Imaging Proteins Docked to Standard Radionuclide Module

描述（由申请人提供）：本项目的总体目标是开发一种标准化系统，用于将锝-99m（99 mTc）“装载”到蛋白质上，该蛋白质识别与人类疾病的发生和进展相关的细胞表面特异性标记物。使用多种标记物进行成像可能会导致更好地表征每个患者，因此，开发个性化的治疗方案和合理选择患者进行实验治疗。然而，用于成像的蛋白质的标记通常需要高度定制的程序，这通常导致蛋白质活性的丧失。一种有吸引力的替代方案是使用标准化的放射性标记的衔接蛋白组装靶向成像复合物，所述衔接蛋白可以与用相应的“对接标签”表达的任何靶向蛋白结合。我们最近开发了一种适配器/对接标签系统的组装复合物的靶向放射性核素成像。我们使用了一个18-127-氨基酸（aa）片段的人核糖核酸酶I（衔接蛋白）放射性标记的锝-99m（99 mTc）作为我们的标准化放射性标记的模块。然后将该模块非共价结合到由RNA酶I的1-15-aa片段（对接标签）和血管内皮因子（VEGF 121）的121- aa同种型组成的融合蛋白，形成99 mTc/VEGF成像复合物。通过这种策略，我们已经实现了小到几毫米的病变中的小鼠肿瘤新血管的选择性和特异性放射性核素成像。我们的实验表明，通过适配器/对接标签系统组装的靶向复合物可用于靶向递送显像剂，特别是用于递送99 mTc。然而，在标准化组装复合物适合临床开发之前，必须实现几个里程碑。首先，我们将开发一种在流通中稳定的适配器/对接标签系统。第二，为了提高信噪比并最大限度地减少对生物活性靶向蛋白的暴露，我们将开发一种成像有效载荷模块，其可以被放射性标记为比目前的99 mTc/蛋白制剂高10-30倍的比活性。第三，使用不同的靶向蛋白，我们将建立靶向复合物可以渗透到肿瘤中的程度。如果这些里程碑得以实现，组装的靶向成像复合物可能成为患者诊断、个体化治疗开发和监测药物治疗反应的有力工具。此外，这种方法将允许任何新发现或构建的细胞靶向蛋白快速和均匀地转化为强大的成像工具。

DESCRIPTION (provided by applicant): The overall goal of this project is to develop a standardized system for "loading" technetium-99m (99mTc) onto proteins that recognizes cell-surface specific markers associated with onset and progression of human diseases. Using multiple markers for imaging might lead to better characterization of each patient and therefore, to development of personalized treatment regiments and rational selection of patients for experimental therapeutics. However, labeling of proteins for imaging frequently requires highly customized procedures that often result in the loss of protein activity. An attractive alternative would be assembly of targeting imaging complexes using a standardized radiolabeled adapter protein that can be bound to any targeting protein expressed with a corresponding "docking tag". We have recently developed an adapter/docking tag system for assembly of complexes for targeted radionuclide imaging. We used an 18-127-amino acid (aa) fragment of human RNase I (adapter protein) radiolabeled with technetium-99m (99mTc) as our standardized radiolabeled module. This module was then noncovalently bound to a fusion protein comprised of a 1-15-aa fragment of RNAase I (docking tag) and the 121- aa isoform of vascular endothelial factor (VEGF121), forming a 99mTc/VEGF imaging complex. With this strategy we have achieved selective and specific radionuclide imaging of mouse tumor neovasculature in lesions as small as several millimeters. Our experiments suggest that targeting complexes assembled via adapter/docking tag system can be used for targeted delivery of imaging agents, and specifically for delivery of 99mTc. However, there are several milestones that have to be achieved before standardized assembled complexes are suitable for clinical development. First, we will develop an adapter/docking tag system that is stable in circulation. Second, to improve signal/noise ratio and to minimize exposure to biologically active targeting proteins, we will develop an imaging payload module that can be radiolabeled to 10-30 fold higher specific activity than current 99mTc /protein preparations. Third, using different targeting proteins we will establish to what extent targeting complexes can penetrate into tumors. If these milestones are achieved, assembled targeting imaging complexes might become a powerful tool in diagnostics of patients, development of individualized treatment, and monitoring responses to drug treatment. Furthermore, this approach would allow rapid and uniform conversion of any newly discovered or constructed cell-targeting protein into a powerful imaging tool.