Davydova Maria, Dewaele Le Roi Guillaume, Adumeau Pierre, Zeglis Brian M
Department of Chemistry, Hunter College of the City University of New York.
Department of Chemistry, Hunter College of the City University of New York; Ph.D. Program in Chemistry, Graduate Center of the City University of New York.
J Vis Exp. 2019 Mar 6(145). doi: 10.3791/59063.
Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent - dubbed 'PODS' - as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.
带有马来酰亚胺的双功能探针已被用于对生物分子（尤其是抗体）中的硫醇进行位点选择性修饰数十年。然而，基于马来酰亚胺的缀合物在体内显示出有限的稳定性，因为琥珀酰亚胺硫醚键可发生逆迈克尔反应。当然，这可能导致放射性载荷的释放或其与循环中含硫醇生物分子的交换。这两个过程都可能导致健康器官中的活性浓度升高以及靶组织中的活性浓度降低，从而导致成像对比度降低和治疗比率降低。2018年，我们报道了一种模块化、稳定且易于获得的苯基恶二唑基甲基砜试剂——称为“PODS”——作为基于硫醇的生物缀合平台。我们已经清楚地证明，基于PODS的位点选择性生物缀合可重复且稳健地产生均匀、明确、高度免疫反应性和高度稳定的放射免疫缀合物。此外，在结直肠癌小鼠模型中的临床前实验表明，与通过基于马来酰亚胺的缀合合成的放射性标记抗体相比，这些位点选择性标记的放射免疫缀合物在体内表现出远 superior 的性能。在本方案中，我们将描述PODS的四步合成、普遍存在的螯合剂DOTA的带有双功能PODS的变体（PODS-DOTA）的创建以及PODS-DOTA与靶向HER2的抗体曲妥珠单抗的缀合。
