Imaging Proteins Docked to Standard Radionuclide Module

描述（由申请人提供）：该项目的总体目标是开发一个标准化系统，用于将锝-99m （99mTc）“装载”到蛋白质上，以识别与人类疾病发病和进展相关的细胞表面特异性标记。使用多种标记物进行成像可能会更好地描述每个患者的特征，从而开发个性化的治疗方案，并合理选择患者进行实验性治疗。然而，用于成像的蛋白质标记经常需要高度定制的程序，这通常会导致蛋白质活性的丧失。一个有吸引力的替代方案是使用一种标准化的放射性标记的适配蛋白组装靶向成像复合物，这种适配蛋白可以与任何带有相应“对接标签”表达的靶向蛋白结合。我们最近开发了一种适配器/对接标签系统，用于组装用于靶向放射性核素成像的复合物。我们使用用锝-99m （99mTc）放射性标记的人类RNase I（适配蛋白）的18-127个氨基酸（aa）片段作为我们的标准化放射性标记模块。然后将该模块非共价结合到由RNAase I的1-15-aa片段（对接标签）和血管内皮因子（VEGF121）的121- aa异构体组成的融合蛋白上，形成99mTc/VEGF成像复合物。利用这种策略，我们已经在小到几毫米的病变中实现了小鼠肿瘤新生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显像剂的靶向递送，特别是99mTc的递送。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，我们将开发一个在流通中稳定的适配器/停泊标签系统。其次，为了提高信噪比并最大限度地减少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，其放射性标记比目前的99mTc /蛋白制剂高10-30倍。第三，使用不同的靶向蛋白，我们将确定靶向复合物在多大程度上可以渗透到肿瘤中。如果达到这些里程碑，组装靶向成像复合物可能成为诊断患者、开发个体化治疗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而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.