Berndorff Dietmar, Borkowski Sandra, Moosmayer Dieter, Viti Francesca, Müller-Tiemann Beate, Sieger Stephanie, Friebe Matthias, Hilger Christoph S, Zardi Luciano, Neri Dario, Dinkelborg Ludger M
Research Laboratories, Schering AG, Berlin, Germany.
J Nucl Med. 2006 Oct;47(10):1707-16.
The aim of this study was to target the angiogenesis-associated extracellular matrix protein ED-B fibronectin for molecular imaging of solid tumors. Recombinant and chemically modified derivatives of the single-chain antibody fragment (scFv) L19, capable of being labeled with 99mTc, were synthesized and radiolabeled. The resulting compounds 99mTc-AP39, 99mTc-L19-His, and 99mTc-L19-Hi20 were assessed for their imaging properties in vivo.
L19 was genetically modified by inserting either the (Gly)3-Cys-Ala (AP39) or a (His)6 tag (L19-His) sequence at the C-terminal end. Chemical modifications were performed by conjugating the bifunctional chelator Hi20 (L19-Hi20) at epsilon-Lys-NH2 residues of the molecule to allow for a direct chelator-based labeling with 99mTc. Tumor-targeting, pharmacokinetic, and scintigraphic imaging properties of the radiolabeled scFvs were evaluated in nude mice bearing murine F9 teratocarcinoma.
99mTc labeling of the L19 derivatives yielded radiochemically pure proteins maintaining high immunoreactivity to ED-B fibronectin, as measured by affinity chromatography. Size-exclusion high-performance liquid chromatographic analysis of labeled L19 derivatives demonstrated either dimeric species (L19-His) or a mixture of predominantly associative dimeric and monomeric species (AP39, L19-Hi20). 99mTc-AP39 showed the most favorable biodistribution and imaging properties with high and fast tumor uptake (8.3 percentage injected dose per gram at 3 h after injection), rapid blood clearance and renal excretion, leading to high signal-to-noise ratios (tumor-to-blood ratio of 6.4 at 3 h after injection), and excellent planar scintigraphy in vivo.
ED-B fibronectin can be efficiently targeted by 99mTc-AP39 and scintigraphically visualized in tumor-bearing mice, providing a potentially useful clinical tool for imaging of angiogenesis-associated ED-B fibronectin-expressing human tumors.
本研究的目的是针对与血管生成相关的细胞外基质蛋白ED-B纤连蛋白进行实体瘤的分子成像。合成了能够用99mTc标记的单链抗体片段（scFv）L19的重组和化学修饰衍生物，并进行放射性标记。对所得化合物99mTc-AP39、99mTc-L19-His和99mTc-L19-Hi20在体内的成像特性进行了评估。
通过在C末端插入（Gly）3-Cys-Ala（AP39）或（His）6标签（L19-His）序列对L19进行基因改造。通过将双功能螯合剂Hi20（L19-Hi20）与分子的ε-Lys-NH2残基偶联进行化学修饰，以便直接用99mTc进行基于螯合剂的标记。在携带鼠F9畸胎癌的裸鼠中评估放射性标记的scFv的肿瘤靶向、药代动力学和闪烁成像特性。
L19衍生物的99mTc标记产生了放射化学纯的蛋白质，通过亲和色谱法测量，其对ED-B纤连蛋白保持高免疫反应性。标记的L19衍生物的尺寸排阻高效液相色谱分析显示为二聚体物种（L19-His）或主要为缔合二聚体和单体物种的混合物（AP39、L19-Hi20）。99mTc-AP39显示出最有利的生物分布和成像特性，具有高且快速的肿瘤摄取（注射后3小时为每克注射剂量的8.3%）、快速的血液清除和肾脏排泄，导致高信噪比（注射后3小时肿瘤与血液的比率为6.4），并且在体内具有出色的平面闪烁成像。
99mTc-AP39可以有效地靶向ED-B纤连蛋白，并在荷瘤小鼠中通过闪烁成像可视化，为血管生成相关的表达ED-B纤连蛋白的人类肿瘤成像提供了一种潜在有用的临床工具。
