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/CD16dimpopulation 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.
单克隆抗体诱导的抗体依赖性细胞介导的细胞毒性（ADCC）作用机制已得到充分阐明。然而，用于量化单克隆抗体诱导ADCC效应的生物测定方法仍需持续开发与优化，以满足监管要求，从而准确评估和比较新开发治疗性单克隆抗体及其生物类似药的效价。本研究以曲妥珠单抗及乳酸脱氢酶（LDH）法ADCC生物测定为模型，系统阐述了效应细胞制备、效靶比、靶细胞选择、检测培养基组分及处理时间等关键参数对ADCC活性数据质量的影响。研究证实，使用冻存外周血单个核细胞（PBMCs）时，需经过4至24小时恢复培养才能重建其对ADCC的应答能力，包括自然杀伤（NK）细胞活性的恢复。进一步研究揭示了恢复培养后ADCC活性重建的细胞机制：在恢复后的NK细胞中，早期激活标志物CD69表达上调，新型CD56dim/CD16dim细胞亚群显著扩增，进而导致穿孔素、颗粒酶B的表达分泌及细胞因子产量增加。本研究为ADCC生物测定优化提供了全面的技术参考，可指导曲妥珠单抗生物类似药的研发。所得结论亦适用于以ADCC为主要作用机制的治疗性单克隆抗体的生物测定方法开发。
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