Molecular Targeting of the Epidermal Growth Factor Receptor

Success of boron neutron capture therapy (BNCT) is dependent on cellular and molecular targeting of sufficient amounts of boron-10 to sustain a lethal B (n, ) Li capture reaction. The purpose of the present study was to determine the efficacy of boronated epidermal growth factor (EGF) either alone or in combination with boronophenylalanine (BPA) as delivery agents for an epidermal growth factor receptor (EGFR) -positive glioma, designated F98EGFR. A heavily boronated precision macromolecule [boronated starburst dendrimer (BSD)] was chemically linked to EGF by heterobifunctional reagents. Either F98 wild-type (F98WT) receptor ( ) or EGFR gene-transfected F98EGFR cells, which expressed 5 10 5 receptor sites/cell, were stereotactically implanted into the brains of Fischer rats, and 2 weeks later biodistribution studies were initiated. For biodistribution studies rats received an intratumoral (i.t.) injection of I-labeled BSD-EGF and were euthanized either 6 or 24 h later. At 6 h, equivalent amounts of BSD-EGF were detected in F98EGFR and F98WT tumors. Persistence of the bioconjugate in F98EGFR tumors was specifically determined by EGFR expression. By 24 h 33.2% of injected dose/g of EGF-BSD was retained by F98EGFR gliomas compared with 9.4% % of injected dose/g in F98WT gliomas, and the corresponding boron concentrations were 21.1 g/g and 9.2 g/g, respectively. Boron concentrations in normal brain, blood, liver, kidneys, and spleen all were at nondetectable levels (<0.5 g/g). On the basis of these results, BNCT was initiated at the Brookhaven National Laboratory Medical Research Reactor. Two weeks after implantation of 10 F98EGFR or F98WT tumor cells, rats received an i.t. injection of BSD-EGF ( 60 g B/ 15 g EGF) either alone or in combination with i.v. BPA (500 mg/kg). Rats were irradiated at the Brookhaven Medical Research Reactor 24 h after i.t. injection, which was timed to coincide with 2.5 h after i.v. injection of BPA for those animals that received both capture agents. Untreated control rats had a mean survival time (MST) SE of 27 1 day, and irradiated controls had a MST of 31 1 day. Animals bearing F98EGFR gliomas, which had received i.t. BSD-EGF and BNCT, had a MST of 45 5 days compared with 33 2 days for animals bearing F98WT tumors (P 0.0032), and rats that received i.t. BSD-EGF in combination with i.v. BPA had a MST of 57 8 days compared with 39 2 days for i.v. BPA alone (P 0.016). Our data are the first to show in vivo efficacy of BNCT using a high molecular weight boronated bioconjugate to target amplified EGFR expressed on gliomas, and they provide a platform for the future development of combinations of high and low molecular weight agents for BNCT. INTRODUCTION BNCT is a binary system based on the selective uptake of sufficient amounts of boron-10 ( 10 atoms/cell) by tumor cells, followed by irradiation with low energy ( 0.025 eV) thermal neutrons. The resulting nuclear capture and fission reactions yield particles and Li nuclei, which have high linear energy transfer and path lengths of 9 m and 5 m, respectively. Each component can be manipulated independently so that the interval between administration of the capture agent and neutron irradiation can be adjusted to an optimal time at which the differential between boron concentration levels in normal tissues and tumor are maximized. For BNCT to be successful, there must be selective accumulation of B in the tumor, low levels in blood, endothelial cells, and normal brain, and a sufficient thermal neutron fluence delivered to the tumor site. These requirements are discussed in detail in several recent reviews (1–3) and a monograph (4). One of the major challenges in effectively treating high-grade brain tumors with BNCT is how to deliver a sufficient amount of B to individual tumor cells to sustain a lethal B(n, )Li capture reaction. We have been interested in the possibility of using combinations of two low molecular weight drugs, BPA and BSH (5, 6), together with high molecular weight targeting agents such as monoclonal or bispecific antibodies (7, 8) and boronated EGF (9, 10). The EGFR gene often is amplified in human glioblastomas and other primary brain tumors but is undetectable or weakly expressed in normal brain. Studies by Bigner et al. (11) revealed that in a series of 33 human glioma biopsies, 15 showed amplification of the EGFR gene. Similar or even higher frequencies of amplification have been observed by others, and this often is associated with increased cell surface receptor expression (12–14). The distribution of EGFR in high-grade gliomas is variable, which probably reflects the cellular heterogeneity of these tumors. Because the number of EGFRs on individual tumor cells can be up to 100 times greater than on normal glial cells (13, 14), the EGFR has been considered as a potential target (15, 16) for the specific delivery of a variety of diagnostic and therapeutic agents, including monoclonal antibodies in patients with brain tumors (17–19). Although there have been a number of reports on the potential use of EGF-based bioconjugates as boron delivery agents (9, 10, 20, 21), to date only low molecular weight boroncontaining drugs have been used for BNCT of experimental brain tumors. We have developed previously a method for linking a heavily boronated precision macromolecule (BSD) to EGF (9) and have shown that after i.t. injection the BSD-EGF bioconjugate could deliver 15.3 g/g of boron (44% ID/g; Ref. 10) to the allogeneic C6 rat Received 1/4/02; accepted 4/1/02. 1 This research was supported by the Biological and Environmental Research Program (BER) United States Department of Energy Grants DE-FG02-90ER6097 and DE-FG0298ER62595, NIH Grants 5R01CA79758 and CA16056-22, the Roswell Park Alliance, and the Jeffrey Wright Fund. Presented in part at the Ninth International Symposium on Neutron Capture Therapy, Osaka, Japan October 2–6, 2000 and the 12 World Congress of Neurosurgery, Sydney, Australia, September 16–20, 2001. 2 To whom requests for reprints should be addressed, at The Ohio State University, Department of Pathology, 165 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210. Phone: (614) 292-2177; Fax: (614) 292-7072; E-mail: [email protected]. 3 Present address: Department of Nuclear Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139. 4 The abbreviations used are: BNCT, boron neutron capture therapy; BPA, boronophenylalanine; BSH, sodium borocaptate; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; BSD, boronated starburst dendrimer; BSD-EGF, bioconjugate of boronated starburst dendrimer and epidermal growth factor; MST, mean survival time; i.t., intratumoral; F98WT, wild-type F98 glioma; F98EGFR, EGFR gene transduced F98 glioma; G418, Geneticin-selective antibiotic; DCP-AES, direct current plasma-atomic emission spectroscopy; BMRR, Brookhaven Medical Research Reactor; b.w., body weight; MW, megawatt; % ID/g, percentage of injected dose per gram; MoAb, monoclonal antibody; CED, convection-enhanced delivery.