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Clinical Cancer Research 13, 1260-1268, February 15, 2007. doi: 10.1158/1078-0432.CCR-06-2399
© 2007 American Association for Cancer Research
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Cancer Therapy: Preclinical

Molecular Targeting and Treatment of an Epidermal Growth Factor Receptor–Positive Glioma Using Boronated Cetuximab

Gong Wu1, Weilian Yang1, Rolf F. Barth1, Shinji Kawabata1, Michele Swindall1, Achintya K. Bandyopadhyaya4, Werner Tjarks4, Behrooz Khorsandi5, Thomas E. Blue5, Amy K. Ferketich6, Ming Yang3, Gregory A. Christoforidis3, Thomas J. Sferra2,7, Peter J. Binns8, Kent J. Riley8, Michael J. Ciesielski9 and Robert A. Fenstermaker9

Authors' Affiliations: Departments of 1 Pathology, 2 Pediatrics, and 3 Radiology, 4 College of Pharmacy, 5 Nuclear Engineering Program, and 6 School of Public Health, The Ohio State University; 7 Children's Research Institute, Columbus, Ohio; 8 Nuclear Reactor Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts; and 9 Department of Neurosurgery, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Rolf F. Barth, Department of Pathology, The Ohio State University, 165 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. Phone: 614-292-2177; Fax: 614-292-7072; E-mail: rolf.barth{at}osumc.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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 µg/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 µg/g, respectively, with normal brain and blood boron values <0.05 µg/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. boronophenylalanine (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 BNCT of brain tumors.

Boron neutron capture therapy (BNCT) is based on the nuclear capture and fission reactions that occur when nonradioactive boron-10 is irradiated with low energy (e ≤ 0.025 eV) thermal neutrons to produce 11B in an unstable form, which undergoes instantaneous nuclear fission to produce {alpha}-particles and recoiling lithium-7 nuclei. These high linear energy transfer particles have a range of 5 to 9 µm, thereby restricting their destructive effects to only those cells containing 10B. To be effective, BNCT requires a sufficient amount (20-30 µg/g) of 10B, homogeneously distributed in the tumor, with concomitantly low 10B concentrations in surrounding normal tissues. These requirements, as well as clinical applications, have been discussed in detail in several recent reviews and monographs (1–5). BNCT primarily has been used to treat high-grade gliomas and either cutaneous primaries or cerebral metastases of melanoma (1). More recently, it has also been used to treat patients with recurrent tumors of the head and neck and colorectal cancer metastatic to the liver (1).

Both the epidermal growth factor (EGF) receptor (EGFR) and its mutant isoform EGFRvIII frequently are overexpressed in human glioblastomas (6–9), which make them attractive targets for the treatment of brain tumors (10). We have investigated molecular targeting of EGFR or EGFRvIII using EGF (11–13), or the monoclonal antibodies (mAb) cetuximab (14, 15) and L8A4 (16, 17), which have been linked to a heavily boronated polyamidoamine dendrimer. Cetuximab (Erbitux), known previously as IMC-C225, is a chimeric mouse-human mAb that originally was produced in the laboratory of Dr. John Mendelsohn (University of Texas M. D. Anderson Cancer Center, Houston, TX; ref. 18). It has greater affinity for EGFR than either EGF or transforming growth factor-{alpha}, and following binding, the receptor-antibody complex is rapidly internalized, thereby eliminating further activation of the receptor (19, 20). Down-regulation of cell surface receptor binding sites and competition of cetuximab for the remaining binding sites can reduce or prevent further activation by ligand. Several mechanisms have been proposed to explain the antitumor activity of cetuximab (21–23). These include cell cycle arrest (24), apoptosis (25), decrease in angiogenesis and cellular adhesion (26, 27), and inhibition of matrix metalloproteinase expression and activity (28). Enhancement of the cytotoxic effects of chemotherapeutic agents (29) and the response to ionizing radiation have also been reported (30). Cetuximab is reactive with both wild-type EGFR and EGFRvIII (14), and recently, it has been approved by the U.S. Food and Drug Administration for use in patients with EGFR-positive colorectal cancer metastatic to the liver and recurrent head and neck cancers (31). Because of its pleiotropic effects, cetuximab is particularly attractive as a boron delivery agent for NCT of gliomas. In the present report, we describe studies to evaluate boronated cetuximab as a delivery agent for BNCT of the F98 rat glioma, which has been transfected with the gene encoding human EGFR (F98EGFR). Our data convincingly show its efficacy for BNCT of this tumor, used either alone or in combination with boronophenylalanine (BPA), a drug that has been used clinically for BNCT of brain tumors (1).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Preparation of the bioconjugate BD-C225. Cetuximab was generously provided to us by Dr. Daniel Hicklin (ImClone Systems, Inc., New York, NY). Site-specific attachment of a heavily boronated polyamidoamine dendrimer was carried out, as described by us in detail elsewhere (14). Briefly, a fifth generation polyamidoamine dendrimer (Sigma-Aldrich, St. Louis, MO), containing 128 terminal amino groups, was reacted with an isocyanato polyhedral borane anion, Na(CH3)3NB10H8NCO. This yielded a heavily boronated macromolecule, which contained ~1,100 boron atoms per molecule of dendrimer. Cetuximab was linked to the boronated dendrimer (BD) by two heterobifunctional linkers, N-succinimidyl 3-(2-pyridyldithio)-propionate and N-(k-maleimido undecanoic acid)-hydrazide (14). Protein content of the bioconjugate was determined spectrophotometrically by means of the Coomassie blue assay reagent (Pierce, Rockford, IL) and boron was quantified by means of direct current plasma-atomic emission spectroscopy (DCP-AES; 32).

In vitro cellular uptake of BD-C225. For in vitro boron uptake studies, F98 glioma cells, expressing 106 human EGFRs per cell (F98fEGFR), were used. These were produced by Dr. Frank Furnari (Ludwig Institute for Cancer Research, La Jolla, CA), who generously provided them to us. Five million F98 wild-type (F98WT), F98fEGFR, or F98npEGFRvIII (17) glioma cells were seeded into T-150 flasks with DMEM containing 10% fetal bovine serum (Life Technologies, Inc., Rockville, MD) supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin. After incubation for 24 h at 37°C, the medium was replaced with DMEM containing 1.68 mg BD-C225 (90 µg boron), and the cells were incubated for an additional 2 h at 37°C. Following this, the medium was decanted and the cells were washed thrice with PBS (pH 7.4), disaggregated by exposure to 0.5 mmol/L EDTA for 5 min, counted, and sedimented. Cells were digested with concentrated sulfuric acid and 50% hydrogen peroxide, and boron uptake was determined by DCP-AES (32).

In vitro neutron irradiation studies. For in vitro neutron irradiation studies, F98fEGFR glioma cells, expressing 106 receptor sites per cell, were cultured until confluent. Cells were harvested by exposure to 0.5 mmol/L EDTA and washed thrice with PBS, and aliquots containing 106 cells were dispensed into 2-mL plastic vials. Except for unirradiated control cells, either cetuximab or BD-C225 was added to all other vials and incubated at 4°C for 90 min following which they were washed thrice with medium. Triplicate samples were irradiated with thermal neutrons at The Ohio State University Research Reactor for 1, 2, 5, or 10 min at a thermal neutron flux of 109 cm–2 sec–1. After irradiation, aliquots of 10,000 cells were taken from each vial and seeded into 96-well microplates (Corning, Corning NY). Cell survival was determined 72 h later by means of the sulforhodamine B assay (33). A clonogenic assay also was carried out following irradiation to assess cell survival (34). Briefly, varying numbers of F98fEGFR cells were seeded into 100-mm Petri dishes and incubated for 7 days at 37°C in an atmosphere containing 95% air and 5% CO2. Following this, the medium was decanted and the cells were fixed by adding 2 to 3 mL of 37% formaldehyde, and then the plates were stained with 3 to 5 mL of saturated crystal violet. The number of colonies containing at least 50 cells was enumerated visually by counting under a dissecting microscope. The surviving fraction was calculated from the number of colonies enumerated / number of cells plated x plating efficiency / 100.

Tumor implantation and biodistribution of BD-C225. F98EGFR glioma cells expressing 105 receptor sites per cell, described previously by us in detail (12), were used for in vivo studies. This cell line, which 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). These cells were used to obviate an immune response directed against human EGFR, which we found occurred when cells expressing 106 receptor sites were implanted i.c. into immunocompetent Fischer rats.10 After i.c. implantation into syngeneic Fischer rats, the F98EGFR glioma forms a progressively growing, infiltrative tumor that invariably results 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 containing 600 µg/mL geneticin (G418; Sigma-Aldrich). F98WT cells were cultured in the same medium, but without G418. Animal studies were done in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC, 1996) and the protocol was approved by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University (Columbus, OH). CD-Fischer rats (Charles River Laboratories, Wilmington, MA), weighing 200 to 220 g, were anesthetized with a 1.2:1 mixture of ketamine/xylazine at a dose of 120 mg of ketamine/20 mg of xylazine/kg body weight. Following this, tumor cells were implanted stereotactically, as originally described by us (36). A small plastic screw (Arrow Machine Manufacturing, Inc., Richmond, VA) with an entry port, which allowed insertion of a 27-gauge needle, was embedded into the calvarium before tumor cell implantation. BD-C225 was given i.c. by means of convection enhanced delivery (CED), using a syringe pump at a rate of 0.33 µL/min for 30 min to deliver a volume of 10 µL (Harvard Apparatus Co., Cambridge, MA) as described previously (12). This technique, completely bypasses the blood-brain barrier, maximizes delivery to the tumor and minimizes uptake by extracranial organs and blood (37, 38). For CED, a plastic cannula was inserted into the entry port and then advanced 5 mm below the dura into the tumor of F98EGFR glioma-bearing rats. Biodistribution studies were carried out in tumor-bearing rats 12 to 14 days following tumor cell implantation. Animals were divided into four experimental groups of four to five rats each. Animals in groups 1 and 2 had F98EGFR gliomas and received 750 µg BD-C225 (40 µg B) by CED at a rate of 0.33 µL/min or intratumoral (i.t.) injection. Rats in groups 3 and 4 received an i.v. injection of BPA (500 mg/kg body weight, equivalent to 27 mg 10B/kg body weight) or BD-C225 by CED with i.v. BPA (Katchem Ltd., Prague, Czech Republic). The biodistribution of BD-C225 was determined at 24 h after CED by measuring concentrations of boron in various tissue samples by DCP-AES (32). Animals were euthanized by an overdose of halothane following which tumors and normal tissues consisting of brain, blood, liver, kidney, and muscle were removed and weighed.

Therapy experiments and dosimetry. Neutron irradiations for these experiments were identical to those reported previously by us using the boronated mAb L8A4 (17). BNCT was done 14 days following stereotactic implantation of 103 F98EGFR glioma cells. Rats were transported approximately 5 to 7 days before irradiation to Massachusetts Institute of Technology (Cambridge, MA) where they were housed in an accredited animal care facility supervised by the Division of Comparative Medicine. Before irradiation at the Nuclear Reactor Laboratory, they were randomized based on weight into experimental groups of 7 to 11 animals each as follows: group 1, untreated controls; group 2, irradiated controls; group 3, i.v. BPA, followed by BNCT; group 4, i.t. of BD-C225 followed by BNCT; group 5, CED of BD-C225 followed by BNCT; and group 6, CED of BD-C225 plus i.v. BPA, followed by BNCT. BNCT was initiated 24 h after CED of 10 µL of 750 µg BD-C225 (40 µg 10B) and 2.5 h after i.v. administration of BPA (500 mg/kg body weight). In a second study, using a different lot number of F98EGFR cells, rats received BD-C225 in combination with either i.v. or intracarotid 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% 6Li 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 x 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 photons, 7.7 cGy/min for thermal neutrons from the nitrogen capture reaction, and 3.4 cGy/min per µg 10B in tissues. The estimated uncertainties on all these dose rates were 5%. Boron concentrations were determined in tumor, normal brain, liver, and blood in a separate group of animals 24 h after CED of BD-C225 and 2.5 h after i.v. injection of BPA to estimate absorbed doses in these tissues. Animal irradiations were done with the reactor operating at a power between 4.0 and 4.8 MW. These took between 6.9 and 8.6 min to deliver a thermal neutron fluence of 2.64 x 1012 n cm–2 that matches previous dose prescriptions (13, 15). After completion of BNCT, the animals were returned to The Ohio State University for clinical monitoring.

Monitoring of clinical status and neuropathologic evaluation. All animals were weighed thrice weekly and their clinical status was evaluated at the same time. Once the animals had progressively growing tumors, as evidenced by the combination of sustained weight loss, ataxia, and periorbital hemorrhage, they were euthanized to minimize discomfort. Survival times were determined by adding 1 day to the time between tumor implantation and euthanization. The brains of all animals in the therapy studies were removed after death, fixed in 10% buffered formalin, and then cut coronally at the level of the optical chiasm and 2 mm anterior and posterior to it. Coronal slices were embedded in paraffin, cut at 4 µm, stained with H&E, and then examined microscopically to assess the histopathologic changes. The tumor size index was determined from H&E-stained coronal sections of brain using a semiquantitative grading scale ranging from 0 to 4. Each section was scored as follows: 0, no tumor; 1, very small (i.e., microscopic, <1 mm); 2, small (approximately 1-3 mm); 3, large (approximately 4-7 mm); and 4, massive (>8 mm); the mean score was calculated for each group.

Magnetic resonance imaging. Magnetic resonance (MR) images of brain tumor–bearing rats were generated on a Bruker Avance scanner (Bruker, Billerica, MA) interfaced with Techron gradient amplifiers (Crown International, Elkhart, IN) and Magnex gradients (Magnex Scientific, Abingdon, England) using a custom-built radio frequency front end. A custom-made, 4 cm in diameter birdcage coil was tuned to the head of the rat at 340 MHz while the rat was in the prone position. Ultrasmall particles of iron oxide (SHU555C, Supravist; Schering AG, Berlin, Germany) were used as a contrast agent. These were given i.v. (2.0 mg Fe/kg) via a right femoral vein catheter after anesthetizing the animals with isoflurane. Each animal was scanned before and after injection of ultrasmall particles of iron oxide using a high resolution T2*-weighted gradient recalled echo sequence with an in-plane resolution of 78 µm. The images were generated with the following pulse-sequence variables: time of repetition, 500 msec; time of echo, 14.6 msec; flip angle, 22.5°; field of view, 4 cm; matrix, 512 x 512; slice thickness/gap, 1/0.1 mm; and acquisition time, 10 min and 14 s.

Statistical evaluation of survival data. The mean survival time (MST), SE, and median survival time were calculated for each group using the Kaplan-Meier method that enabled Kaplan-Meier Survival and Cox proportional hazard survival curves to be plotted (44). The hypotheses involved comparing each BD-C225–treated animal to each irradiated control. A log-rank test was used for these comparisons, with a Bonferroni method of adjustment for the multiple comparisons (45). Because five comparisons were tested for statistical significance within the BD-C225 tests, an {alpha} = 0.0125 was used.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
In vitro uptake and irradiation studies. To show that the bioconjugate was selectively taken up by EGFR-positive glioma cells, F98WT, F98fEGFR, and F98npEGFRvIII cells were incubated with 1.68 mg BD-C225 (90 µg B) for 2 h at 37°C. As determined by DCP-AES, 41.8 µg B were taken up by 109 F98fEGFR cells, 19.6 µg by F98npEGFRvIII cells, and 9.1 µg by F98WT cells (Fig. 1 ), which was a 4.6- to 2.2-fold difference between EGFR-positive and EGFR-negative cells. It is noteworthy 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, followed by 10 min neutron irradiation (3.8 Gy), had 20.7 ± 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 ± 2.6% for irradiated controls compared with 5.4 ± 0.4% for cells that had been exposed to BD-C225 before irradiation.


Figure 1
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  		Fig. 1. Cellular uptake of BD-C225 by F98fEGFR, F98npEGFRvIII, and F98WT glioma cells. BD-C225 (90 µg boron) were incubated with glioma cells at 37°C for 2 h and then washed with medium for three times. Cells were digested, and boron content was determined by DCP-AES.

 

Figure 2
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  		Fig. 2. In vitro neutron irradiation studies. F98EGFR glioma cells were exposed to cetuximab (bullet), BD-C225 ({blacktriangleup}), or medium alone ({square}) for 90 min at 4°C following which they were washed with medium and then irradiated with thermal neutron for 0 to 10 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.

 
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 ± 14.8 µg B/g compared with 50.8 ± 5.7 µg 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 µg B/g, which was the background limit of detection. The tumor boron concentration in rats that received i.v. BPA was 10.7 ± 1.7 µg/g compared with 87.9 ± 16.5 µg B/g in animals 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. The reported absorbed dose was based on mean boron concentrations measured in tumor, brain, and blood at 24 h following CED of BD-C225 and 2.5 h after i.v. administration of BPA using a separate group of untreated animals. Based on these total boron concentrations, the mean absorbed doses delivered to F98EGFR tumors were 19.5 Gy following CED of BD-C225, 4.2 Gy following i.v. administration of BPA alone, and 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 doses were expressed without biological weighting factors due to uncertainty relating in their determination that relate to the chemical form of the 10B (i.e., BD-C225 versus BPA).


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  		Table 1. Boron concentrations and calculated absorbed radiation doses following administration of BD-C225 to F98EGFR glioma-bearing rats

 
Therapeutic response of glioma-bearing rats following BNCT. All animals in a pilot study to determine tolerance to BNCT following CED of BD-C225 lost weight within 7 to 10 days after treatment. Rats that received CED of 40 µg 10B/750 µg BD-C225 and 500 mg/kg body weight of BPA i.v. lost <10% of their body weight but regained it within 2 weeks. Based on these results, a dose of 40 µg 10B/750 µg BD-C225 was selected. This was given by CED, either alone or in combination with i.v. BPA. BNCT was initiated at the MITR-II reactor 14 days following i.c. implantation of 103 F98EGFR glioma cells. All rats tolerated BNCT without any untoward effects, and 3 to 7 days later, they were returned to Columbus, Ohio. A Cox proportional hazards regression model was fit to the data and the proportional hazards assumption was checked. Because this was met, the log-rank test was used to test for significance between survival curves, and the survival data of the treatment groups were significantly different from the irradiated controls and untreated controls (P < 0.001). Survival data following BNCT are summarized in Tables 2 and 3 and Kaplan-Meier and Cox survival plots for BNCT-treated animals and irradiated controls are shown in Figs. 3 , 4 and 5 . Untreated control rats had a MST ± SE of 26.3 ± 1.6 days compared with a modest increase of 30.3 ± 1.6 days for the irradiated controls. Animals bearing F98EGFR gliomas, which had received CED of BD-C225, either alone or in combination with i.v. BPA, had a MST of 54.5 ± 4.3 days and 70.9 ± 11.1 days, respectively, with one rat surviving more than 180 days compared with 40.1 ± 2.2 days for animals that received i.v. BPA alone. The corresponding mean percentages increase in life span were 170% for the combination versus 107% for CED of BD-C225 alone and 52% for i.v. BPA alone. The results from these comparisons indicated that CED of BD-C225 plus i.v. BPA was significantly different from the i.v. BPA alone group (P = 0.0002), and the difference between the groups that received BD-C225 alone or in combination with BPA was also significant (P = 0.017). In the second study, the efficacy of BD-C225 was evaluated in combination with either i.v. or intracarotid administration of BPA plus BSH (Fig. 5; Table 3). The MSTs of animals that received BD-C225 plus BPA and BSH by the i.v. or intracarotid route were equivalent (67.1 ± 21.6 and 75.8 ± 28.4 days, respectively; P = 0.261), and these MSTs were equivalent (P = 0.893) to those of animals that received BD-C225 plus i.v. BPA (70.9 ± 11.1 days; Table 2). However, MST of animals that received the combination of i.v. BPA plus BSH and BD-C225 was significantly different from those that received BD-C225 alone (P = 0.034).


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  		Table 2. Survival times of F98EGFR glioma-bearing rats following CED of BD-C225 with or without i.v. BPA and BNCT

 

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  		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

 

Figure 3
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  		Fig. 3. Kaplan-Meier survival plots for F98EGFR glioma-bearing rats. Survival times in days after implantation have been plotted for untreated animals ({circ}); irradiated controls (bullet); and those that received i.v. BPA alone ({blacktriangleup}), i.t. of BD-C225 ({triangledown}), CED of BD-C225 alone ({blacksquare}), or in combination with i.v. BPA (+) followed by BNCT.

 

Figure 4
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  		Fig. 4. Cox survival plots for F98EGFR glioma-bearing rats. Survival time in days after implantation have been plotted for untreated animals ({circ}), irradiation control (bullet), i.v. BPA + BNCT ({blacktriangleup}), i.t. BD-C225 ({triangledown}) and CED of BD-C225 alone ({blacksquare}) or in combination with i.v. BPA (+) followed by BNCT.

 

Figure 5
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  		Fig. 5. Kaplan-Meier survival plots for F98EGFR glioma-bearing rats. Survival times in days after implantation have been plotted for untreated animals ({circ}); irradiated controls (bullet); and those that received i.v. BPA + BSH ({square}); CED of BD-C225 alone ({blacktriangleup}) or plus either i.v. BPA and BSH ({triangledown}); or intracarotid BPA and BSH ({blacksquare}) followed by BNCT.

 
MR imaging and neuropathologic evaluation. The tumor sizes indices of animals that received BD-C225 by either CED or i.t. injection, followed by BNCT, were equivalent (5.1 ± 1.5 and 5.3 ± 1.6 mm) and not different from that of animals that received CED of BD-C225 and i.v. BPA (4.75 ± 1.39 mm). The MR images of an animal from the untreated group was taken 1 day before euthanization. As shown in Fig. 6A , the tumor vascular bed was significantly enhanced after i.v. administration of ultrasmall particles of iron oxide, and the tumor margins could be clearly delineated in the right cerebral 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.


Figure 6
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  		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 F98EGFR glioma. A, MR imaging of a tumor-bearing untreated rat. B, MR imaging of a BNCT-treated rat at 35 d posttumor implantation. C, MR imaging of a BNCT-treated rat at 49 d posttumor implantation. D, H&E-stained coronal section of the brain of untreated rate shown in (A).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The purpose of the present study was to evaluate the potential use of the anti-EGFR mAb cetuximab as a 10B delivery agent for BNCT of the F98EGFR glioma. Cetuximab was covalently and site specifically linked to a heavily boronated polyamidoamine dendrimer by means of two heterobifunctional linkers, N-succinimidyl 3-(2-pyridyldithio)-propionate and N-(k-maleimido undecanoic acid)-hydrazide. The resulting bioconjugate showed both in vitro and in vivo specificity for targeting the F98EGFR glioma. Based on these findings, in vivo BNCT studies were initiated. Animals that received BD-C225 by CED in combination with i.v. BPA had a MST of 70.9 ± 11.1 days compared with 54.5 ± 4.3 days for those that received the bioconjugate alone and 30.3 ± 1.6 days for irradiated controls. These survival data were superior to those that we have obtained in other studies using either i.v. BPA (40.1 ± 2.2 days) alone or in combination with BSH (39, 40). Furthermore, they were comparable with those obtained following intracarotid administration of both drugs (73 days), although among these animals, there was a subset of long-term survivors (39, 40). Equivalent survival data were obtained using BD-C225 in combination with BPA and BSH, given by either i.v. or intracarotid injection. This is of particular significance because, clinically, it would be much easier to give BPA by i.v. injection rather than by the i.v. or intracarotid administration of BPA and BSH. The present data, and those recently reported by us in other studies using boronated L8A4 (17) and EGF (13), establish proof of principle that gliomas expressing either EGFR or EGFRvIII can be selectively targeted with boronated mAbs or EGF, given by either i.t. injection or CED, and that a significant therapeutic gain can be obtained following BNCT.

The combination of i.v. BPA, and BD-C225 significantly 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 bioconjugate 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 experimental animals, but to date, none has advanced to clinical biodistribution studies in humans. There are a variety of reasons for this, but probably one of the most important is 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 available: two low molecular weight tyrosine kinase inhibitors, gefitinib (Iressa) and erlotinib (Tarceva), and the mAb cetuximab (56). Tyrosine kinase inhibitors block binding of ATP to the catalytic site of the EGFR tyrosine kinase domain, thereby preventing receptor autophosphorylation and activation 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 complex signaling cascade that otherwise would have been initiated with receptor activation. BNCT kills tumor cells by the production of high linear energy transfer {alpha}-particles and heavy ions, which are generated instantaneously by the 10B capture and fission reactions following neutron irradiation. Therefore, by using BD-C225 as the boron delivery agent, it is possible that tumor cell killing could occur by both high linear energy transfer radiation and blockade of the EGFR signaling pathway. However, the F98EGFR transfectants used for our in vivo studies had a nonfunctional (i.e., weakly phosphorylated) receptor11. These cells were chosen to obviate the immune response that otherwise would have been evoked if a large enough number (106 per cell) of human EGFRs were expressed on the rat tumor cells. Better survival data might have been obtainable if the transfectants expressed a functional receptor, such as the F98fEGFR cell line. However, such studies would have to be carried out in immunologically deficient nude rats to obviate the antihuman EGFR immune response.

Preliminary studies to assess the effects of cetuximab, given by CED, in combination with external beam photon irradiation (15 Gy, given in 5 Gy fractions) have been carried out in F98EGFR gliomas-bearing Fischer rats.12 Because these transfectants 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. administration 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. Based on all of our published data, which have shown equivalent efficacy for boronated EGF, L8A4, and cetuximab, however, we have concluded that the latter would be the best choice for boron delivery because it is the only agent that has been approved for clinical use (61). Conceivably, BD-C225 could be moved into clinical biodistribution studies within a short period time, if those clinicians who are treating patients with EGFR-positive tumors with BNCT were so inclined.


   Footnotes
 
Grant support: NIH grants 1R01 CA098945 (R.F. Barth) and 1R01 NS39071 (T.J. Sferra); the Roswell Park Alliance Foundation (R.A. Fenstermaker); and U.S. Department of Energy through the program of Innovations in Nuclear Infrastructure and Education, Office of Nuclear Energy, Science and Technology (contract nos. DE-FG07-02ID14420 and DE-FG07-02 [K14420]) and the Office of Environmental and Biological Research (contract no. DE-FG02-02ER63358).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Current address for T.J. Sferra: Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK; current address for S. Kawabata: Department of Neurosurgery, Osaka Medical College, Takatsuki City, Osaka, Japan.

Presented in part at the 12th International Symposium on Neutron Capture Therapy, Takamatsu, Japan, October 9-12, 2006.

10 R.F. Barth and W. Yang, unpublished data. Back

11 F. Furnari, et al. unpublished data. Back

12 R.F. Barth, et al., unpublished data. Back

Received 9/29/06; accepted 11/10/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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