Fully automated enzymatic radiolabeling of biomolecules

项目总结/摘要 尽管[18F]-蛋白放射性示踪剂具有固有的潜力，但在临床前和临床环境中使用[18F]-蛋白放射性示踪剂是不可能的。 由于缺乏可靠的放射性标记技术而受阻。我们的目标是通过开发一个完全自动化的 放射性标记方法是位点特异性的，在温和的水性条件下快速发生，并且仅需要 少量前体肽用于高放射性标记产率。 最常见的蛋白质放射性标记方法是将N-琥珀酰亚胺基4-[18F]-氟苯甲酸酯缀合至 赖氨酸残基，受到冗长的多步合成、低产率、对标记位点（其 可导致免疫反应性降低），以及经常需要大量的蛋白质前体。 据报道，还有其他假体组，但迄今为止没有一个能解决所有这些问题。缺乏一个 最佳的，广泛适用的，蛋白质的放射性标记策略促使我们研究酶， 放射性标记催化剂。 初步的概念验证数据显示，酶硫辛酸连接酶可以位点特异性地连接[18F]- 与用13个氨基酸的受体序列（“LAP-标签”）标记的模型蛋白质是假体。将反应 在中性pH和环境温度下的高产率，确保蛋白质的生物活性的完全保留。 在本提案中，我们将在这些研究的基础上开发第二代假肢， 放射合成和改善的代谢稳定性。至关重要的是，所有放射性合成步骤都将在 自动放射合成器，可实现再现性并促进标签的立即转移 不同站点之间的协议。在具体目标1中，我们将合成第二代芳基[18F]-氟化物 修复，确认其连接到模型蛋白质-β-内酰胺构建体，并纯化所得的放射性示踪剂。在特定 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 无线电合成器最后，将对放射性示踪剂的纯度、比活度、生物活性保留率和生物活性进行表征。 活性和代谢稳定性。在完成这些研究后，我们预计将有一个完全优化的 放射性同位素方案与我们的模型蛋白的概念验证数据一起沿着。 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型的蛋白质，包括 抗体、纳米抗体、双抗体和Fab抗体片段，目的是定义其全部范围和 以该领域目前的黄金标准为基准， 用于蛋白质放射性定量的自动化的、商业上可获得的、基于试剂盒的方法。

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.