Site Selection: a Case Study in the Identification of Optimal Cysteine Engineered Antibody Drug Conjugates
Site Selection: a Case Study in the Identification of Optimal Cysteine Engineered Antibody Drug Conjugates
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DOI：
10.1208/s12248-017-0083-7
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发表时间：
2017-07-01
期刊：
影响因子：
4.5
通讯作者：
Tchistiakova, Lioudmila
中科院分区：
文献类型：
--
作者：
Tumey, L. Nathan;Li, Fengping;Tchistiakova, Lioudmila
随着抗体药物偶联物（ADC）领域不断转向位点特异性偶联技术，人们越来越需要了解偶联物的位点如何影响ADC的生物物理和生物学特性。为了满足这一需求，我们精心挑选了一系列工程半胱氨酸adc，并系统地评估了它们的效力、稳定性和PK暴露。偶联位点对adc的热稳定性和体外细胞毒性没有显著影响。然而，我们证明了组织蛋白酶介导的连接体切割速率严重依赖于位点，并与ADC疏水性密切相关，从而证实了最近关于这一现象的其他报道。有趣的是，具有高组织蛋白酶介导的连接物裂解率的偶联物并没有表现出血浆稳定性的下降。事实上，血浆不稳定的主要来源是逆转录michael介导的解偶联。这一过程被琥珀酰亚胺水解所阻碍，因此，我们进行了一系列突变实验，证明位于共轭位点附近的基本残基可能是琥珀酰亚胺环打开的重要驱动因素。最后，我们发现大鼠总抗体PK暴露与ADC疏水性呈松散相关。我们希望这些观察结果将有助于ADC社区建立“设计规则”，从而能够更有效地执行下一代ADC发现程序。
As the antibody drug conjugate (ADC) community continues to shift towards site-specific conjugation technology, there is a growing need to understand how the site of conjugation impacts the biophysical and biological properties of an ADC. In order to address this need, we prepared a carefully selected series of engineered cysteine ADCs and proceeded to systematically evaluate their potency, stability, and PK exposure. The site of conjugation did not have a significant influence on the thermal stability and in vitro cytotoxicity of the ADCs. However, we demonstrate that the rate of cathepsin-mediated linker cleavage is heavily dependent upon site and is closely correlated with ADC hydrophobicity, thus confirming other recent reports of this phenomenon. Interestingly, conjugates with high rates of cathepsin-mediated linker cleavage did not exhibit decreased plasma stability. In fact, the major source of plasma instability was shown to be retro-Michael mediated deconjugation. This process is known to be impeded by succinimide hydrolysis, and thus, we undertook a series of mutational experiments demonstrating that basic residues located nearby the site of conjugation can be a significant driver of succinimide ring opening. Finally, we show that total antibody PK exposure in rat was loosely correlated with ADC hydrophobicity. It is our hope that these observations will help the ADC community to build "design rules" that will enable more efficient prosecution of next-generation ADC discovery programs.