Late Stage Pharmaceutical Development, Genentech, South San Francisco, California 94080.
Protein Analytical Chemistry, Genentech, South San Francisco, California 94080.
J Pharm Sci. 2019 Jan;108(1):133-141. doi: 10.1016/j.xphs.2018.10.063. Epub 2018 Nov 17.
A new class of highly potent biopharmaceutical drugs, antibody-drug conjugates (ADCs), has been proven to be clinically effective to treat oncologic diseases. ADCs contain 3 major components: the monoclonal antibody, cytotoxic drug, and chemical linker. THIOMAB™ drug conjugates and interchain-cysteine ADCs are common ADC platforms that apply thiol-maleimide chemistry via Michael addition to conjugate linker-drugs to cysteine residues. However, the resulting succinimide ring in the linker is susceptible to ring-opening reactions via hydrolysis, especially at high pH and elevated temperatures. Once the succinimide ring is opened, in vivo stability of the ADCs can be changed and the therapeutic activity will be altered. In this study, we investigated the impact of conjugation sites on succinimide ring opening for ADCs. A new methodology based on imaged capillary isoelectric focusing was developed to monitor the formation of succinimide ring-opened products. In addition, a reverse-phase high-performance liquid chromatography method was used to monitor site-specific ring-opening reactions. Our data confirmed that succinimide ring-opening rates in ADCs are conjugation-site dependent. With a good understanding of the conjugation site impact on final product's stability, it is potentially feasible to modify ring-opening rates in vitro to achieve desirable in vivo stability and biological activity.
一类新型的高效生物制药药物，抗体药物偶联物（ADC），已被证明在治疗肿瘤疾病方面具有临床疗效。ADC 包含 3 个主要成分：单克隆抗体、细胞毒性药物和化学连接子。THIOMAB™ 药物偶联物和链间半胱氨酸 ADC 是常见的 ADC 平台，它们通过迈克尔加成反应将连接子-药物与半胱氨酸残基连接，应用巯基-马来酰亚胺化学。然而，连接子中的琥珀酰亚胺环容易通过水解发生开环反应，特别是在高 pH 值和高温下。一旦琥珀酰亚胺环打开，ADC 的体内稳定性就会发生变化，治疗活性也会发生改变。在这项研究中，我们研究了缀合部位对半琥珀酰亚胺开环 ADC 的影响。开发了一种基于成像毛细管等电聚焦的新方法来监测琥珀酰亚胺开环产物的形成。此外，还使用反相高效液相色谱法监测特定部位的开环反应。我们的数据证实，ADC 中的琥珀酰亚胺开环速率与缀合部位有关。通过很好地了解缀合部位对最终产物稳定性的影响，有可能在体外改变开环速率，以达到理想的体内稳定性和生物活性。
