1. Cancer Therapy: Preclinical
Molecular Targeting and Treatment of an Epidermal Growth Factor
Receptor ^ Positive Glioma Using Boronated Cetuximab
Gong Wu,1Weilian Yang,1Rolf F. Barth,1Shinji Kawabata,1Michele Swindall,1Achintya K. Bandyopadhyaya,4
Werner Tjarks,4 Behrooz Khorsandi,5 Thomas E. Blue,5 Amy K. Ferketich,6 Ming Yang,3
Gregory A. Christoforidis,3 Thomas J. Sferra,2,7 Peter J. Binns,8 Kent J. Riley,8
Michael J. Ciesielski,9 and Robert A. Fenstermaker9
Abstract Purpose: The purpose of the present study was to evaluate the anti ^ epidermal growth factor
monoclonal antibody (mAb) cetuximab (IMC-C225) as a delivery agent for boron neutron
capture therapy (BNCT) of a human epidermal growth factor receptor (EGFR) gene-transfected
rat glioma, designated as F98EGFR.
Experimental Design: A heavily boronated polyamidoamine dendrimer was chemically linked
to cetuximab by means of the heterobifunctional reagents N-succinimidyl 3-(2-pyridyldithio)-
propionate and N-(k-maleimido undecanoic acid)-hydrazide. The bioconjugate, designated as
BD-C225, was specifically taken up by F98EGFR glioma cells in vitro compared with receptor-
negative F98 wild-type cells (41.8 versus 9.1 Ag/g). For in vivo biodistribution studies, F98EGFR
cells were implanted stereotactically into the brains of Fischer rats, and 14 days later, BD-C225
was given intracerebrally by either convection enhanced delivery (CED) or direct intratumoral
(i.t.) injection.
Results: The amount of boron retained by F98EGFR gliomas 24 h following CED or i.t. injection
was 77.2 and 50.8 Ag/g, respectively, with normal brain and blood boron values <0.05 Ag/g.
Boron neutron capture therapy was carried out at the Massachusetts Institute of Technology
Research Reactor 24 h after CED of BD-C225, either alone or in combination with i.v. boronophe-
nylalanine (BPA). The corresponding mean survival times (MST) were 54.5 and 70.9 days
(P = 0.017), respectively, with one long-term survivor (more than 180 days). In contrast, the MSTs
of irradiated and untreated controls, respectively, were 30.3 and 26.3 days. In a second study, the
combination of BD-C225 and BPA plus sodium borocaptate, given by either i.v. or intracarotid
injection, was evaluated and the MSTs were equivalent to that obtained with BD-C225 plus i.v.
BPA.
Conclusions: The survival data obtained with BD-C225 are comparable with those recently
reported by us using boronated mAb L8A4 as the delivery agent.This mAb recognizes the mutant
receptor, EGFRvIII. Taken together, these data convincingly show the therapeutic efficacy of
molecular targeting of EGFR using a boronated mAb either alone or in combination with BPA
and provide a platform for the future development of combinations of high and low molecular
weight delivery agents for BNCTof brain tumors.
Boron neutron capture therapy (BNCT) is based on the nuclear instantaneous nuclear fission to produce a-particles and
capture and fission reactions that occur when nonradioactive recoiling lithium-7 nuclei. These high linear energy transfer
boron-10 is irradiated with low energy (e V 0.025 eV) thermal particles have a range of 5 to 9 Am, thereby restricting their
neutrons to produce 11B in an unstable form, which undergoes destructive effects to only those cells containing 10B. To be
Authors’ Affiliations: Departments of 1Pathology, 2Pediatrics, and 3Radiology, The costs of publication of this article were defrayed in part by the payment of page
4
College of Pharmacy, 5Nuclear Engineering Program, and 6School of Public charges. This article must therefore be hereby marked advertisement in accordance
Health, The Ohio State University; 7Children’s Research Institute, Columbus, Ohio; with 18 U.S.C. Section 1734 solely to indicate this fact.
8
Nuclear Reactor Laboratory, Massachusetts Institute of Technology, Cambridge, Note: Current addressfor T.J. Sferra:Departmentof Pediatrics, Universityof Oklahoma
Massachusetts; and 9Department of Neurosurgery, Roswell Park Cancer Institute, Health Sciences Center, Oklahoma City, OK; current address for S. Kawabata: Department
Buffalo, NewYork of Neurosurgery, Osaka Medical College, Takatsuki City, Osaka, Japan.
Received 9/29/06; accepted 11/10/06. Presented in part at the 12th International Symposium on Neutron CaptureTherapy,
Grant support: NIH grants 1R01CA098945 (R.F. Barth) and 1R01NS39071 (T.J. Takamatsu, Japan, October 9-12, 2006.
Sferra); the Roswell Park Alliance Foundation (R.A. Fenstermaker); and U.S. Requests for reprints: Rolf F. Barth, Department of Pathology, The Ohio State
Department of Energy through the program of Innovations in Nuclear Infrastructure University, 165 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. Phone:
and Education, Office of Nuclear Energy, Science andTechnology (contract nos. DE- 614-292-2177; Fax: 614-292-7072; E-mail: rolf.barth@ osumc.edu.
FG07-02ID14420 and DE-FG07-02 [K14420]) and the Office of Environmental and F 2007 American Association for Cancer Research.
Biological Research (contract no. DE-FG02-02ER63358). doi:10.1158/1078-0432.CCR-06-2399
Clin Cancer Res 2007;13(4) February 15, 2007 1260 www.aacrjournals.org

2. Molecular Targeting of EGFR-Positive Gliomas
effective, BNCT requires a sufficient amount (20-30 Ag/g) of propionate and N-(k-maleimido undecanoic acid)-hydrazide (14).
10 Protein content of the bioconjugate was determined spectrophotomet-
B, homogeneously distributed in the tumor, with concomi-
tantly low 10B concentrations in surrounding normal tissues. rically by means of the Coomassie blue assay reagent (Pierce, Rockford,
IL) and boron was quantified by means of direct current plasma-atomic
These requirements, as well as clinical applications, have been
emission spectroscopy (DCP-AES; 32).
discussed in detail in several recent reviews and monographs
In vitro cellular uptake of BD-C225. For in vitro boron uptake
(1 – 5). BNCT primarily has been used to treat high-grade studies, F98 glioma cells, expressing 106 human EGFRs per cell
gliomas and either cutaneous primaries or cerebral metastases (F98fEGFR), were used. These were produced by Dr. Frank Furnari
of melanoma (1). More recently, it has also been used to treat (Ludwig Institute for Cancer Research, La Jolla, CA), who generously
patients with recurrent tumors of the head and neck and provided them to us. Five million F98 wild-type (F98WT), F98fEGFR, or
colorectal cancer metastatic to the liver (1). F98npEGFRvIII (17) glioma cells were seeded into T-150 flasks with
Both the epidermal growth factor (EGF) receptor (EGFR) and DMEM containing 10% fetal bovine serum (Life Technologies, Inc.,
its mutant isoform EGFRvIII frequently are overexpressed in Rockville, MD) supplemented with 100 units/mL penicillin and 100
human glioblastomas (6 – 9), which make them attractive Ag/mL streptomycin. After incubation for 24 h at 37jC, the medium
was replaced with DMEM containing 1.68 mg BD-C225 (90 Ag boron),
targets for the treatment of brain tumors (10). We have
and the cells were incubated for an additional 2 h at 37jC. Following
investigated molecular targeting of EGFR or EGFRvIII using
this, the medium was decanted and the cells were washed thrice with
EGF (11 – 13), or the monoclonal antibodies (mAb) cetuximab PBS (pH 7.4), disaggregated by exposure to 0.5 mmol/L EDTA for
(14, 15) and L8A4 (16, 17), which have been linked to a 5 min, counted, and sedimented. Cells were digested with concentrated
heavily boronated polyamidoamine dendrimer. Cetuximab sulfuric acid and 50% hydrogen peroxide, and boron uptake was
(Erbitux), known previously as IMC-C225, is a chimeric determined by DCP-AES (32).
mouse-human mAb that originally was produced in the In vitro neutron irradiation studies. For in vitro neutron irradiation
laboratory of Dr. John Mendelsohn (University of Texas studies, F98fEGFR glioma cells, expressing 106 receptor sites per cell,
M. D. Anderson Cancer Center, Houston, TX; ref. 18). It has were cultured until confluent. Cells were harvested by exposure to 0.5
greater affinity for EGFR than either EGF or transforming mmol/L EDTA and washed thrice with PBS, and aliquots containing 106
cells were dispensed into 2-mL plastic vials. Except for unirradiated
growth factor-a, and following binding, the receptor-antibody
control cells, either cetuximab or BD-C225 was added to all other vials
complex is rapidly internalized, thereby eliminating further
and incubated at 4jC for 90 min following which they were washed
activation of the receptor (19, 20). Down-regulation of cell thrice with medium. Triplicate samples were irradiated with thermal
surface receptor binding sites and competition of cetuximab for neutrons at The Ohio State University Research Reactor for 1, 2, 5, or
the remaining binding sites can reduce or prevent further 10 min at a thermal neutron flux of 109 cmÀ2 secÀ1. After irradiation,
activation by ligand. Several mechanisms have been proposed aliquots of 10,000 cells were taken from each vial and seeded into 96-
to explain the antitumor activity of cetuximab (21 – 23). These well microplates (Corning, Corning NY). Cell survival was determined
include cell cycle arrest (24), apoptosis (25), decrease in 72 h later by means of the sulforhodamine B assay (33). A clonogenic
angiogenesis and cellular adhesion (26, 27), and inhibition assay also was carried out following irradiation to assess cell survival
of matrix metalloproteinase expression and activity (28). (34). Briefly, varying numbers of F98fEGFR cells were seeded into 100-
mm Petri dishes and incubated for 7 days at 37jC in an atmosphere
Enhancement of the cytotoxic effects of chemotherapeutic
containing 95% air and 5% CO2. Following this, the medium was
agents (29) and the response to ionizing radiation have also
decanted and the cells were fixed by adding 2 to 3 mL of 37%
been reported (30). Cetuximab is reactive with both wild-type formaldehyde, and then the plates were stained with 3 to 5 mL of
EGFR and EGFRvIII (14), and recently, it has been approved by saturated crystal violet. The number of colonies containing at least 50
the U.S. Food and Drug Administration for use in patients with cells was enumerated visually by counting under a dissecting
EGFR-positive colorectal cancer metastatic to the liver and microscope. The surviving fraction was calculated from the number of
recurrent head and neck cancers (31). Because of its pleiotropic colonies enumerated / number of cells plated  plating efficiency / 100.
effects, cetuximab is particularly attractive as a boron delivery Tumor implantation and biodistribution of BD-C225. F98EGFR
agent for NCT of gliomas. In the present report, we describe glioma cells expressing 105 receptor sites per cell, described previously
studies to evaluate boronated cetuximab as a delivery agent for by us in detail (12), were used for in vivo studies. This cell line, which
BNCT of the F98 rat glioma, which has been transfected with the has been stable for over 5 years, was produced by transfecting F98 wild-
type (F98WT) cells with the human gene encoding wild-type EGFR (11).
gene encoding human EGFR (F98EGFR). Our data convincingly
These cells were used to obviate an immune response directed against
show its efficacy for BNCT of this tumor, used either alone or in human EGFR, which we found occurred when cells expressing 106
combination with boronophenylalanine (BPA), a drug that has receptor sites were implanted i.c. into immunocompetent Fischer rats.10
been used clinically for BNCT of brain tumors (1). After i.c. implantation into syngeneic Fischer rats, the F98EGFR glioma
forms a progressively growing, infiltrative tumor that invariably results
Materials and Methods in the death of the host with an inoculum as few as 1,000 cells (35).
Cells were maintained and propagated in vitro in supplemented DMEM
Preparation of the bioconjugate BD-C225. Cetuximab was gener- containing 600 Ag/mL geneticin (G418; Sigma-Aldrich). F98WT cells
ously provided to us by Dr. Daniel Hicklin (ImClone Systems, Inc., were cultured in the same medium, but without G418. Animal studies
New York, NY). Site-specific attachment of a heavily boronated were done in accordance with the Guide for the Care and Use of
polyamidoamine dendrimer was carried out, as described by us in Laboratory Animals (National Academy Press, Washington, DC, 1996)
detail elsewhere (14). Briefly, a fifth generation polyamidoamine and the protocol was approved by the Institutional Laboratory Animal
dendrimer (Sigma-Aldrich, St. Louis, MO), containing 128 terminal Care and Use Committee of The Ohio State University (Columbus,
amino groups, was reacted with an isocyanato polyhedral borane OH). CD-Fischer rats (Charles River Laboratories, Wilmington, MA),
anion, Na(CH3)3NB10H8NCO. This yielded a heavily boronated weighing 200 to 220 g, were anesthetized with a 1.2:1 mixture of
macromolecule, which contained f1,100 boron atoms per molecule
of dendrimer. Cetuximab was linked to the boronated dendrimer (BD)
10
by two heterobifunctional linkers, N-succinimidyl 3-(2-pyridyldithio)- R.F. Barth and W. Yang, unpublished data.
www.aacrjournals.org 1261 Clin Cancer Res 2007;13(4) February 15, 2007

3. Cancer Therapy: Preclinical
ketamine/xylazine at a dose of 120 mg of ketamine/20 mg of xylazine/ were determined in tumor, normal brain, liver, and blood in a separate
kg body weight. Following this, tumor cells were implanted stereo- group of animals 24 h after CED of BD-C225 and 2.5 h after i.v.
tactically, as originally described by us (36). A small plastic screw injection of BPA to estimate absorbed doses in these tissues. Animal
(Arrow Machine Manufacturing, Inc., Richmond, VA) with an entry irradiations were done with the reactor operating at a power between 4.0
port, which allowed insertion of a 27-gauge needle, was embedded into and 4.8 MW. These took between 6.9 and 8.6 min to deliver a thermal
the calvarium before tumor cell implantation. BD-C225 was given i.c. neutron fluence of 2.64 Â 1012 n cmÀ2 that matches previous dose
by means of convection enhanced delivery (CED), using a syringe prescriptions (13, 15). After completion of BNCT, the animals were
pump at a rate of 0.33 AL/min for 30 min to deliver a volume of 10 AL returned to The Ohio State University for clinical monitoring.
(Harvard Apparatus Co., Cambridge, MA) as described previously (12). Monitoring of clinical status and neuropathologic evaluation. All
This technique, completely bypasses the blood-brain barrier, maximizes animals were weighed thrice weekly and their clinical status was
delivery to the tumor and minimizes uptake by extracranial organs and evaluated at the same time. Once the animals had progressively growing
blood (37, 38). For CED, a plastic cannula was inserted into the entry tumors, as evidenced by the combination of sustained weight loss,
port and then advanced 5 mm below the dura into the tumor of ataxia, and periorbital hemorrhage, they were euthanized to minimize
F98EGFR glioma-bearing rats. Biodistribution studies were carried out in discomfort. Survival times were determined by adding 1 day to the time
tumor-bearing rats 12 to 14 days following tumor cell implantation. between tumor implantation and euthanization. The brains of all
Animals were divided into four experimental groups of four to five rats animals in the therapy studies were removed after death, fixed in 10%
each. Animals in groups 1 and 2 had F98EGFR gliomas and received buffered formalin, and then cut coronally at the level of the optical
750 Ag BD-C225 (40 Ag B) by CED at a rate of 0.33 AL/min or intra- chiasm and 2 mm anterior and posterior to it. Coronal slices were
tumoral (i.t.) injection. Rats in groups 3 and 4 received an i.v. injection embedded in paraffin, cut at 4 Am, stained with H&E, and then
of BPA (500 mg/kg body weight, equivalent to 27 mg 10B/kg body examined microscopically to assess the histopathologic changes. The
weight) or BD-C225 by CED with i.v. BPA (Katchem Ltd., Prague, Czech tumor size index was determined from H&E-stained coronal sections of
Republic). The biodistribution of BD-C225 was determined at 24 h after brain using a semiquantitative grading scale ranging from 0 to 4. Each
CED by measuring concentrations of boron in various tissue samples by section was scored as follows: 0, no tumor; 1, very small (i.e.,
DCP-AES (32). Animals were euthanized by an overdose of halothane microscopic, <1 mm); 2, small (approximately 1-3 mm); 3, large
following which tumors and normal tissues consisting of brain, blood, (approximately 4-7 mm); and 4, massive (>8 mm); the mean score was
liver, kidney, and muscle were removed and weighed. calculated for each group.
Therapy experiments and dosimetry. Neutron irradiations for these Magnetic resonance imaging. Magnetic resonance (MR) images of
experiments were identical to those reported previously by us using the brain tumor – bearing rats were generated on a Bruker Avance scanner
boronated mAb L8A4 (17). BNCT was done 14 days following stereo- (Bruker, Billerica, MA) interfaced with Techron gradient amplifiers
tactic implantation of 103 F98EGFR glioma cells. Rats were transported (Crown International, Elkhart, IN) and Magnex gradients (Magnex
approximately 5 to 7 days before irradiation to Massachusetts Institute Scientific, Abingdon, England) using a custom-built radio frequency
of Technology (Cambridge, MA) where they were housed in an front end. A custom-made, 4 cm in diameter birdcage coil was tuned to
accredited animal care facility supervised by the Division of Compar- the head of the rat at 340 MHz while the rat was in the prone position.
ative Medicine. Before irradiation at the Nuclear Reactor Laboratory, Ultrasmall particles of iron oxide (SHU555C, Supravist; Schering AG,
they were randomized based on weight into experimental groups of 7 to Berlin, Germany) were used as a contrast agent. These were given i.v.
11 animals each as follows: group 1, untreated controls; group 2, (2.0 mg Fe/kg) via a right femoral vein catheter after anesthetizing the
irradiated controls; group 3, i.v. BPA, followed by BNCT; group 4, i.t. of animals with isoflurane. Each animal was scanned before and after
BD-C225 followed by BNCT; group 5, CED of BD-C225 followed injection of ultrasmall particles of iron oxide using a high resolution
by BNCT; and group 6, CED of BD-C225 plus i.v. BPA, followed by T2*-weighted gradient recalled echo sequence with an in-plane
BNCT. BNCT was initiated 24 h after CED of 10 AL of 750 Ag BD-C225 resolution of 78 Am. The images were generated with the following
(40 Ag 10B) and 2.5 h after i.v. administration of BPA (500 mg/kg body pulse-sequence variables: time of repetition, 500 msec; time of echo,
weight). In a second study, using a different lot number of F98EGFR cells, 14.6 msec; flip angle, 22.5j; field of view, 4 cm; matrix, 512 Â 512; slice
rats received BD-C225 in combination with either i.v. or intracarotid thickness/gap, 1/0.1 mm; and acquisition time, 10 min and 14 s.
administration of sodium borocaptate (BSH), another drug that has
been used in both experimental (39, 40) and clinical studies (1) of
BNCT. All irradiated rats were anesthetized with a mixture of ketamine
and xylazine. Irradiations were carried out at the MITR-II nuclear reactor
in the M011 irradiation facility (41). This produces a thermal neutron
beam of high purity and intensity with no measurable fast neutron
component (42). Rats were positioned two at a time in a lithiated (95%
6
Li enriched) polyethylene box that provided whole-body shielding
from the thermal neutrons during irradiation. The head of each animal
was aligned in the middle of a 13 Â 2 cm2 aperture, machined in the box
lid, which served as the beam delimiter. The output generated by four
fission counters, located at the periphery of the 15 cm circular field,
automatically controlled beam delivery and provided real-time data on
the relative neutron fluence during an irradiation and was used to
automatically control beam delivery that was reproducible to within 1%.
The beam monitors were calibrated against dosimetric measurements,
which were carried out on both euthanized rats and phantoms made
from type 6 nylon, using bare gold foils and a graphite-walled ionization
chamber (V = 0.1 cm3) flushed with reagent grade CO2 (43). The
measured dose rates in brain (2.2% nitrogen by weight), normalized to
the reactor operating at a power of 5 MW, were 18.5 cGy/min for
Fig. 1. Cellular uptake of BD-C225 by F98fEGFR, F98npEGFRvIII, and F98WT glioma
photons, 7.7 cGy/min for thermal neutrons from the nitrogen capture
cells. BD-C225 (90 Ag boron) were incubated with glioma cells at 37jC for 2 h and
reaction, and 3.4 cGy/min per Ag 10B in tissues. The estimated then washed with medium for three times. Cells were digested, and boron content
uncertainties on all these dose rates were 5%. Boron concentrations was determined by DCP-AES.
Clin Cancer Res 2007;13(4) February 15, 2007 1262 www.aacrjournals.org

4. Molecular Targeting of EGFR-Positive Gliomas
between EGFR-positive and EGFR-negative cells. It is notewor-
thy that the bioconjugate targeted both wild-type EGFR and its
most common mutant, EGFRvIII. Based on these results,
in vitro neutron irradiation studies were initiated at The Ohio
State University Research Reactor. As determined by the
sulforhodamine B assay, cells preexposed to BD-C225, fol-
lowed by 10 min neutron irradiation (3.8 Gy), had 20.7 F
1.0% survival compared with 85% for irradiated controls
(Fig. 2). Similar results were also obtained using a clonogenic
assay with a surviving fraction of 42 F 2.6% for irradiated
controls compared with 5.4 F 0.4% for cells that had been
exposed to BD-C225 before irradiation.
In vivo biodistribution studies and dosimetry. Biodistribution
data of BD-C225 following i.c. administration to F98EGFR
glioma-bearing rats are summarized in Table 1. At 24 h
following CED, the mean tumor boron concentration was
77.2 F 14.8 Ag B/g compared with 50.8 F 5.7 Ag B/g following
i.t. injection, which was a 52% increase. Boron concentrations
in the blood and the nontumor-bearing cerebral hemisphere
were <0.5 Ag B/g, which was the background limit of detection.
Fig. 2. In vitro neutron irradiation studies. F98EGFR glioma cells were exposed
to cetuximab (.), BD-C225 (E), or medium alone (5) for 90 min at 4jC following The tumor boron concentration in rats that received i.v. BPA was
which they were washed with medium and then irradiated with thermal neutron for 10.7 F 1.7 Ag/g compared with 87.9 F 16.5 Ag B/g in animals
0 to10 min.The cells there were plated into 96-well microplates and cultured for
72 h following which the cell survival was determined by the sulforhodamine B assay.
that received the combination of i.v. BPA and CED of BD-C225
in combination. Liver, kidneys, spleen, and skin all had
undetectable levels of boron following CED or i.t. injection.
Statistical evaluation of survival data. The mean survival time The reported absorbed dose was based on mean boron
(MST), SE, and median survival time were calculated for each group
concentrations measured in tumor, brain, and blood at 24 h
using the Kaplan-Meier method that enabled Kaplan-Meier Survival and
following CED of BD-C225 and 2.5 h after i.v. administration
Cox proportional hazard survival curves to be plotted (44). The
hypotheses involved comparing each BD-C225 – treated animal to each of BPA using a separate group of untreated animals. Based on
irradiated control. A log-rank test was used for these comparisons, with these total boron concentrations, the mean absorbed doses
a Bonferroni method of adjustment for the multiple comparisons (45). delivered to F98EGFR tumors were 19.5 Gy following CED of
Because five comparisons were tested for statistical significance within BD-C225, 4.2 Gy following i.v. administration of BPA alone, and
the BD-C225 tests, an a = 0.0125 was used. 21.9 Gy when in combination with CED of BD-C225 (Table 1).
The normal brain doses ranged from 1.9 to 2.7 Gy. Absorbed
Results doses were expressed without biological weighting factors due
to uncertainty relating in their determination that relate to
In vitro uptake and irradiation studies. To show that the the chemical form of the 10B (i.e., BD-C225 versus BPA).
bioconjugate was selectively taken up by EGFR-positive glioma Therapeutic response of glioma-bearing rats following
cells, F98WT, F98fEGFR, and F98npEGFRvIII cells were incubated BNCT. All animals in a pilot study to determine tolerance to
with 1.68 mg BD-C225 (90 Ag B) for 2 h at 37jC. As BNCT following CED of BD-C225 lost weight within 7 to 10
determined by DCP-AES, 41.8 Ag B were taken up by 109 days after treatment. Rats that received CED of 40 Ag 10B/750 Ag
F98fEGFR cells, 19.6 Ag by F98npEGFRvIII cells, and 9.1 Ag by BD-C225 and 500 mg/kg body weight of BPA i.v. lost <10% of
F98WT cells (Fig. 1), which was a 4.6- to 2.2-fold difference their body weight but regained it within 2 weeks. Based on
Table 1. Boron concentrations and calculated absorbed radiation doses following administration of BD-C225
to F98EGFR glioma-bearing rats
Group Boron concentration (Mg/g)* Physical dose (Gy)c
b
Tumor Brain Blood Tumor Brain Blood
CED BD-C225 77.2 F 14.8 <0.5 <0.5 19.5 <1.9 <1.9
i.t. BD-C225 50.8 F 5.7 <0.5 <0.5 13.4 <1.9 <1.9
i.v. BPA 10.7 F 1.7 3.8 F 1.1 5.2 F 1.3 4.2 2.6 2.9
CED BD-C225 + i.v. BPA 87.9 F 16.5 4.3 F 1.5 5.7 F 1.3 21.9 2.7 3.1
*Boron content was quantified by means of DCP-AES. These values were obtained from rats that had received BD-C225 (40 Ag 10B/750 Ag
BD-225) by either CED or i.t. 24 h earlier either alone or in combination with i.v. BPA (500 mg/kg body weight), which was given 2.5 h before
euthanization.
cAbsorbed doses include contributions from g photons, 14N (n,p) 14C, and 10B(n,a) 7Li reactions.
bBoron concentration in the tumor-bearing cerebral hemisphere after excision of the tumor.
www.aacrjournals.org 1263 Clin Cancer Res 2007;13(4) February 15, 2007

5. Cancer Therapy: Preclinical
Table 2. Survival times of F98EGFR glioma-bearing rats following CED of BD-C225 with or without i.v. BPA and
BNCT
Group n* Survival times (d) % Increased life spanc
Range Mean F SE Median Mean Median
CED BD-C225 11 39-88 54.5 F 4.3 50 107 92
CED BD-C225 + i.v. BPA 11 42 to >180 (1)b 70.9 F 11.1 58 170 123
i.t. BD-C225 9 33-58 42.7 F 2.6 41 62 58
i.v. BPA 8 32-52 40.1 F 2.2 40 52 54
Irradiated controls 7 24-37 30.3 F 1.6 30 15 15
Untreated controls 7 21-34 26.3 F 1.6 26 — —
*ndesignates the number of animals per group.
cPercentageof increased life span was defined relative to mean and median survival times of untreated controls.
bThe number of rats surviving longer than 180 d.
these results, a dose of 40 Ag 10B/750 Ag BD-C225 was selected. that CED of BD-C225 plus i.v. BPA was significantly different
This was given by CED, either alone or in combination with i.v. from the i.v. BPA alone group (P = 0.0002), and the difference
BPA. BNCT was initiated at the MITR-II reactor 14 days between the groups that received BD-C225 alone or in
following i.c. implantation of 103 F98EGFR glioma cells. All rats combination with BPA was also significant (P = 0.017).
tolerated BNCT without any untoward effects, and 3 to 7 days In the second study, the efficacy of BD-C225 was evaluated in
later, they were returned to Columbus, Ohio. A Cox propor- combination with either i.v. or intracarotid administration of
tional hazards regression model was fit to the data and the BPA plus BSH (Fig. 5; Table 3). The MSTs of animals that
proportional hazards assumption was checked. Because this received BD-C225 plus BPA and BSH by the i.v. or intracarotid
was met, the log-rank test was used to test for significance route were equivalent (67.1 F 21.6 and 75.8 F 28.4 days,
between survival curves, and the survival data of the treatment respectively; P = 0.261), and these MSTs were equivalent (P =
groups were significantly different from the irradiated controls 0.893) to those of animals that received BD-C225 plus i.v. BPA
and untreated controls (P < 0.001). Survival data following (70.9 F 11.1 days; Table 2). However, MST of animals that
BNCT are summarized in Tables 2 and 3 and Kaplan-Meier and received the combination of i.v. BPA plus BSH and BD-C225
Cox survival plots for BNCT-treated animals and irradiated was significantly different from those that received BD-C225
controls are shown in Figs. 3, 4 and 5. Untreated control rats alone (P = 0.034).
had a MST F SE of 26.3 F 1.6 days compared with a modest MR imaging and neuropathologic evaluation. The tumor
increase of 30.3 F 1.6 days for the irradiated controls. Animals sizes indices of animals that received BD-C225 by either CED or
bearing F98EGFR gliomas, which had received CED of BD-C225, i.t. injection, followed by BNCT, were equivalent (5.1 F 1.5
either alone or in combination with i.v. BPA, had a MST of and 5.3 F 1.6 mm) and not different from that of animals that
54.5 F 4.3 days and 70.9 F 11.1 days, respectively, with one rat received CED of BD-C225 and i.v. BPA (4.75 F 1.39 mm). The
surviving more than 180 days compared with 40.1 F 2.2 days MR images of an animal from the untreated group was taken
for animals that received i.v. BPA alone. The corresponding 1 day before euthanization. As shown in Fig. 6A, the tumor
mean percentages increase in life span were 170% for the vascular bed was significantly enhanced after i.v. administra-
combination versus 107% for CED of BD-C225 alone and 52% tion of ultrasmall particles of iron oxide, and the tumor
for i.v. BPA alone. The results from these comparisons indicated margins could be clearly delineated in the right cerebral
Table 3. Survival times of F98EGFR glioma-bearing rats following CED of BD-C225 either alone or in
combination with either i.v. or intracarotid BPA plus BSH
Group n* Survival times (d) % Increased life spanc
Range Mean F SE Median Mean Median
CED BD-C225 9 43-80 56.4 F 13.7 51 65 46
i.v. BPA + BSH 9 42-76 50.9 F 11.0 47 50 34
CED BD-C225 BPA + BSH i.v. 10 48 to >120b 67.1 F 21.6 64 97 83
CED BD-C225 BPA + BSH i.c. 9 47 to >120x 75.8 F 28.4 68 120 94
Irradiated controls 8 34-46 40.3 F 3.6 40 18 14
Untreated controls 10 28-40 34.4 F 3.9 35 — —
*n designates the number of animals per group.
cPercentage of increased life span was defined relative to mean and median survival times of untreated controls.
bThis includes one animal surviving more than 120 d.
x This includes two animals surviving more than 120 d.
Clin Cancer Res 2007;13(4) February 15, 2007 1264 www.aacrjournals.org

6. Molecular Targeting of EGFR-Positive Gliomas
hemisphere. This correlated well with a H&E-stained section of
the tumor (Fig. 6D), which showed a central zone of necrosis
surrounded by viable tumor cells and capillary proliferation at
the periphery of the tumor. The MR images of rats that had
received BD-C225 and i.v. BPA, given by CED, are shown in Fig.
6B and C. These were taken at 35 and 49 days following tumor
implantation that showed no tumor but only the plastic screw,
which had been embedded in the calvarium. These images
clearly showed that ultrahigh field, high-resolution 8.0 Tesla
MR imaging with the aid of the blood pool contrast agent,
ultrasmall particles of iron oxide, could be used to delineate not
only tumor size but also the neovasculature. This could be a
useful tool to sequentially study the effects of BNCT on tumor
neovasculature from initial cell killing to eventual regrowth and
progression.
Discussion
Fig. 4. Cox survival plots for F98EGFR glioma-bearing rats. Survival time in days
The purpose of the present study was to evaluate the after implantation have been plotted for untreated animals (o), irradiation control
potential use of the anti-EGFR mAb cetuximab as a 10B delivery (.), i.v. BPA + BNCT (E), i.t. BD-C225 (5) and CED of BD-C225 alone (n) or
in combination with i.v. BPA (+) followed by BNCT.
agent for BNCT of the F98EGFR glioma. Cetuximab was
covalently and site specifically linked to a heavily boronated
polyamidoamine dendrimer by means of two heterobifunc-
tional linkers, N-succinimidyl 3-(2-pyridyldithio)-propionate (39, 40). Equivalent survival data were obtained using BD-
and N-(k-maleimido undecanoic acid)-hydrazide. The resulting C225 in combination with BPA and BSH, given by either i.v.
bioconjugate showed both in vitro and in vivo specificity for or intracarotid injection. This is of particular significance
targeting the F98EGFR glioma. Based on these findings, in vivo because, clinically, it would be much easier to give BPA by i.v.
BNCT studies were initiated. Animals that received BD-C225 by injection rather than by the i.v. or intracarotid administration
CED in combination with i.v. BPA had a MST of 70.9 F 11.1 of BPA and BSH. The present data, and those recently reported
days compared with 54.5 F 4.3 days for those that received the by us in other studies using boronated L8A4 (17) and EGF
bioconjugate alone and 30.3 F 1.6 days for irradiated controls. (13), establish proof of principle that gliomas expressing
These survival data were superior to those that we have either EGFR or EGFRvIII can be selectively targeted with
obtained in other studies using either i.v. BPA (40.1 F 2.2 boronated mAbs or EGF, given by either i.t. injection or CED,
days) alone or in combination with BSH (39, 40). Furthermore, and that a significant therapeutic gain can be obtained
they were comparable with those obtained following intra- following BNCT.
carotid administration of both drugs (73 days), although The combination of i.v. BPA, and BD-C225 significantly
among these animals, there was a subset of long-term survivors increased the MST compared with that of animals that received
the bioconjugate alone (P = 0.017) and the largest number of
long-term survivors was seen among those animals that
received both agents. Transfected cells expressing EGFR were
used in the present study, but we cannot exclude the
possibility that some of these cells may have either lost or
down-regulated receptor expression (10). In this case, BPA
could have been taken up by both receptor-negative and
receptor-positive cells. Furthermore, i.v. administration of BPA
could have delivered 10B to more remote clusters of tumor cells
that otherwise would not have been targeted by the
bioconjugate. Because survival following BNCT has been
shown by us to be linearly related to the total tumor boron
concentration (17, 39), the effect would have been an additive
one. An important advantage of direct i.c. delivery of the
capture agent, whether it be a high molecular weight bio-
conjugate or a low molecular weight agent, such as the
boronated nucleoside, N5-2OH (46), is that the tumor boron
concentration can be increased without a concomitant increase
in the blood concentration.
As recently reviewed by us (47), a variety of high molecular
weight boron delivery agents have been evaluated in
Fig. 3. Kaplan-Meier survival plots for F98EGFR glioma-bearing rats. Survival times experimental animals, but to date, none has advanced to
in days after implantation have been plotted for untreated animals (o); irradiated
controls (.); and those that received i.v. BPA alone (E), i.t. of BD-C225 (5), CED clinical biodistribution studies in humans. There are a variety
of BD-C225 alone (n), or in combination with i.v. BPA (+) followed by BNCT. of reasons for this, but probably one of the most important is
www.aacrjournals.org 1265 Clin Cancer Res 2007;13(4) February 15, 2007

7. Cancer Therapy: Preclinical
that these agents must be delivered i.c. Although CED is
being used clinically to deliver high molecular weight
therapeutic agents, such as radiolabeled antibodies (48) and
toxin fusion proteins (49, 50), these studies have all been
carried out by highly interdisciplinary clinical teams that
would find it difficult to function in the setting of a nuclear
reactor, unless it was specifically dedicated to BNCT. Two
examples are the Technical Research Center of Finland (VTT)
Research Reactor (FiR1), which is located in Helsinki, Finland
(51), and the small in-hospital neutron irradiator, IHNI-I,
which has been designed and is under construction in
Beijing, China (52). Another alternative would be to use
accelerator-based neutron sources, which currently are in the
design stage (53) and could be easily sited in a hospital. As
recently reported by Nigg (54), the most promising of these is
a gantry-mounted neutron source that is under construction
in Belgium (55).
Currently, three EGFR targeting agents are clinically avail-
able: two low molecular weight tyrosine kinase inhibitors,
gefitinib (Iressa) and erlotinib (Tarceva), and the mAb Fig. 6. Monitoring tumor growth by ultrasmall particles of iron oxide enhanced
8T MR images in untreated and BNCT-treated rats bearing i.c. implants of the
cetuximab (56). Tyrosine kinase inhibitors block binding of F98EGFR glioma. A, MR imaging of a tumor-bearing untreated rat. B, MR imaging
ATP to the catalytic site of the EGFR tyrosine kinase domain, of a BNCT-treated rat at 35 d posttumor implantation. C, MR imaging of a
thereby preventing receptor autophosphorylation and activa- BNCT-treated rat at 49 d posttumor implantation. D, H&E-stained coronal
section of the brain of untreated rate shown in (A).
tion of downstream signaling. On the other hand, cetuximab
exerts its tumoristatic and/or tumoricidal effects by blocking
the binding of EGF to the receptor, thereby disrupting the lated) receptor11. These cells were chosen to obviate the
complex signaling cascade that otherwise would have been immune response that otherwise would have been evoked if a
initiated with receptor activation. BNCT kills tumor cells by the large enough number (106 per cell) of human EGFRs were
production of high linear energy transfer a-particles and heavy expressed on the rat tumor cells. Better survival data might
ions, which are generated instantaneously by the 10B capture have been obtainable if the transfectants expressed a functional
and fission reactions following neutron irradiation. Therefore, receptor, such as the F98fEGFR cell line. However, such studies
by using BD-C225 as the boron delivery agent, it is possible would have to be carried out in immunologically deficient
that tumor cell killing could occur by both high linear energy nude rats to obviate the antihuman EGFR immune response.
transfer radiation and blockade of the EGFR signaling Preliminary studies to assess the effects of cetuximab, given
pathway. However, the F98EGFR transfectants used for our by CED, in combination with external beam photon irradiation
in vivo studies had a nonfunctional (i.e., weakly phosphory- (15 Gy, given in 5 Gy fractions) have been carried out in
F98EGFR gliomas-bearing Fischer rats.12 Because these trans-
fectants expressed a nonfunctional receptor, it was not
surprising that there were no differences in MST of animals
that received X-irradiation alone or in combination with
cetuximab. Recently published data indicate that i.p. adminis-
tration of cetuximab in combination with external beam
photon irradiation significantly enhanced the survival of nude
mice bearing i.c. implants of two different human glioma cell
lines (30). These results support our hypothesis that the use of
BD-C225 to target cells with a functional receptor might result
in a significant improvement in survival data compared with
those that we have obtained with a nonfunctional receptor.
The Food and Drug Administration recently has approved
cetuximab for use in the treatment of recurrent EGFR (+)
squamous cell carcinomas of the head and neck. Using i.v. BPA
as the capture agent, BNCT has been used to treat patients with
therapeutically refractory head and neck cancer and striking
clinical responses have been observed (57 – 59). Because these
tumors strongly express EGFR (60), even better and more
durable responses might be obtainable if an EGFR targeting
agent, such as BD-C225, were used in combination with BPA.
Fig. 5. Kaplan-Meier survival plots for F98EGFR glioma-bearing rats. Survival times
in days after implantation have been plotted for untreated animals (o); irradiated
controls (.); and those that received i.v. BPA + BSH (5); CED of BD-C225 alone
11
(E) or plus either i.v. BPA and BSH (5); or intracarotid BPA and BSH (n) followed F. Furnari, et al. unpublished data.
12
by BNCT. R.F. Barth, et al., unpublished data.
Clin Cancer Res 2007;13(4) February 15, 2007 1266 www.aacrjournals.org

8. Molecular Targeting of EGFR-Positive Gliomas
Based on all of our published data, which have shown been approved for clinical use (61). Conceivably, BD-C225
equivalent efficacy for boronated EGF, L8A4, and cetuximab, could be moved into clinical biodistribution studies within a
however, we have concluded that the latter would be the best short period time, if those clinicians who are treating patients
choice for boron delivery because it is the only agent that has with EGFR-positive tumors with BNCT were so inclined.
References
1. Barth RF, Coderre JA, Vicente MG, Blue TE. Boron epidermal growth factor receptors. Mol Biol Med nylalanine for neutron capture therapy by means of
neutron capture therapy of cancer: current status 1983;1:511 ^ 29. intracarotid injection and blood-brain barrier disrup-
and future prospects. Clin Cancer Res 2005;11: 20. Gill GN, Kawamoto T, Cochet C, et al. Monoclonal tion. Neurosurgery 1996;38:985 ^ 92.
3987 ^ 4002. anti-epidermal growth factor receptor antibodies 37. Morrison PF, Laske DW, Bobo H, Oldfield EH,
2. Zamenhof RG, Coderre JA, Rivard MJ, Patel H. which are inhibitors of epidermal growth factor Dedrick RL. High-flow microinfusion: tissue penetra-
Topics in neutron capture therapy. Proceedings of the binding and antagonists of epidermal growth factor tion and pharmacodynamics. Am J Physiol 1994;266:
Eleventh World Congress on Neutron Capture Thera- stimulated tyrosine protein kinase activity. J Biol R292 ^ 305.
py. Appl Radiat Isot 2004;61:731 ^ 1130. Chem 1984;259:7755 ^ 60. 38. Laske DW, Morrison PF, Lieberman DM, et al.
3. Barth RF, editor. A critical assessment of boron neu- 21. Mendelsohn J, Baselga J. Status of epidermal Chronic interstitial infusion of protein to primate brain:
tron capture therapy. J Neurooncol 2003;62:1 ^ 210. growth factor receptor antagonists in the biology and determination of drug distribution and clearance with
4. Vicente MGH, editor. Boron in medicinal chemistry. treatment of cancer. J Clin Oncol 2003;21:2787 ^ 99. single-photon emission computerized tomography
Anti-Cancer Agents Med Chem 2006;6:73 ^ 181. 22. Castillo L, Etienne-Grimaldi MC, Fischel JL, et al. imaging. J Neurosurg 1997;87:586 ^ 94.
5. NakagawaY, Kobayashi T, Fukuda H, editors. Advan- Pharmacological background of EGFR targeting. Ann 39. Barth RF,YangW, RotaruJH, et al. Boronneutroncap-
ces in Neutron Capture Therapy. Proceedings 12th In- Oncol 2004;15:1007 ^ 12. ture therapy of brain tumors: enhanced survival follow-
ternational Congress on Neutron Capture Therapy, 23. Sartor CI. Mechanisms of disease: radiosensitiza- ing intracarotid injection of either sodium borocaptate
Takamatsu, Japan. October 9 ^ 12. 2006. tion by epidermal growth factor receptor inhibitors. or boronophenylalanine with or without blood-brain
6. Bigner SH, Humphrey PA,Wong AJ, et al. Character- Nat Clin Pract Oncol 2004;1:80 ^ 7. barrier disruption. Cancer Res1997;57:1129 ^ 36.
ization of the epidermal growth factor receptor in hu- 24. Kiyota A, Shintani S, Mihara M, et al. Anti-epidermal 40. Barth RF, Yang W, Rotaru JH, et al. Boron neutron
man glioma cell lines and xenografts. Cancer Res growth factor receptor monoclonal antibody 225 capture therapy of brain tumors: enhanced survival
1990;50:8017 ^ 22. upregulates p27(KIP1) and p15(INK4B) and induces and cure following blood-brain barrier disruption and
7. Sauter G, Maeda T, Waldman FM, Davis RL, Feuerstein G1 arrest in oral squamous carcinoma cell lines. Oncol- intracarotid injection of sodium borocaptate and boro-
BG. Patterns of epidermal growth factor receptor am- ogy 2002;63:92 ^ 8. nophenylalanine. Int J Radiat Oncol Biol Phys 2000;
plification in malignant gliomas. AmJPathol1996;148: 25.Wu X, Fan Z, Masui H, Rosen N, Mendelsohn J. Ap- 47:209 ^ 18.
1047 ^ 53. optosis induced by an anti-epidermal growth factor 41. Harling OK, Riley KJ, Binns PJ, Patel H, Coderre JA.
8. Schwechheimer K, Huang S, Cavenee WK. EGFR receptor monoclonal antibody in a human colorectal The MIT user center for neutron capture therapy re-
gene amplification-rearrangement in human glioblas- carcinoma cell line and its delay by insulin. J Clin Invest search. Radiat Res 2005;164:221 ^ 9.
tomas. Int J Cancer 1995;62:145 ^ 8. 1995;95:1897 ^ 905. 42. Harling OK, Riley KJ. Fission reactor neutron sour-
9. Frederick L, Wang XY, Eley G, James CD. Diversity 26. Huang SM, Harari PM. Modulation of radiation re- ces for neutron capture therapy-a critical review.
and frequency of epidermal growth factor receptor sponse after epidermal growth factor receptor block- J Neurooncol 2003;62:7 ^ 17.
mutations in human glioblastomas. Cancer Res 2000; ade in squamous cell carcinomas : inhibition of 43. Rogus RD, Harling OK, Yanch JC. Mixed field do-
60:1383 ^ 7. damage repair, cell cycle kinetics, and tumor angio- simetry of epithermal neutron beams for boron neu-
10. Mendelsohn J, Baselga J. Epidermal growth factor genesis. Clin Cancer Res 2000;6:2166 ^ 74. tron capture therapy at the MITR-II research reactor.
receptor targeting in cancer. Semin Oncol 2006;33: 27. Perrotte P, MatsumotoT, Inoue K, et al. Anti-epider- Med Phys 1994;21:1611 ^ 25.
369 ^ 85. mal growth factor receptor antibody C225 inhibits an- 44. Klein JP, Moeschberger ML. Survival analysis: tech-
11. Barth RF, Yang W, Adams DM, et al. Molecular tar- giogenesis in human transitional cell carcinoma niques for censored and truncated data. 2nd ed. New
geting of the epidermal growth factor receptor for growing orthotopically in nude mice. Clin Cancer Res York (NY): Springer; 2003.
neutron capture therapy of gliomas. Cancer Res 1999;5:257 ^ 65. 45. Madsen RW, Moeschberger ML. Statistical con-
2002;62:3159 ^ 66. 28. Huang SM, Li J, Harari PM. Molecular inhibition of cepts. Englewood Cliffs (NJ): Prentice-Hall; 1986.
12. Yang W, Barth RF, Adams DM, et al. Convection- angiogenesis and metastatic potential in human 46. Barth RF, Yang W, Al-Madhoun AS, et al. Boron-
enhanced delivery of boronated epidermal growth squamous cell carcinomas after epidermal growth containing nucleosides as potential delivery agents
factor for molecular targeting of EGF receptor-positive factor receptor blockade. Mol Cancer Ther 2002;1: for neutron capture therapy of brain tumors. Cancer
gliomas. Cancer Res 2002;62:6552 ^ 8. 507 ^ 14. Res 2004;64:6287 ^ 95.
13. Yang W, Barth RF, Wu G, et al. Boronated epidermal 29. Sung FL, Poon TC, Hui EP, et al. Antitumor effect 47. Wu G, Barth RF, Yang W, et al. Boron containing
growth factor as a delivery agent for neutron capture and enhancement of cytotoxic drug activity by cetux- macromolecules and nanovehicles as delivery agents
therapy of EGF receptor positive gliomas. Appl Radiat imab in nasopharyngeal carcinoma cells. InVivo 2005; for neutron capture therapy. Anti-Cancer Agents Med
Isot 2004;61:981 ^ 5. 19:237 ^ 45. Chem 2006;6:167 ^ 84.
14. Wu G, Barth RF, Yang W, et al. Site-specific conju- 30. Eller JL, Longo SL, Kyle MM, et al. Anti-epidermal 48. Reardon DA, Akabani G, Coleman RE, et al. Salvage
gation of boron-containing dendrimers to anti-EGF growth factor receptor monoclonal antibody cetuxi- radioimmunotherapy with murine iodine-131-labeled
receptor monoclonal antibody cetuximab (IMC- mab augments radiation effects in glioblastoma mul- antitenascin monoclonal antibody 81C6 for patients with
C225) and its evaluation as a potential delivery agent tiforme in vitro and in vivo. Neurosurgery 2005; recurrent primary and metastatic malignant brain tumors:
for neutron capture therapy. Bioconjug Chem 2004; 56:155 ^ 62. phase II study results. JClin Oncol 2006;24:115 ^ 22.
15:185 ^ 94. 31. Harding J, Burtness B. Cetuximab: an epidermal 49. Laske DW, Youle RJ, Oldfield EH. Tumor regression
15. Barth RF,Wu G,Yang W, et al. Neutron capture ther- growth factor receptor chemeric human-murine with regional distribution of the targeted toxin TF-
apy of epidermal growth factor (+) gliomas using monoclonal antibody. Drugs Today (Barc) 2005;41: CRM107 in patients with malignant brain tumors. Nat
boronated cetuximab (IMC-C225) as a delivery 107 ^ 27. Med 1997;3:1362 ^ 8.
agent. Appl Radiat Isot 2004;61:899 ^ 903. 32. Barth RF, Adams DM, Soloway AH, et al. Determi- 50. Parney IF, Kunwar S, McDermott M, et al. Neurora-
16. Yang W, Barth RF, Wu G, et al. Development of a nation of boron in tissues and cells using direct- diographic changes following convection-enhanced
syngeneic rat brain tumor model expressing EGFRvIII current plasma atomic emission spectroscopy. Anal delivery of the recombinant cytotoxin interleukin 13-
and its use for molecular targeting studies with Chem 1991 ;63:890 ^ 3. PE38QQR for recurrent malignant glioma. J Neuro-
monoclonal antibody L8A4. Clin Cancer Res 2005; 33. Papazisis KT, Geromichalos GD, Dimitriadis KA, surg 2005;102:267 ^ 75.
11:341 ^ 50. Kortsaris AH. Optimizationof the sulforhodamine Bcol- 51. Joensuu H, Kankaanranta L, Seppala T, et al. Boron
« «
17. Yang W, Barth RF, Wu G, et al. Molecular targeting orimetric assay. JImmunol Methods1997;208:151 ^ 8. neutron capture therapy of brain tumors: clinical trials
and treatment of EGFRvIII-positive gliomas using 34. Pauwels B, Korst AE, de Pooter CM, et al. Compar- at the Finnish facility using boronophenylalanine.
boronated monoclonal antibody L8A4. Clin Cancer ison of the sulforhodamine B assay and the clono- J Neurooncol 2003;62:123 ^ 34.
Res 2006;12:3792 ^ 802. genic assay for in vitro chemoradiation studies. 52. Zhou Y, Gao Z, Li Y, Guo C, Liu X. Design and
18. Goldstein NI, Prewett M, Zuklys K, Rockwell P, Cancer Chemother Pharmacol 2003;51:221 ^ 6. construction of the in-hospital neutron irradiator-1
Mendelsohn J. Biological efficacy of a chimeric anti- 35. Barth RF. Rat brain tumor models in experimental <IHNI-I>. In: Nakagawa Y, Kobayashi T, Fukuda H,
body to the epidermal growth factor receptor in a hu- neuro-oncology: the 9L, C6, T9, F98, RG2 (D74), editors. Advances in Neutron Capture Therapy 2006.
man tumor xenograft model. Clin Cancer Res 1995;1: RT-2, and CNS-1 gliomas. J Neurooncol 1998;36: Proceedings 12th International Congress on Neutron
1311 ^ 8. 91 ^ 102. Capture Therapy; 2006 October 9 ^ 12; Takamatsu,
19. Sato JD, Kawamoto T, Le AD, et al. Biological 36. Yang W, Barth RF, Carpenter DE, Moeschberger Japan. pp. 557 ^ 560.
effects in vitro of monoclonal antibodies to human ML, Goodman JH. Enhanced delivery of boronophe- 53. Blue TE, Yanch JC. Accelerator-based epithermal
www.aacrjournals.org 1267 Clin Cancer Res 2007;13(4) February 15, 2007

9. Cancer Therapy: Preclinical
neutron sources for boron neutron capture therapy of ceedings 12th International Congress on Neutron 59. Kato I, Ono K, Sakurai F. Boron neutron capture
brain tumors. J Neurooncol 2003;62:19 ^ 31. Capture Therapy; 2006 October 9 ^ 12; Takamatsu, therapy for recurrent head and neck malignancies. In:
54. Nigg D. Neutron sources and applications in radiothe- Japan. pp. 308 ^ 311. Nakagawa, Y, Kobayashi T, Fukuda H, editors. Advan-
rapyöa brief history, and current trends. In: Nakagawa Y, 56. Ciardiello F. Epidermal growth factor receptor inhibi- ces in Neutron Capture Therapy 2006. 12th Interna-
Kobayashi T, Fukuda H, editors. Advances In Neutron tors in cancer treatment. Future Oncol 2005;1:221 ^ 34. tional Congress on Neutron Capture Therapy; 2006
CaptureTherapy 2006. Proceedings 12th International 57. Obayashi S, Kato I, Ono K, et al. Delivery of 10boron October 9 ^ 12,Takamatsu, Japan. pp. 1 ^ 4.
Congress on Neutron CaptureTherapy; 2006 October to oral squamous cell carcinoma using boronopheny- 60. Ford AC, Grandis JR. Targeting epidermal growth
9 ^ 12;Takamatsu, Japan. lalanine and borocaptate sodium for boron neutron factor receptor in head and neck cancer. Head Neck
55. Stichelbaut F, Forton E, JongenY. Design of a beam capture therapy. Oral Oncol 2004;40:474 ^ 82. 2003;25:67 ^ 73.
shaping assembly for an accelerator-based BNCT sys- 58. Kato I, Ono K, Sakurai Y, et al. Effectiveness of 61. Frieze DA, McCune JS. Current status of cetuximab
tem. In: Nakagawa Y, Kobayashi T, Fukuda H, editors. BNCT for recurrent head and neck malignancies. Appl for the treatment of patients with solid tumors. Ann
Advances In Neutron Capture Therapy 2006. Pro- Radiat Isot 2004;61:1069 ^ 73. Pharmacother 2006;40:241 ^ 50.
Clin Cancer Res 2007;13(4) February 15, 2007 1268 www.aacrjournals.org