In vivo metal-free cycloaddition chemistry driven pretargeted cancer radiotherapy

描述（由申请人提供）：拟议研究的目标是开发用于癌症放射免疫治疗（RIT）的新型无金属“点击化学”预靶向方法。据推测，新的体内环加成化学将极大地促进预靶向抗体作为癌症治疗药物的使用，克服其相对缓慢的体内药代动力学特性，并使肿瘤高度定位于正常组织。单克隆抗体（mAb） CC49将被用作靶向剂。CC49结合tag72粘蛋白抗原，该抗原在包括结肠癌在内的许多癌症中过表达。CC49单抗将由反式环烯（TCO）衍生，并在注射治疗探针之前通过两步预靶向方法靶向肿瘤。双（吡啶基）四嗪（四嗪）共轭金属将用？-发射极212Bi或其母体212Pb或？-发射器177Lu，并在注射后数小时至数天内注射抗肿瘤抗体。在体内，肿瘤靶向抗体上的TCO与放射性标记螯合物四氮的四氮部分之间会发生快速和高度特异性的环加成反应，产生共价偶联物。未反应的放射性标记螯合剂四氮会迅速从体内清除，导致肿瘤对正常组织定位的高放射性。本研究的目的如下：目标1，预靶向化学的发展，包括第二代环烯、螯合物-四嗪缀合物和清除剂的合成和表征。目标2，放射化学和偶联研究，将重点关注螯合剂-四嗪偶联物的放射化学标记效率和放射化学稳定性，这些螯合剂-四嗪偶联物将被212Bi， 212Pb和177Lu放射性标记。TCO-CC49和标记的四氮螯合剂的生物活性将在表达TAG72的细胞株上进行评估。目的3，评估新的预靶向成分，将优化对肿瘤的剂量，同时减少对正常组织的剂量，包括新开发的四嗪探针的血液动力学和生物分布。Aim4，治疗研究，将采取最好的组合？然后呢?发射器预靶向分子并检测其在结肠癌异种移植小鼠模型中的治疗效果。该项目的里程碑包括将体内环加成反应提高10倍，目标是使用预先靶向的？RIT和5倍的目标？罗切斯特理工学院的航拍。最后一个里程碑将是证明预靶向治疗研究将在统计学上显著延长小鼠结肠癌模型的生存时间。这种新的体内环加成化学在癌症放疗的预靶向方法中的应用是高度创新的。烯烃与四嗪在体内的环加成反应由于其前所未有的反应性，是第一次在体内有效地用于临床相关条件，并且第一次有可能将这种类型的有机化学扩展到人类。本项目将首次在体内应用tco -四嗪环加成化学与预靶向？粒子放射治疗。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop novel metal-free "click chemistry" pretargeted approaches for cancer radioimmunotherapy (RIT). It is hypothesized that novel in vivo cycloaddition chemistry will greatly facilitate the use of pretargeted antibodies as cancer treatment agents, overcoming their relatively slow in vivo pharmacokinetic properties and yielding high tumor to normal tissue localization. The monoclonal antibody (mAb) CC49 will be used as the targeting agent. CC49 binds the-TAG72 mucin antigen that is over expressed on a number of cancers including colon carcinomas. The CC49 mAb will be derivatized with trans-cyclooctene (TCO) and used to target the tumor prior to injection of the therapeutic probe in a 2-step pretargeting approach. A bis(pyridinyl)tetrazine (tetrazine) conjugated metal will be radiolabeled with the ?-emitter 212Bi or its parent 212Pb or the ?-emitter 177Lu and injected hours to a few days post injection of the tumor-avid antibody. In vivo, a rapid and highly specific cycloaddition reaction will occur between the TCO on the tumor- targeted antibody and the tetrazine moiety of the radiolabeled chelator-tetrazine yielding a covalent conjugate. Unreacted radiolabeled chelator-tetrazine will be rapidly cleared from the body resulting in a high tumor to normal tissue localization of radioactivity. The aims of the proposed research are as follows. Aim1, pretargeting chemistry development, which encompassed second generation cyclooctene, chelator-tetrazine conjugate and clearing agent synthesis and characterization. Aim 2, radiochemistry and conjugation studies, will focus on the radiochemical labeling efficiencies and radiochemical stabilities of the chelator-tetrazine conjugates that will be radiolabled with 212Bi, 212Pb and 177Lu. The bioacitivties of TCO-CC49 and the labeled tetrazine-chelators will be evaluated with TAG72 expressing cell lines. Aim 3, evaluation of new pretargeting components, will optimize the dose to the tumor while reducing the dose to normal tissues, including blood kinetics and biodistribution of newly developed tetrazine probes. Aim4, therapy studies, will take the best combination of ? and ? emitter pretargeting molecules and examine their therapeutic efficacy in a colon cancer xenograft mouse model. Milestones for the project include, improving in vivo cycloaddition reaction by 10 fold, targeting a greater that five-fold improvement in dose to tumor versus normal tissue using pretargeted ? RIT and a five-fold improvement in targeted ? RIT. The final milestone will be to demonstrate that pretargeted therapy studies will yield statistically significantly extension in survival in a mouse colon cancer model. This application of novel in vivo cycloaddition chemistry in a pretargeted approach for cancer radiotherapy is highly innovative. The cycloaddition reaction of olefins with tetrazines in vivo is the first of its kind to be used effectively at clincally relevant conditions in vivo due to its unprecedented reactivity, and for the first time has the potential to extend this type of organic chemistry in to man. This project would be the first of it kind in vivo application of TCO-tetrazine cycloaddition chemistry with pretargeted ?-particle radiotherapy.