尽管它们在前药应用中的价值,由于人酯酶的普遍存在和混杂性质,因此通常避免了在抗体-药物-缀合物(ADC)有效载荷和接头中使用酯.ADC通常具有长的循环半衰期(3-7天),这使得它们对酯酶介导的代谢敏感。此外,在ADC内化后,溶酶体和细胞溶质酯酶是否会裂解含酯的接头,目前尚不清楚.由于我们对靶向递送免疫调节剂的兴趣,我们的团队最近准备了一系列酯连接的地塞米松ADC.在这里,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,酯在细胞摄取后选择性地但低效地裂解,可能是细胞溶质酯酶。溶酶体分解代谢研究表明,尽管有很强的蛋白水解活性,在溶酶体中很少发生含酯接头的裂解。然而,带有酯连接的有效载荷的ADC在各种免疫抑制测定中具有活性,表明胞质分裂正在发生。这通过细胞裂解后有效负载的LCMS定量得到证实。最后,在小鼠和人血浆中评价酯键的稳定性。我们发现,类似于其他报道,对切割有显著的位点依赖性。酯附着在高度暴露的地方,例如443C,在血浆中迅速裂解,而酯在更受阻的部位,在334C,不是。一起,这些结果有助于解开酯掺入ADC接头的复杂性,并为它们在ADC应用中的应用铺平了道路。
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.