Wen X, Wu Q P, Lu Y, Fan Z, Charnsangavej C, Wallace S, Chow D, Li C
Department of Diagnostic Radiology and Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Bioconjug Chem. 2001 Jul-Aug;12(4):545-53. doi: 10.1021/bc0001443.
Several biological barriers, including significant liver uptake, limit the clinical application of radiolabeled antibodies in radioimmunoscintigraphy. Here, a general approach is described for radiolabeling of monoclonal antibodies conjugated with poly(ethylene glycol) (PEG). This strategy is demonstrated with C225, a monoclonal antibody directed against epidermal growth factor (EGF) receptor. We synthesized a heterofunctional PEG with one end attached to a radiometal chelator, diethylenetriaminepentaacetic acid (DTPA), and the other end to a protected thiol group, S-acetylthioacetate. After a deprotection step, the resulting DTPA-PEG-SH was conjugated to maleimide-activated C225 to yield DTPA-PEG-C225 conjugate. Characterization of DTPA-PEG-C225 with immunoprecipitation and Western blot analysis revealed that the conjugate was biologically active in binding to the EGF receptor in A431 cells. Competitive EGF receptor binding assay in MDA-MB-468 cells showed that DTPA-PEG-C225, with up to 60% of the amino groups in C225 substituted, retained 66% of C225's binding affinity. Moreover, DTPA-PEG-C225 with increasing degrees of NH(2) substitution from 20% to 70% retained the activity of C225 to induce apoptosis in DiFi cells. More importantly, DTPA-PEG-C225 demonstrated less nonspecific interaction than DTPA-C225. Pharmacokinetic analysis using (111)In-labeled compounds revealed narrower steady-state distribution of (111)In-DTPA-PEG-C225 than (111)In-DTPA-C225, probably due to reduced nonspecific binding of PEG-modified antibody to tissues. The terminal half-life (t(1/2,)(gamma)) of (111)In-DTPA-PEG-C225, 21.1 h, was shorter than that of (111)In-DTPA-C225, 52.9 h. These data suggest that (111)In-DTPA-PEG-C225 may provide better imaging characteristics than (111)In-DTPA-C225, and that using PEG as a linker between the monoclonal antibody and DTPA may be a promising strategy in optimizing the imaging characteristics of immunoscintigraphic agents.
包括肝脏显著摄取在内的多种生物屏障限制了放射性标记抗体在放射免疫闪烁成像中的临床应用。在此，描述了一种用于对与聚乙二醇（PEG）偶联的单克隆抗体进行放射性标记的通用方法。以针对表皮生长因子（EGF）受体的单克隆抗体C225为例展示了该策略。我们合成了一种异功能PEG，一端连接放射性金属螯合剂二亚乙基三胺五乙酸（DTPA），另一端连接受保护的巯基，即S - 乙酰硫代乙酸酯。经过脱保护步骤后，所得的DTPA - PEG - SH与马来酰亚胺活化的C225偶联，得到DTPA - PEG - C225偶联物。通过免疫沉淀和蛋白质印迹分析对DTPA - PEG - C225进行表征，结果表明该偶联物在与A431细胞中的EGF受体结合方面具有生物活性。在MDA - MB - 468细胞中进行的竞争性EGF受体结合试验表明，C225中高达60%的氨基被取代的DTPA - PEG - C225保留了C225 66%的结合亲和力。此外，氨基取代度从20%增加到70%的DTPA - PEG - C225保留了C225诱导DiFi细胞凋亡的活性。更重要的是，DTPA - PEG - C225的非特异性相互作用比DTPA - C225少。使用铟 - 111（¹¹¹In）标记化合物进行的药代动力学分析表明，¹¹¹In - DTPA - PEG - C225的稳态分布比¹¹¹In - DTPA - C225更窄，这可能是由于PEG修饰的抗体与组织的非特异性结合减少所致。¹¹¹In - DTPA - PEG - C225的终末半衰期（t(1/2,)(γ)）为21.1小时，短于¹¹¹In - DTPA - C225的52.9小时。这些数据表明，¹¹¹In - DTPA - PEG - C225可能比¹¹¹In - DTPA - C225具有更好的成像特性，并且使用PEG作为单克隆抗体与DTPA之间的连接体可能是优化免疫闪烁成像剂成像特性的一种有前景的策略。
