Fully automated enzymatic radiolabeling of biomolecules

项目摘要/摘要 尽管有其固有的潜力，[18F]-蛋白质放射性示踪剂在临床前和临床环境中的使用是 由于缺乏可靠的无线电标记技术而受到阻碍。我们的目标是通过开发一种全自动的 放射性标记方法是特定部位的，在温和的含水条件下快速发生，只需要 少量前体多肽，可获得高放射性标记产率。 最常见的蛋白质放射性氟化方法，N-琥珀酰亚胺4-[18F]-氟苯甲酸酯偶联为 赖氨酸残基，受限于长时间的多步骤合成，低得率，对标记位置的控制不佳( 可能导致免疫反应性降低)，以及对大量蛋白质前体的频繁需求。 其他假体小组也有报道，然而到目前为止还没有解决所有这些问题的小组。缺乏一种 最佳的、广泛适用的蛋白质放射性标记策略促使我们研究酶作为 放射性标记催化剂。 初步的概念验证数据表明，硫辛酸连接酶可以定点连接a[18F]- 假体到带有13个氨基酸受体序列的模型蛋白(‘LAP-Tag’)。当时的反应是 在中性pH和环境温度下高产率，确保充分保持蛋白质的生物活性。 在这项提案中，我们将在这些研究的基础上，开发出具有流线型假体的第二代假体 放射性合成和改善的代谢稳定性。至关重要的是，所有的放射合成步骤都将在 自动放射合成仪，以实现重复性和促进标签的即时转移 不同站点之间的协议。在特定目标1，我们将合成第二代芳基[18F]-氟化物 假体，确认其连接到模型蛋白质-LAP结构，并纯化得到的放射性示踪剂。具体而言 目标2，我们将把这个方案转移到加州大学旧金山分校，在那里放射性标记将在他们的 放射合成器。最后，将对放射性示踪剂的纯度、比活度、生物保留率进行表征 活跃性和代谢稳定性。在完成这些研究后，我们预计将拥有一个完全优化的 手头有放射性氟化方案以及我们的模型蛋白质的概念验证数据。 在这个项目的第二阶段，我们将把我们的方法应用于几种不同类别的蛋白质，包括 亲和抗体、纳米抗体、抗体和Fab抗体片段，目的是定义其全部范围和 将其业绩与该领域当前的黄金标准进行基准比较，以期生产出全面的 自动化的、商用的、基于试剂盒的蛋白质放射性氟化方法。

PROJECT SUMMARY/ABSTRACT Despite their inherent potential, the use of [18F]-protein radiotracers in both preclinical and clinical settings is hampered by a lack of robust radiolabeling techniques. We aim to address this by developing a fully automated radiolabeling methodology that is site-specific, occurs rapidly under mild aqueous conditions, and requires only small amounts of precursor peptide for high radiolabeling yields. The most frequent protein radiofluorination approach, conjugation of N-succinimidyl 4-[18F]-fluorobenzoate to lysine residues, is limited by a lengthy multi-step synthesis, low yields, poor control over labeling site (which can lead to reduced immunoreactivity), and the frequent requirement for large amounts of protein precursor. Other prosthetic groups have been reported, however to date none address all of these issues. The lack of an optimal, broadly applicable, radiolabeling strategy for proteins motivated us to investigate enzymes as radiolabeling catalysts. Preliminary, proof-of-concept data shows that the enzyme lipoic acid ligase can site-specifically ligate a [18F]- prosthetic to a model protein tagged with a 13-amino acid acceptor sequence (`LAP-tag'). The reaction was high yielding at neutral pH and ambient temperature, ensuring full retention of the protein's biological activity. In this proposal, we will build on these studies by developing a second generation prosthetic with a streamlined radiosynthesis and improved metabolic stability. Crucially, all radiosynthetic steps will be performed on an automated radiosynthesizer to enable reproducibility and to facilitate the immediate transfer of labeling protocols between different sites. In Specific Aim 1 we will synthesize the second generation aryl [18F]-fluoride prosthetic, confirm its ligation to a model protein-LAP construct, and purify the resulting radiotracer. In Specific Aim 2, we will transfer this protocol to UCSF where the radiolabeling will be reproduced on their radiosynthesizer. Finally, the radiotracer will be characterized for purity, specific activity, retention of biological activity, and metabolic stability. Upon completion of these studies we anticipate having a fully optimized radiofluorination protocol in hand along with proof-of-concept data for our model protein. In Phase II of this project, we will apply our methodology to several different classes of protein, including affibodies, nanobodies, diabodies, and Fab antibody fragments, with the goal of defining its full scope and benchmarking its performance against the current gold standard in the field in anticipation of producing a fully automated, commercial available, kit-based approach for protein radiofluorination.