Wu Gong, Barth Rolf F, Yang Weilian, Chatterjee Madhumita, Tjarks Werner, Ciesielski Michael J, Fenstermaker Robert A
Department of Pathology and College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, USA.
Bioconjug Chem. 2004 Jan-Feb;15(1):185-94. doi: 10.1021/bc0341674.
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.
编码表皮生长因子受体（EGFR）的基因在人类胶质瘤中常发生扩增，该受体本身被视为将治疗药物特异性递送至脑肿瘤的潜在靶点。本研究的目的是探讨抗EGFR和EGFRvIII的嵌合单克隆抗体西妥昔单抗（IMC-C225）作为硼传递剂用于脑肿瘤中子俘获治疗（NCT）的情况。通过¹²⁵I-西妥昔单抗放射性配体结合试验测定，已转染编码EGFR基因（F98EGFR）的F98大鼠胶质瘤细胞表达1.60±0.13×10⁵个受体位点/细胞，解离常数（Ka）=1.64±0.32×10⁸ M⁻¹。转染编码EGFR突变形式（称为F98EGFRvIII胶质瘤）基因的F98细胞表达1.07±0.10×10⁵个受体位点/细胞，Ka=2.18±0.54×10⁹ M⁻¹，而F98野生型细胞（F98WT）表达的是背景水平。一种高度硼化的第五代聚酰胺-胺（PAMAM或“星爆”）树枝状大分子G5-B1100，通过异双功能试剂N-琥珀酰亚胺基-3-(2-吡啶二硫代)丙酸酯（SPDP）和N-(κ-马来酰亚胺十一酸)酰肼（KMUH）与远离西妥昔单抗抗原结合位点的寡糖部分相连。得到的生物缀合物称为C225-G5-B1100，使用Sephacryl S-300柱与未结合的树枝状大分子分离。根据硼与蛋白质的相对浓度比，每分子西妥昔单抗约有1100个硼原子，且Ka仅有轻微降低。在直接瘤内（i.t.）注射24小时后，测定C225-G5-B1100或G5-B1100在植入F98EGFR或F98WT胶质瘤的大鼠体内的定位，此时F98EGFR胶质瘤中肿瘤定位的硼为92.3±23.3微克硼/克肿瘤，而F98WT胶质瘤中为36.5±18.8微克硼/克肿瘤，正常脑中为13.4±6.1微克硼。相比之下，i.t.注射后F98EGFR胶质瘤中G5-B1100仅定位有6.7±3.6微克硼/克肿瘤，从而证明了EGFR的特异性分子靶向作用。基于这些数据，将在携带F98EGFR胶质瘤的大鼠中启动硼中子俘获治疗（BNCT）研究，以评估C225-G5-B1100治疗脑内脑肿瘤的效果。
