Novel Reagents for Rapid and Stable Thiol-Based Bioconjugations

项目总结/摘要 三十多年来，带有马来酰亚胺的探针和硫醇之间的连接一直是生物化学的基石。 合成位点特异性标记的抗体。然而，尽管这种生物结合的方法很受欢迎， 有严重的局限性。基于马来酰亚胺的缀合物在体内显示出有限的稳定性，因为它们的琥珀酰亚胺基是聚乙二醇。 硫醚键可以经历逆迈克尔反应，其导致货物或其衍生物的解离。 与循环的生物分子交换。在核成像和放射免疫治疗的背景下，这种逆转录- 迈克尔反应可导致放射性有效载荷的释放和放射性同位素的体内放射性标记。 内源性生物分子，导致对健康组织的较高辐射剂量，降低成像对比度， 和较低的治疗比率。为了规避这一障碍，我们开发了一个模块化的，稳定的， 容易获得的苯基恶二唑基甲基砜试剂-“PODS”-作为硫醇基 生物共轭我们已经证明，PODS可以用于可重复地创建均匀的，良好的- 明确的、高度免疫反应性的和高度稳定的放射性免疫缀合物， 与通过传统的基于马来酰亚胺的方法合成的类似探针相比， 该提案的核心是对该技术的扩展和优化。我们将寻求建立 通过优化PODS-巯基连接的快速性和选择性并评估 用89 Zr、177 Lu、131 I和225 Ac标记的基于PODS的放射性免疫缀合物的体内性能。 具体目标1（SA 1）将集中在第二个- 第二代PODS试剂，总体目标是鉴定具有最有利的 稳定性、溶解性和反应性的组合。具体目标2（SA 2）将集中在以下方面的评价： 使用PODS合成的放射免疫缀合物的体内性能。最有希望的第二名- 来自SA 1的第二代PODS试剂将用于合成89 Zr-和177 Lu-标记的放射免疫缀合物， 并且将在小鼠癌症模型中评估它们的体内性能，并与 使用基于马来酰亚胺的生物缀合产生的放射性标记的抗体。具体目标3（SA 3）将侧重于 开发基于PODS的辅基，用于用131 I对抗体进行位点特异性放射性标记， 225Ac。使用这些辅基合成的131 I和225 Ac标记的抗体的体内性能 将在小鼠癌症模型中进行评估，并与使用目前的“金”合成的类似物进行比较， 标准策略。我们认为，这项建议可能会对临床护理产生重大的近期影响， 开发和验证用于合成高度稳定的放射性免疫缀合物的工具， 性能，从而改善成像协议、治疗方案和患者结果。此外，委员会认为， 我们认为，这项工作可能会从根本上改变生物共轭化学的范式， 改变了生物分子药物在实验室和临床上的合成方式。

Project Summary/Abstract For over three decades, the ligation between maleimide-bearing probes and thiols has been a cornerstone of the synthesis of site-specifically labeled antibodies. Yet despite its popularity, this approach to bioconjugation has serious limitations. Maleimide-based conjugates display limited stability in vivo because their succinimidyl thioether linkage can undergo a retro-Michael reaction that leads to the dissociation of the cargo or its exchange with circulating biomolecules. In the context of nuclear imaging and radioimmunotherapy, this retro- Michael reaction can lead to the release of the radioactive payload and the in vivo radiolabeling of endogeneous biomolecules, resulting in higher radiation doses to healthy tissues, reduced imaging contrast, and lower therapeutic ratios. In order to circumvent this obstacle, we have developed a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent — `PODS' — as a platform for thiol-based bioconjugations. We have demonstrated that PODS can be used to reproducibly create homogenous, well- defined, highly immunoreactive, and highly stable radioimmunoconjugates with far superior in vivo performance compared to analogous probes synthesized via traditional, maleimide-based approaches. This proposal is centered upon the expansion and optimization of this technology. We will seek to build upon our preliminary work by optimizing the rapidity and selectivity of the PODS-thiol ligation and evaluating the in vivo performance of PODS-based radioimmunoconjugates labeled with 89Zr, 177Lu, 131I, and 225Ac. Specific Aim 1 (SA1) will be focused on the design, synthesis, and characterization of a library of second- generation PODS reagents, with the overall goal of identifying a single construct with the most favorable combination of stability, solubility, and reactivity. Specific Aim 2 (SA2) will be centered on the evaluation of the in vivo performance of radioimmunoconjugates synthesized using PODS. The most promising second- generation PODS reagent from SA1 will be used to synthesize 89Zr- and 177Lu-labeled radioimmunoconjugates, and their in vivo performance will be assessed in mouse models of cancer and compared to that of radiolabeled antibodies created using maleimide-based bioconjugations. Specific Aim 3 (SA3) will focus on the development of PODS-based prosthetic groups for the site-specific radiolabeling of antibodies with 131I and 225Ac. The in vivo performance of 131I- and 225Ac-labeled antibodies synthesized using these prosthetic groups will be evaluated in murine models of cancer and compared to analogues synthesized using current `gold- standard' strategies. We believe that this proposal could have a significant near-term impact on clinical care by developing and validating tools for the synthesis of highly stable radioimmunoconjugates with excellent in vivo performance, thereby improving imaging protocols, treatment regimens, and patient outcomes. Furthermore, we contend that this work could have a paradigm-shifting influence on bioconjugation chemistry, fundamentally changing the way biomolecular medicines are synthesized in the laboratory and clinic.