In silico screening of toxin payloads typically employed in ADCs revealed a wide range of hydrophobicities and sizes as measured by logP and topological polar surface area (tPSA) values. These descriptors were used to identify three nontoxic surrogate payloads that encompass the range of hydrophobicity defined by the ADC toxin training set. The uniform drug to antibody ratio (DAR) ADCs were prepared for each surrogate payload by conjugation to the interchain cysteine residues of a model IgG1 subtype mAb. Linkage of these surrogate payloads to a common mAb with a matched DAR value allowed for preliminary analytical interrogation of the influence of payload hydrophobicity on global structure, self-association, and aggregation properties. The results of differential scanning fluorimetry and dynamic light scattering experiments clearly revealed a direct correlation between the destabilization of the native mAb structure and the increasing payload hydrophobicity. Also, self-association/aggregation propensity examined by self-interaction biolayer interferometry or size exclusion HPLC was consistent with increased conversion of the monomeric mAb to higher order aggregated species, with the degree of conversion directly proportional to the payload hydrophobicity. In summary, these findings prove that the payload-dependent structure destabilization and enhanced propensity to self-associate/aggregate driven by the increasing payload hydrophobicity contribute to reduced ADC stability and more complex behavior when assessing exposure and safety/efficacy relationships.

中文翻译：

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有效载荷疏水性对抗体-药物结合物稳定性的影响

在计算机上对ADC中通常使用的毒素有效载荷进行的筛查发现，疏水性和大小范围很广，如log P所示和拓扑极性表面积（tPSA）值。这些描述符用于识别三种无毒替代有效载荷，这些有效载荷涵盖了ADC毒素训练集定义的疏水性范围。通过缀合至模型IgG1亚型mAb的链间半胱氨酸残基，为每种替代有效载荷制备了统一的药物抗体比（DAR）ADC。将这些替代有效载荷链接到具有匹配DAR值的通用mAb，可以对有效载荷疏水性对全局结构，自缔合和聚集特性的影响进行初步分析。差示扫描荧光法和动态光散射实验的结果清楚地表明，天然mAb结构的不稳定与有效载荷疏水性的增加之间存在直接的关系。还，通过自相互作用生物层干涉术或尺寸排阻HPLC检查的自缔合/聚集倾向与单体mAb向更高阶聚集物种的转化率提高相一致，转化程度与有效载荷疏水性成正比。总而言之，这些发现证明，当评估暴露和安全/功效关系时，有效载荷依赖性结构的去稳定作用和由不断增加的有效载荷疏水性驱动的自缔合/聚集体倾向增强，导致ADC稳定性下降，行为更加复杂。转化率与有效载荷疏水性成正比。总而言之，这些发现证明，当评估暴露和安全/功效关系时，有效载荷依赖性结构的去稳定作用和由不断增加的有效载荷疏水性驱动的自缔合/聚集体倾向增强，导致ADC稳定性下降，行为更加复杂。转化率与有效载荷疏水性成正比。总而言之，这些发现证明，在评估暴露和安全/功效关系时，有效载荷依赖性结构的去稳定作用以及由不断增加的有效载荷疏水性驱动的自缔合/聚集体倾向增强，导致ADC稳定性下降，行为更为复杂。