Site-Specific Conjugation of Boron-Containing Dendrimers to Anti-EGF Receptor Monoclonal Antibody Cetuximab (IMC-C225) and Its Evaluation as a Potential Delivery Agent for Neutron Capture Therapy

Abstract

The gene encoding EGFR often is amplified in human gliomas, and the receptor itself has been considered as a potential target for the specific delivery of therapeutic agents to brain tumors. The purpose of the present study was to investigate the use of the chimeric MoAb cetuximab (IMC-C225), which is directed against EGFR and EGFRvIII, as a boron delivery agent for neutron capture therapy (NCT) of brain tumors. As determined by 125I-cetuximab radioligand binding assays, F98 rat glioma cells, which had been transfected with the gene encoding EGFR (F98EGFR), expressed 1.60 +/- 0.13 x 10(5) receptor sites/cell with a Ka = 1.64 +/- 0.32 x 10(8) M-1). F98 cells transfected with the gene encoding a mutant form of EGFR, designated the F98EGFRvIII glioma, expressed 1.07 +/- 0.10 x 10(5) receptor sites/cell with a Ka = 2.18 +/- 0.54 x 10(9) M-1 compared to background levels expressed on F98 wild-type cells (F98WT). A heavily boronated, fifth generation polyamidoamine (PAMAM or "starburst") dendrimer, G5-B1100, was linked to oligosaccharide moieties, which were distant from antigen binding sites of cetuximab, by means of the heterobifunctional reagents N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and N-(k-maleimidoundecanoic acid) hydrazide (KMUH). The resulting bioconjugate, designated C225-G5-B1100, was separated from the unconjugated dendrimer using a Sephacryl S-300 column. On the basis of the relative concentration ratios of boron and protein, there were approximately 1100 boron atoms per molecule of cetuximab with only a slight reduction of Ka. The localization of C225-G5-B1100 or G5-B1100 in rats bearing intracerebral implants of either F98EGFR or F98WT gliomas was determined 24 h following direct intratumoral (i.t.) injection at which time 92.3 +/- 23.3 micrograms B/g tumor was localized in F98EGFR gliomas versus 36.5 +/- 18.8 micrograms B/g tumor in F98WT gliomas and 13.4 +/- 6.1 micrograms in normal brain. In contrast, only 6.7 +/- 3.6 micrograms B/g tumor of G5-B1100 was localized in F98EGFR gliomas following i.t. injection, thereby demonstrating specific molecular targeting of EGFR. Based on these data, BNCT studies will be initiated in F98EGFR glioma bearing rats to evaluate C225-G5-B1100 for the treatment of intracerebral brain tumors.

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

“…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.…”

Dendrimers as tunable vectors of drug delivery systems and biomedical and ocular applications

“…A heavily boronated fifth-generation PAMAM dendrimer (G5-B1100) linked to C225 and boron neutron capture therapy has been examined and it is being assessed for the treatment of intracerebral brain tumors. 50 In addition, the covalent conjugation of DOX, a putative anticancer drug to a three-arm poly (ethylene oxide) dendrimer hybrid has also been examined. The study revealed a longer release time for the polymer-DOX conjugate compared with the free DOX.…”

Cationic lipid-coated magnetic nanoparticles associated with transferrin for gene delivery

“…Human EGFR cDNA transfected F98 EGFR glioma cell line was kindly provided by Dr. Rolf F. Barth (Department of Pathology, The Ohio State University, Columbus, OH) (Wu et al, 2004), and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented 10% FBS. All cell lines were cultured continuously as a monolayer in a humidified atmosphere containing 5% CO 2 at 37 °C.…”