Novel Reagents for Rapid and Stable Thiol-Based Bioconjugations

项目摘要/摘要 三十多年来，含马来酰亚胺的探针与硫醇之间的连接一直是 定点标记抗体的合成。然而，尽管它很受欢迎，这种生物结合的方法 有严重的局限性。基于马来酰亚胺的偶联物在体内表现出有限的稳定性，因为它们的琥珀酰亚胺 硫醚键可以进行逆迈克尔反应，从而导致货物或其 与循环生物分子交换。在核成像和放射免疫治疗的背景下，这种追溯- Michael反应可导致放射性有效载荷的释放和体内放射性标记 内源性生物分子，导致健康组织受到更高的辐射剂量，成像对比度降低， 和较低的治疗比率。为了绕过这一障碍，我们开发了一种模块化、稳定和 易于获得的苯并二唑甲基磺酸试剂--‘Pods’--作为硫醇类化合物的平台 生物结合物。我们已经证明，豆荚可以用来重复地创造均匀的、良好的- 明确、高度免疫活性和高度稳定的放射免疫结合物，在体内表现出极大的优势 与传统的以马来酰亚胺为基础的方法合成的类似探针相比，其性能更佳。 这项建议是围绕这项技术的扩展和优化而提出的。我们将努力建设 在前期工作的基础上，通过优化豆荚-硫醇连接的快速性和选择性并进行评价 ~(89)Zr、~(177)Lu、~(131)I和~(225)Ac标记的豆荚类放射免疫结合物的体内性能 具体目标1(SA1)将集中在设计、合成和表征第二个- 一代豆荚试剂，总体目标是确定具有最有利的单一结构 稳定性、溶解性和反应性的结合。具体目标2(SA2)将集中于对 利用豆荚合成的放射免疫结合物的体内性能。最有希望的第二个- 从SA1产生的豆荚试剂将用于合成89Zr和177Lu标记的放射免疫结合物， 它们在体内的表现将在小鼠癌症模型中进行评估，并与 使用以马来酰亚胺为基础的生物结合物创建的放射性标记抗体。具体目标3(SA3)将重点放在 ~(131)I-~(131)I和~(131)I-~(131)I标记抗体的豆荚基团的研制 225 Ac。~(131)I和~(225)Ac标记抗体在体内的应用 将在小鼠癌症模型中进行评估，并与使用目前的“金-金”合成的类似物进行比较。 标准的战略。我们认为，这项提议可能会对临床护理产生重大的近期影响，具体方式如下 体内高稳定性放射性免疫结合物合成工具的开发与验证 性能，从而改善成像方案、治疗方案和患者结果。此外， 我们认为，这项工作可能会从根本上改变生物结合化学的范式。 改变生物分子药物在实验室和临床合成的方式。

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.