The Tetrazine Ligation for Efficient 18F Labeling and Pretargeted Imaging/Radiotherapy of Cancer

描述（由申请人提供）：提出了一种简单、快速和通用的策略，用于将放射性核素掺入生物分子中，用于癌症成像和预靶向免疫治疗。该策略基于Fox实验室开发的生物共轭反应：四嗪和反式环辛烯（TCO）之间的反应。“四嗪-TCO连接”的快速动力学（k2 e 250，000 L*mol/s-1）使得能够在几分钟内在低微摩尔浓度下快速反应，并且没有过量的任一反应物。这种快速反应性为蛋白质和抗体修饰以及在体内进行反应提供了前所未有的机会。该提案涉及以下方面的合作努力： 南加州大学（USC）的分子成像中心（MIC）和福克斯集团（UD）。本申请的一个目的是使用四嗪连接来开发用于构建具有用于癌症成像的高比活性的18F标记蛋白的有效方法。此外，我们还将开发一种新的预靶向成像和放射免疫治疗方法，该方法将允许用成像/治疗同位素对单克隆抗体（mAb）进行通用和直接的标记，而不会对正常器官造成严重的辐射暴露。为了实现这些目标，我们将首先开发基于四嗪-TCO连接的具有快速动力学和稳健的体内稳定性的生物缀合反应。由于四嗪-TCO的快速动力学 连接能够在几分钟内在低微摩尔浓度下快速反应，并且没有过量的反应物，我们将把这种反应转化为有效的方法，可以用于构建具有高比活性的18F标记蛋白。最后，我们还将建立一种基于四嗪-TCO连接和抗EphB 4抗体的化学预靶向方法，用于癌症的预靶向成像/放射免疫治疗。总之，在本申请中，我们正在开发用18F标记蛋白质的有效方法，以及可用于乳腺癌的预靶向成像和放射免疫治疗的化学预靶向方法。将用EphB 4抗体、F（ab '）2和Fab测试这些方法的可行性。这些新的成像和治疗方法的成功可以使乳腺癌诊断成为可能，使直接监测对治疗干预的反应成为可能，并可能显着提高乳腺癌的治疗效果。此外，这些新开发的方法可能在许多其他癌症类型中具有重要的应用，从而对大量癌症患者具有显著的临床影响。

DESCRIPTION (provided by applicant): Proposed is the development of a simple, fast and universal strategy for incorporating radionuclides into biomolecules for applications for cancer imaging and pretargeted immunotherapy. The strategy is based on a bioconjugation reaction developed in the Fox laboratories: the reaction between tetrazines and trans-cyclooctenes (TCO). The fast kinetics of the 'tetrazine-TCO ligation' (k2 e 250,000 L*mol/s-1) enable fast reactivity at low micromolar concentrations within minutes and without an excess of either reactant. This fast reactivity provides unprecedented opportunities for protein and antibody modification and for performing reactions in vivo. This proposal involves collaborative effort from the Molecular Imaging Center (MIC) at University of Southern California (USC) and the Fox group (UD). An objective of this application is to use the tetrazine ligation to develop efficient methods for the construction of 18F labeled proteins with high specific activity for cancer imaging. Moreover, we will also develop a novel pretargeted imaging and radioimmunotherapy method that will allow for universal and straightforward tagging of monoclonal antibodies (mAbs) with an imaging/therapeutic isotope without severe radiation exposure towards normal organs. To achieve these objectives, we will first develop bioconjugation reactions with fast kinetics and robust in vivo stability based on the tetrazine-TCO ligation. As the fast kinetics of tetrazine-TCO ligation enable fast reactivity at low micromolar concentrations within minutes and without an excess of either reactant, we will transform this reaction into efficient methods that could be used for the construction of 18F labeled proteins with high specific activity. At last, we will als establish a chemical pretargeting approach that for pretargeted imaging/radioimmunotherapy of cancer based on the tetrazine-TCO ligation and anti-EphB4 antibody. In summary, in this application, we are developing efficient methods for proteins labeling with 18F, and a chemical pretargeting approach that could be used for pretargeted imaging and radioimmunotherapy of breast cancer. The feasibility of these approaches will be tested with EphB4 antibody, F(ab')2, and Fab. The success of these novel imaging and therapy approached could enable breast cancer diagnosis, make possible direct monitoring of responses to therapeutic interventions, and may significantly improve breast cancer treatment efficacy. Moreover, these newly developed approaches could have important applications in many other cancer types, and thus have a significant clinical impact on a very large number of cancer patients