% at 16 h, suggesting that [125 I]SGMIB-NZ-1 was alsointernalized effectively into D2159MG xenograft cells.
3.5. Biodistribution of NZ-1 in D2159MG xenograft:
Iodogen vs. SGMIB
A paired-label biodistribution experiment was performed
in athymic mice bearing D2159MG human glioblastoma
xenograft to directly compare the tumor and normal organ
distribution of an anti-podoplanin mAb NZ-1 radioiodinatedusing Iodogen and SGMIB methods. The immunoreactive
fraction was 62% and 55%, respectively. No growth of these
D2159MG xenografts was seen during the course of theexperiments. The mean tumor weight harvested at necropsy
on the first day was 0.46±0.20 g, whereas that on theFig. 3. Paired-label internalization of 125I-NZ-1(Iodogen) and [131I]SGMIBNZ-1 in LN319 cells. LN319 cells were incubated with 125I-NZ-1(Iodogen)(A) or [131I]SGMIB-NZ-1 (B) at 4°C for 1 h and then washed with fresh
medium to remove unbound radioactivity. Cells were cultured at 0, 1, 2, 4, 8,
16 and 24 h at 37°C and processed at various time periods.

Fig. 4. Internalization of [125I]SGMIB-NZ-1 in D2159MG xenograft cells.
D2159MG cells were incubated with [125I]SGMIB-NZ-1 at 4°C for 1 h
and washed with fresh medium to remove unbound radioactivity. Cells
were cultured at 0, 1, 2, 4, 8, 16 and 24 h at 37°C and processed at various
time periods.

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Y. Kato et al. / Nuclear Medicine and Biology 37 (2010) 785–794

eighth day was 0.45±0.19 g. As shown in Fig. 5, thetumor accumulation of radioiodine activity (%ID/g) of[131 I]SGMIB-NZ-1 was significantly higher than that of
125
I-NZ-1(Iodogen) (Pb.05) at 24 h (39.9±8.8 %ID/g vs.
29.7±6.1 %ID/g) and 48 h (34.4±5.2 %ID/g vs. 27.4±4.0 %ID/g). Data for uptake in normal tissues are given in Table 1.
Except at early time points for a few tissues, the uptake ofradioactivity from [131I]SGMIB-NZ-1 was generally lower.
Liver and spleen demonstrated slightly higher uptake at 6 h.
The thyroid uptake of radioactivity from [131 I]SGMIB-NZ1 was substantially lower than that from 125 I-NZ-1(Iodogen) at all time points. Higher uptake in tumor andconcomitant lower uptake in normal tissues resulted inhigher tumor-to-tissue ratios for [131I]SGMIB-NZ-1 (data for
selected tissues given in Fig. 6).
To determine the specificity of uptake in D2159MG
tumor in vivo, the biodistribution assay of NZ-1 labeled with
SGMIB ([125I]SGMIB-NZ-1) was repeated. The immunoreactive fraction of [125 I]SGMIB-NZ-1 was 75%. No growthof these D2159MG xenografts was seen during the course of
the experiments. The mean tumor weight harvested at
necropsy on the first day was 0.98±0.19 g, whereas it was
0.68±0.29 g on the second day. As shown in Fig. 7, thetumor accumulation of radioiodine activity from [125I]
SGMIB-NZ-1 was significantly reduced at all time points

Fig. 5. Paired-label uptake of 125I-NZ-1(Iodogen) or [131I]SGMIB-NZ-1 inathymic mice hosting D2159MG xenografts. Values given are %ID/g (mean±S.D.). An asterisk indicates that differences were statistically significant(Pb.05) by an unpaired Student's t test.

Table 1
Biodistribution of 125I-NZ-1(Iodogen) and [131I]SGMIB-NZ-1 in athymic mice bearing D2159MG xenograftsa
Tissue

6h

24 h

48 h

72 h

144 h

192 h

I-NZ-1(Iodogen)Liver
SpleenLungs
Heart
Kidneys
StomachSmall intestine
Large intestine
Thyroid
Muscle
Blood
Bone
Brain

6.85±0.76
4.20±0.68
8.90±0.93
6.01±0.95
6.71±0.88
4.11±1.23
2.35±0.34
1.27±0.18
0.63±0.13
0.89±0.26
20.62±1.55
1.57±0.51
0.38±0.13

3.91±1.06
2.47±0.58
7.77±2.14
4.89±0.99
4.39±1.71
1.63±0.56
1.54±0.35
0.96±0.19
0.46 ±0.47
1.38±0.20
14.95±3.48
1.49±0.37
0.26±0.09

2.46±0.77
1.76±0.27
4.46±0.88
3.36±0.38
2.25±0.64
0.95±0.30
0.85±0.10
0.53±0.04
1.01±0.29
0.93±0.05
10.14±1.79
1.17±0.42
0.25±0.07

1.34±0.47
1.14±0.61
2.98±1.45
1.88±0.96
1.69±0.79
0.56±0.25
0.51±0.26
0.33±0.14
1.20±0.19
0.52±0.19
6.32±2.41
0.72±0.27
0.23±0.13

1.08±0.27
0.97±0.12
2.07±0.82
1.71±0.23
1.35±0.34
0.64±0.08
0.49±0.05
0.29±0.05
1.86±0.45
0.47±0.07
5.55±0.80
0.58±0.03
0.16±0.02

0.93±0.28
0.61±0.18
1.63±0.56
1.18±0.20
0.71±0.18
0.26±0.08
0.28±0.09
0.18±0.05
1.36±0.14
0.29±0.10
3.76±0.89
0.35±0.09
0.12±0.03[131I]SGMIB-NZ-1
Liver
SpleenLungs
Heart
Kidneys
StomachSmall intestine
Large intestine
Thyroid
Muscle
Blood
Bone
Brain

8.38±1.32
4.78±1.04
7.48±0.59
5.62±0.86
6.54±1.01
1.25±0.16
2.63±0.36
1.87±0.24
0.45±0.15
0.72±0.21
17.54±1.49
1.50±0.46
0.31±0.11

3.98±1.21
2.55±0.59
6.46±1.81
4.10±0.94
3.95±1.56
0.72±0.30
1.49±0.41
1.07±0.20
0.14±0.17
1.15±0.20
11.25±3.26
1.33±0.25
0.21±0.06

2.39±1.06
1.83±0.24
3.13±0.79
2.52±0.36
1.78±0.58
0.25±0.09
0.74±0.14
0.57±0.08
0.15±0.05
0.72±0.08
6.84±1.67
1.04±0.40
0.17±0.06

1.02±0.42
1.01±0.54
1.90±1.13
1.20±0.79
1.12±0.71
0.12±0.08
0.38±0.24
0.32±0.13
0.11±0.05
0.34±0.17
3.36±1.83
0.53±0.24
0.14±0.10

0.83±0.20
0.77±0.16
1.17±0.51
1.02±0.19
0.84±0.26
0.24±0.07
0.36±0.02
0.28±0.04
0.11±0.04
0.29±0.04
3.04±0.48
0.38±0.03
0.10±0.01

0.60±0.18
0.48±0.09
0.88±0.37
0.63±0.12
0.38±0.12
0.08±0.03
0.18±0.05
0.19±0.09
0.08±0.05
0.17±0.08
1.86±0.52
0.24±0.06
0.07±0.02

125aValues are mean %ID/g±S.D. (n=5 for 6, 24 and 48 h; n=4 for 72, 144 and 192 h) except for thyroid for which %ID/organ is used.

Y. Kato et al. / Nuclear Medicine and Biology 37 (2010) 785–794

791

Table 2
Biodistribution of [125I]SGMIB-NZ-1 and [125I]SGMIB-NZ-1+-NZ-1 in athymic mice bearing D2159MG xenograftsa
TissuePecent injected dose per gram
6h

a

48 h

125

[ I]SGMIB-NZ-1 +cold NZ-1

125

[

125

[ I]SGMIB-NZ-1 +cold NZ-1[125I]SGMIB-NZ-1[125I]SGMIB-NZ-1 +cold NZ-1

5.92±2.40
4.75±2.34
11.35±5.16
4.05±1.22
4.40±0.70
0.94±0.40
1.52±0.13
1.31±0.15
0.40±0.19
1.03±0.27
15.20±2.39
1.41±0.21
0.48±0.07

5.75±1.27
3.97±0.66
11.27±2.98
5.23±0.81
5.56±0.66
1.20±0.39
2.31±0.43
1.62±0.43
0.54±0.18
1.05±0.15
19.03±2.54
1.72±0.40
0.60±0.08

2.68±0.74
2.00±0.46
5.23±0.97
2.56±0.54
2.61±0.77
0.41±0.19
0.83±0.21
0.63±0.17
0.20± 0.11
0.96±0.18
8.35±2.20
1.12±0.44
0.31±0.10

2.89±0.52
2.44±0.48
6.73±0.99
3.66±1.06
3.46±0.71
0.44±0.16
1.03±0.15
0.68±0.14
0.24±0.10
1.28±0.16
12.08±2.29
1.33±0.23
0.42±0.10

1.68±0.57
1.38±0.58
3.67±1.44
1.78±0.78
1.78±0.70
0.28±0.13
0.64±0.26
0.45±0.16
0.17±0.05
0.67±0.21
5.68±2.18
0.82±0.21
0.23±0.04

2.42±0.40
1.77±0.23
5.21±0.91
2.47±0.27
2.76±0.28
0.54±0.12
0.97±0.09
0.69±0.18
0.24±0.06
0.93±0.10
9.13±0.71
1.08±0.20
0.33±0.12[
Liver
SpleenLungs
Heart
Kidneys
StomachSmall intestine
Large intestine
Thyroid
Muscle
Blood
Bone
Brain

24 h

125

I]SGMIB-NZ-1

I]SGMIB-NZ-1

Values are mean %ID/g±S.D. (n=5) except for thyroid for which %ID/organ is used.

when mice were co-injected with 100-fold excess of
unlabeled NZ-1 (Pb.01). Data for uptake in normal tissues
are given in Table 2.

4. Discussion
The purpose of this study was to determine the affinity ofanti-podoplanin antibody NZ-1 and to investigate whether
NZ-1 is suitable for therapy against malignant gliomas byperforming internalization assays in vitro using glioblastoma
cells as well as biodistribution experiments in vivo using
glioblastoma xenograft models. To determine the affinity of
NZ-1, we performed a kinetic analysis of the interaction of
NZ-1 with podoplanin peptide by surface plasmon resonanceand we also performed Scatchard analysis using podoplaninpositive glioblastoma cells. KD was determined to be about
0.1 nM in BIAcore and about 1 nM in Scatchard analysis,
indicating that NZ-1 has high affinity against both
podoplanin peptide and glioblastoma cells. This high affinity
of NZ-1 should be sufficient for antibody-based immunotherapy, because the affinity of NZ-1 was much higher
than that of anti-epidermal growth factor receptor-vIII(EGFRvIII) scFv MR1-1 [38] and was similar to that ofmouse anti-EGFRvIII mAb L8A4 [39]. The binding sitebarrier is an important theoretical concept in antibody
targeting because it suggests that if the on-rate for binding to
the tumor molecular target is too high, it will impede deliveryof the antibody to regions of the tumor that are distant fromvascular supply [40]. Although the affinity of NZ-1 binding
to podoplanin peptide was 0.12 nM, the KD determined bycell-based Scatchard analysis was an order of magnitude
higher, 1.0–2.6 nM. It is difficult to determine whether adissociation constant in this range will result in inhomogeneous delivery to tumor due to binding site barrier-related
effects. However, the impact of this potential issue should be

minimized in the clinical settings of minimum residual
disease where we anticipate applying radiolabeled NZ-1–
post-surgical glioblastoma and neoplastic meningitis.
Initially, NZ-1 was radioiodinated by the direct electrophilic method using Iodogen as the oxidant. It is not known
whether the NZ-1–podoplanin complex will undergointernalization once NZ-1 binds to podoplanin. To investigate this and to have a stably radioiodinated NZ-1, it was alsoradioiodinated using SGMIB, one of the residualizing agentsthat we have developed for radiolabeling mAbs that undergo
extensive internalization [34]. Indeed, it was possible toradioiodinate NZ-1 in reasonable yields with the preservation of immunoreactivity.
Multiple models were utilized to confirm that the behavior
of NZ-1 was not unique to a particular cell line. Internalization experiments were performed using two podoplaninpositive glioblastoma cell lines, D397MG and LN319, as
well as cells obtained by dissociation of one podoplaninpositive glioblastoma xenograft, D2159MG. These threeglioblastoma cells have different characters both in vitro andin vivo. For example, LN319 cells grow two to three times
faster than both D397MG and D2159MG cells in vitro.
LN319 has no tumorigenicity, whereas D397MG and
D2159MG cells grow very well in vivo. However, thepodoplanin expression of D397MG cells was extremely
decreased in vivo, making it impractical to perform in vivo
targeting experiments with this cell line. As shown in Fig. 2,
125
I-NZ-1(Iodogen) was internalized into D397MG and
LN319 cells. Interestingly, cell surface-bound counts in
LN319 gradually decreased, accompanied by a slow increasein counts in the cell culture supernatant (Figs. 2B and 3),
whereas D397MG showed little change over time (Fig. 2A),
which might reflect differences in growth rate, metabolism,shedding or recycling. We further compared the internalization of 125 I-NZ-1(Iodogen) with that of [131I]SGMIB-NZ-1
using LN319, because we have previously demonstrated the792

Y. Kato et al. / Nuclear Medicine and Biology 37 (2010) 785–794

Fig. 6. Tumor-to-tissue ratios observed in athymic mice bearing D2159MG xenografts and injected with 125I-NZ-1(Iodogen) (square) and [131I]SGMIB-NZ-1 (circle).

Y. Kato et al. / Nuclear Medicine and Biology 37 (2010) 785–794

Fig. 7. Biodistribution of [125I]SGMIB-NZ-1 in D2159MG. Uptake of[125I]SGMIB-NZ-1 and [125I]SGMIB-NZ-1+100-fold nonlabeled NZ-1 inathymic mice hosting D2159MG xenografts. Values given are %ID/g (mean±S.D.). An asterisk indicates that differences were statistically significant(Pb.01) by unpaired Student's t test.

advantages of labeling with SGMIB for the efficient deliveryof halogen radionuclides attached to mAbs that undergointernalization in tumors cells [34]. For example, paired-label
in vitro internalization assays using EGFRvIII-expressing
U87MG cells demonstrated that the amount of radioactivity
retained in cells after internalization for EGFRvIII-specific
L8A4 labeled with [131 I]SGMIB was three to four times
higher than that for L8A4 labeled with 125 I using Iodogen.
Likewise, the internalization of radioiodine activity of [131I]
SGMIB-NZ-1 (26.3±0.8% at 8 h) was significantly higher
than that of 125 I-NZ-1(Iodogen) (10.0±0.2% at 8 h) in LN319
cells with more than 2.5 times higher intracellular activity
observed with SGMIB labeling at later time points (Fig. 3A
and B). The internalization of [125I]SGMIB-NZ-1 into
D2159MG xenograft cells was also considerably higher,
with 30.7±2.0% of initially bound radioactivity in theintracellular compartments at the 16-h time point (Fig. 4).
These results are similar to results seen with the antiEGFRvIII mAb L8A4 radioiodinated with this prosthetic
group and other cell lines [41], suggesting that NZ-1 does
indeed become internalized once it binds to podoplanin.
Next, we performed biodistribution of NZ-1 in athymic
mice bearing the D2159MG glioblastoma xenograft. We
could not obtain LN319 xenograft in athymic mice becauseof its low tumorigenicity, and the podoplanin expression was
extremely decreased in D397MG xenograft. Both 125I-NZ-1(Iodogen) and [131I]SGMIB-NZ-1 were well accumulated
into tumors, and the tumor accumulation of radioiodine
activity of [131 I]SGMIB-NZ-1 was significantly higher(Pb.05) than that of 125I-NZ-1(Iodogen) at time points of
24 and 48 h (Fig. 5), which is consistent with the dataobtained from internalization assay in vitro. The absolutetumor uptake of [131I]SGMIB-NZ-1 is similar to that seen
earlier for mAb L8A4 labeled using SGMIB in a different
xenograft model [42]. Furthermore, reduction in tumor
uptake when a large excess of unlabeled NZ-1 was present793

indicates that the uptake is indeed due to the specific bindingof NZ-1 to podoplanin (Fig. 7). As seen in the past for mAbs
radioiodinated with acylation agents in general, and SGMIB
in particular, the thyroid uptake of radioactivity from [131 I]
SGMIB-NZ-1 was substantially lower than that from 125 INZ-1(Iodogen), which again demonstrates inertness of thisprosthetic group for in vivo dehalogenation. Lower uptake inblood and other normal tissues, along with the resultant
higher tumor-to-normal tissue ratios for [131I]SGMIB-NZ-1,
makes this mAb a potential tool in the targeted immunotherapy of gliomas (Fig. 6). Collectively, these results suggestthat NZ-1 should be suitable for targeted radioimmunotherapy of glioblastomas.
In conclusion, the rat anti-podoplanin mAb NZ-1 showed
high affinity against not only podoplanin peptide but also
glioblastoma cells. Furthermore, 125I-NZ-1(Iodogen) wasinternalized into glioblastoma cells in vitro. Paired-label
internalization assays indicated that [131 I]SGMIB-NZ-1
showed higher retention of radioactivity compared to 125 INZ-1(Iodogen). Likewise, tumor uptake of radioactivity inathymic mice bearing D2159MG xenografts in vivo wassignificantly higher for [131I]SGMIB-NZ-1 than for 125 I-NZ1(Iodogen). Taken together, these results demonstrate thepotential utility of an anti-podoplanin antibody NZ-1 inantibody-based therapy against glioblastoma.
Acknowledgments
We thank Donna Affleck, Philip Welsh, Xiao-Guang
Zhao and Scott E. Szafranski of Duke University Medical
Center for their excellent technical assistance. We also thankChifumi Kitanaka of Yamagata University School of
Medicine for his helpful suggestions.
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