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

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 I-125-cetuximab radioligand binding assays, F98 rat glioma cells, which had been transfected with the gene encoding EGFR (F98(EGFR)), expressed 1.60 +/- 0.13 x 10(5) receptor sites/cell with a K-a = 1.64 +/- 0.32 x 10(8) M-1. F98 cells transfected with the gene encoding a mutant form of EGFR, designated the F98(EGFRvIII) glioma, expressed 1.07 0.10 x 105 receptor sites/ cell with a K-a = 2.18 +/- 0.54 x 10(9) M-1 compared to background levels expressed on F98 wild-type cells (F98(WT)). A heavily boronated, fifth generation polyamidoamine (PAMAM or starburst) dendrimer, G5-B-1100, 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-B-1100, 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 similar to1100 boron atoms per molecule of cetuximab with only a slight reduction of K-a. The localization of C225G5-B-1100 or G5-B-1100 in rats bearing intracerebral implants of either F98(EGFR) or F98(WT) gliomas was determined 24 h following direct intratumoral (i.t.) injection at which time 92.3 +/- 23.3 mug B/g tumor was localized in F98(EGFR) gliomas versus 36.5 +/- 18.8 mug B/g tumor in F98(WT) gliomas and 13.4 +/- 6.1 mug in normal brain. In contrast, only 6.7 +/- 3.6 mug B/g tumor of G5-B-1100 was localized in F98(EGFE) gliomas following i.t. injection, thereby demonstrating specific molecular targeting of EGFR. Based on these data, BNCT studies will be initiated in F98(EGFR) glioma bearing rats to evaluate C225-G5-B-1100 for the treatment of intracerebral brain tumors.