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Boronated Cetuximab CCR tumor targeting in BNCT


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Boronated Cetuximab CCR tumor targeting in BNCT

  1. 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@ 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
  2. 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. 1261 Clin Cancer Res 2007;13(4) February 15, 2007
  3. 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
  4. 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. 1263 Clin Cancer Res 2007;13(4) February 15, 2007
  5. 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
  6. 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 1265 Clin Cancer Res 2007;13(4) February 15, 2007
  7. 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
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