In vivo metal-free cycloaddition chemistry driven pretargeted cancer radiotherapy

描述(由申请人提供)：拟议研究的目标是为癌症放射免疫治疗(RIT)开发新的无金属“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，克服其在体内相对缓慢的药代动力学特性，并产生较高的肿瘤对正常组织的定位。将使用单抗CC49作为靶向剂。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上过度表达。CC49单抗将被反式环辛烯(TCO)衍生，并在以两步预靶向方法注射治疗性探针之前用于靶向肿瘤。双(吡啶基)四嗪(四嗪)共轭金属将被放射性标记~(212)Bi或其母体~(212Pb)或~(177)Lu，并在注射肿瘤亲和抗体后数小时至数天内注射。在体内，肿瘤靶向抗体上的TCO将与放射性标记的螯合剂-四嗪部分发生快速且高度特异的环加成反应，产生共价结合物。未反应的放射性标记的螯合剂-四嗪将迅速从体内清除，导致肿瘤组织到正常组织的高放射性定位。拟议研究的目的如下。前置靶向化学进展，包括第二代环辛烯、螯合剂-四嗪共轭化合物和清洗剂的合成和表征。目的2，放射化学和偶联研究，将重点放在将被~(212)Bi、~(212)Pb和~(177)Lu辐射修饰的螯合剂-四嗪偶联物的放化标记效率和放化稳定性。TCO-CC49和标记的四嗪螯合剂的生物活性将用表达TAG72的细胞株进行评估。目的3，评价新的前靶向成分，将优化肿瘤的剂量，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。目的4、治疗研究，是否会采取最佳组合？然后呢？发射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，目标是使用预靶向？RIT和五倍的目标改进？好的。最后一个里程碑将是证明预靶向治疗研究将在统计上显着延长小鼠结肠癌模型的存活率。这种新型体内环加成化学在癌症放射治疗中的应用具有很高的创新性。由于其前所未有的反应活性，体内烯烃与四氮杂环加成反应是第一个在临床相关条件下被有效利用的此类反应，并首次有可能将这种类型的有机化学推广到人类身上。该项目将是TCO-四氮杂环加成化学与预靶向粒子放射治疗在体内的第一次应用。

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.