For antibody-drug conjugates to be efficacious and safe, they must be stable in circulation to carry the payload to the site of the targeted cell. Several components of a drug-conjugated antibody are known to influence stability: 1) the site of drug attachment on the antibody, 2) the linker used to attach the payload to the antibody, and 3) the payload itself. In order to support the design and optimization of a high volume of drug conjugates and avoid unstable conjugates prior to testing in animal models, we wanted to proactively identify these potential liabilities. Therefore, we sought to establish an in vitro screening method that best correlated with in vivo stability. While traditionally plasma has been used to assess in vitro stability, our evaluation using a variety of THIOMABTM antibody-drug conjugates revealed several disconnects between the stability assessed in vitro and the in vivo outcomes when using plasma. When drug conjugates were incubated in vitro for 24 h in mouse whole blood rather than plasma and then analyzed by affinity capture LC-MS, we found an improved correlation to in vivo stability with whole blood (R2 = 0.87, coefficient of determination) compared to unfrozen or frozen mouse plasma (R2 = 0.34, 0.01, respectively). We further showed that this whole blood assay was also able to predict in vivo stability of other preclinical species such as rat and cynomolgus monkey, as well as in human. The screening method utilized short (24 h) incubation times, as well as a custom analysis software, allowing increased throughput and in-depth biotransformation characterization. While some instabilities that were more challenging to identify remain, the method greatly enhanced the process of screening, optimizing, and lead candidate selection, resulting in the substantial reduction of animal studies.

中文翻译：

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使用体外全血检测可改善偶联抗体的稳定性翻译。

为了使抗体-药物偶联物有效且安全，它们必须在循环中稳定以将有效载荷携带到靶细胞的位置。已知药物偶联抗体的几种成分会影响稳定性：1）药物在抗体上的附着位点； 2）用于将有效负载附着到抗体的连接子；以及3）有效负载本身。为了支持大量药物偶联物的设计和优化并避免在动物模型中进行测试之前出现不稳定的偶联物，我们希望主动识别这些潜在的负债。因此，我们寻求建立一种与体内稳定性最相关的体外筛选方法。传统上，血浆已用于评估体外稳定性，我们使用多种THIOMABTM抗体-药物偶联物进行的评估显示，使用血浆时，体外评估的稳定性与体内结果之间存在一些脱节。当药物偶联物在小鼠全血而不是血浆中体外孵育24小时，然后通过亲和捕获LC-MS分析时，我们发现与全血相比，其与体内稳定性的相关性得到了改善（R2 = 0.87，测定系数）。未冷冻或冷冻的小鼠血浆（R2分别为0.34、0.01）。我们进一步表明，这种全血测定法还能够预测其他临床前物种，例如大鼠和食蟹猴以及人类的体内稳定性。筛选方法利用了较短的（24 h）孵育时间以及定制的分析软件，允许增加通量和深入的生物转化表征。尽管仍然存在一些更具挑战性的不确定性，但该方法大大增强了筛选，优化和引导候选对象选择的过程，从而大大减少了动物研究。