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首页> 外文期刊>Analytical chemistry >Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma
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Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma

机译：Linker-Payload去哪里了？血浆-马来酰亚胺连接体与抗体-药物偶联物释放的连接体-有效载荷目的地的定量研究

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The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzurnab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to antibody ratio (DAR), as measured by liquid chromatography mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload irnmunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.
机译：半胱氨酸的反应性硫醇通常用于将含马来酰亚胺的接头有效负载偶联至抗体，从而导致抗体药物偶联物（ADC）的产生。当前，药物开发中的许多ADC是通过将连接子有效负载与抗体上的天然或工程半胱氨酸残基偶联而制得的。在本研究中，通过铰链半胱氨酸偶联到auristatin有效负载的ADC被用作模型，以了解马来酰亚胺接头对ADC稳定性的影响。有效负载通过蛋白酶可裂解的接头马来酰亚胺基-己酰基-缬氨酸-瓜氨酸-对氨基-苄氧基羰基（mcVC-PABC）缀合至曲妥珠单抗。在血浆稳定性测定中，当ADC（Trastuzumab-mcVC-PABC-Auristatin-0101）与血浆在144小时的时间范围内孵育时，观察到测得的释放的有效载荷浓度与测得的药物损失之间存在差异抗体比（DAR），通过液相色谱质谱（LC-MS）测定。我们发现，在144 h时，从ADC耗尽的人类血浆中酶促释放出的有效载荷几乎可以解决DAR损失的100％。完整的蛋白质质量分析显示，在144小时的时间点上，ADC耗尽的人血浆中主要蛋白质的质量比从人血浆中提取的天然白蛋白多了1347 Da，与接头有效负载的质量完全匹配。此外，蛋白质凝胶电泳显示，与0 h血浆免疫沉淀样品相比，在144 h ADC耗尽和抗有效载荷免疫沉淀的血浆样品中只有一种富集蛋白质，并且这种富集蛋白质的质量略重于质量。血清白蛋白。此外，还在体内食蟹猕猴血浆中96小时和168小时后鉴定出白蛋白加合物。这些结果强烈表明，大多数解偶联的mc-VC-PABC-auristatin最终都转移到血清白蛋白上，形成了长寿命的白蛋白-接头-有效负载加合物。据我们所知，这是第一份定量表征接头-有效负载转移至血清白蛋白程度的报告，并且是体内形成白蛋白-接头-有效负载加合物的第一个明确实例。

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