Stéen E Johanna L, Jørgensen Jesper T, Denk Christoph, Battisti Umberto M, Nørregaard Kamilla, Edem Patricia E, Bratteby Klas, Shalgunov Vladimir, Wilkovitsch Martin, Svatunek Dennis, Poulie Christian B M, Hvass Lars, Simón Marina, Wanek Thomas, Rossin Raffaella, Robillard Marc, Kristensen Jesper L, Mikula Hannes, Kjaer Andreas, Herth Matthias M
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.
Department of Clinical Physiology, Nuclear Medicine & PET, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark.
ACS Pharmacol Transl Sci. 2021 Feb 16;4(2):824-833. doi: 10.1021/acsptsci.1c00007. eCollection 2021 Apr 9.
The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of pretargeted chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted -cyclooctene (TCO)-modified antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their performance. In particular, high rate constants (>50 000 M s) for the reaction with TCO and low calculated log values (below -3) of the tetrazine were identified as strong indicators for successful pretargeting. Radiolabeling gave access to a set of selected F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for application and will thereby assist the clinical translation of bioorthogonal pretargeting.
用于连接和裂解的高选择性且快速的生物相容性反应的发展，为预靶向化学的新诊断和治疗应用铺平了道路。生物正交预靶向的概念引起了相当大的兴趣，特别是在放射性核素和药物的靶向递送方面。在核医学中，与传统方法相比，预靶向可以在早期时间点提供更高的靶本底比。这减少了对健康组织的辐射负担，并且根据所选的放射性核素，能够实现更好的成像对比度或更高的治疗效率。此外，生物正交触发的预靶向抗体 - 药物偶联物的裂解代表了一种实现控释和局部提高药物浓度的新兴策略。在过去十年中，生物正交反应的工具箱有了显著扩展，四嗪连接反应是最快且最通用的化学方法之一。然而，该领域的进展在很大程度上依赖于（放射性）标记化合物的开发和评估，这阻碍了使用化合物库进行系统研究。因此，由于对结构参数对连接性能影响的理解有限，四嗪探针和触发剂的合理设计受到了阻碍。在这项工作中，我们描述了一种预靶向阻断试验的开发，该试验允许研究45种未标记四嗪的结构多样文库的命运，以及它们在荷瘤小鼠中与预靶向的反式环辛烯（TCO）修饰抗体到达并反应的能力。这项研究使我们能够评估四嗪的点击反应性和亲脂性与其性能之间的相关性。特别是，与TCO反应的高反应速率常数（>50000 M⁻¹s⁻¹）和四嗪的低计算log P值（低于 -3）被确定为成功预靶向的有力指标。放射性标记得到了一组选定的¹⁸F标记四嗪，包括高反应性支架，这些用于预靶向PET成像研究以确认阻断研究的结果。因此，这些见解能够实现用于应用的四嗪探针的合理设计，并将有助于生物正交预靶向的临床转化。
