In spite of their value in prodrug applications, the use of esters in antibody-drug-conjugate (ADC) payloads and linkers has generally been avoided due to the ubiquitous and promiscuous nature of human esterases. ADCs generally have a long circulating half life (3–7 days) that makes them susceptible to esterase-mediated metabolism. Moreover, it is largely unclear whether lysosomal and cytosolic esterases cleave ester-containing linkers upon ADC internalization. Due to our interest in the targeted delivery of immune-modulators, our team has recently prepared a series of ester-linked dexamethasone ADCs. Herein, we report our studies of the functional activity of these ADCs, with a particular focus on their catabolism in various biological milieu. We found that esters are selectively but inefficiently cleaved upon cellular uptake, likely by cytosolic esterases. Lysosomal catabolism studies indicate that, in spite of the strong proteolytic activity, very little cleavage of ester-containing linkers occurs in the lysosome. However, ADCs bearing the ester-linked payloads are active in various immune-suppressive assays, suggesting that cytosolic cleavage is taking place. This was confirmed through LCMS quantitation of the payload following cell lysis. Finally, the stability of the ester linkage was evaluated in mouse and human plasma. We found, similar to other reports, there is a significant site-dependence on the cleavage. Esters attached at highly exposed sites, such as 443C, were rapidly cleaved in plasma while esters at more hindered sites, such at 334C, were not. Together, these results help to unravel the complexities of ester-incorporation into ADC linkers and pave a path forward for their utility in ADC applications.

尽管它们在前药应用中具有价值，但由于人类酯酶的普遍存在和混杂性，通常避免在抗体-药物偶联物 (ADC) 有效载荷和接头中使用酯。ADC 通常具有较长的循环半衰期（3-7 天），这使得它们易受酯酶介导的代谢影响。此外，在很大程度上不清楚溶酶体和胞质酯酶是否会在 ADC 内化后切割含酯接头。由于我们对免疫调节剂的靶向递送感兴趣，我们的团队最近准备了一系列酯联地塞米松 ADC。在此，我们报告了我们对这些 ADC 功能活性的研究，特别关注它们在各种生物环境中的分解代谢。我们发现酯类在细胞摄取时被选择性但低效地切割，可能是通过胞质酯酶。溶酶体分解代谢研究表明，尽管具有很强的蛋白水解活性，但在溶酶体中很少发生含酯接头的裂解。然而，带有酯连接有效载荷的 ADC 在各种免疫抑制试验中都很活跃，表明细胞溶质裂解正在发生。这通过细胞裂解后有效载荷的 LCMS 定量得到证实。最后，在小鼠和人血浆中评估了酯键的稳定性。我们发现，与其他报告类似，裂解具有显着的位点依赖性。附着在高暴露位点（如 443C）的酯在血浆中迅速裂解，而位阻较大的位点（如 334C）的酯则不会。一起，