PDF(Pubmed)
Abstract：
The mechanisms of mAb-induced ADCC have been well established. However, the ADCC bioassays used to quantify mAb-induced ADCC require continued development/refinement to properly assess and compare the potency of newly developed therapeutic mAbs and biosimilars to meet regulatory requirements. We used trastuzumab and a lactate dehydrogenase (LDH)-based ADCC bioassay as a model to define critical parameters of the ADCC bioassay, describing how several bioassay parameters, including preparation of effector cells, E/T ratio, target cell selection, bioassay media components, and treatment time can influence the data quality of the ADCC activity. We confirm that a 4 to 24 h recovery cultivation is required to restore peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cell activity toward ADCC when using cryopreserved PBMCs. Furthermore, we delineated the cellular mechanisms underlying the restored ADCC activity following the recovery cultivation. We observed that CD69, an early marker of NK cell activation, was upregulated and a new subset CD56dim/CD16dim population was dramatically increased in the recovered NK cells, which led to an increase in expression and secretion of perforin, granzyme B, and cytokine production. This study provides comprehensive technical insights into ADCC bioassay optimization to inform trastuzumab biosimilar development. The knowledge gained from this study can also be leveraged to guide bioassay development for therapeutic mAbs with ADCC as the primary mechanism of action.
摘要：
mAb诱导ADCC的机制已经很好地建立。然而,用于量化mAb诱导的ADCC的ADCC生物测定法需要继续开发/改进,以正确评估和比较新开发的治疗性mAb和生物仿制药的效力,以满足监管要求.我们使用曲妥珠单抗和基于乳酸脱氢酶（LDH）的ADCC生物测定作为模型来定义ADCC生物测定的关键参数。描述了几个生物测定参数，包括效应细胞的制备，E/T比，目标小区选择,生物测定培养基成分，处理时间会影响ADCC活性的数据质量。我们确认，当使用冷冻保存的PBMC时，需要4至24小时的恢复培养才能恢复外周血单核细胞（PBMC）和自然杀伤（NK）细胞对ADCC的活性。此外，我们描述了恢复培养后恢复ADCC活性的细胞机制。我们观察到CD69，NK细胞活化的早期标志物，在恢复的NK细胞中，一个新的亚群CD56dim/CD16dim群体急剧增加，导致穿孔素的表达和分泌增加，颗粒酶B，和细胞因子的产生。这项研究为ADCC生物测定优化提供了全面的技术见解，为曲妥珠单抗生物仿制药的开发提供了信息。从这项研究中获得的知识也可以用来指导以ADCC为主要作用机制的治疗性mAb的生物测定开发。
