Division of Monoclonal Antibody Products, National Institu-tes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing 102629, China.
Division of Monoclonal Antibody Products, National Institu-tes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, Beijing 102629, China.
Int Immunopharmacol. 2021 Nov;100:108112. doi: 10.1016/j.intimp.2021.108112. Epub 2021 Sep 11.
More than 100 monoclonal antibodies (mAbs) have been approved by FDA. The mechanism of action (MoA) involves in neutralization of a specific target via the Fab region and Fc effector functions through Fc region, while the latter include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). ADCP has been recognized one of the most important MoAs, especially for anti-cancer mAbs in recent years. However, traditional bioassays measuring ADCP always introduced primary macrophages and flow cytometry, which are difficult to handle and highly variable. In this study, we engineered a monoclonal Jurkat/NFAT/CD32a-FcεRIγ effector cell line that stably expresses CD32a-FcεRIγ chimeric receptor and NFAT-controlled luciferase. The corresponding mAb could bind with the membrane antigens on the target cells with its Fab fragment and CD32a-FcεRIγ on the effector cells with its Fc fragment, leading to the crosslinking of CD32a-FcεRIγ and the resultant expression of subsequent NFAT-controlled luciferase, which represents the bioactivity of ADCP based on the MoA of the mAb. With rituximab as the model mAb, Raji cells as the target cells, and Jurkat/NFAT/CD32a-FcεRIγ cells as the effector cells, we adopted the strategy of Design of Experiment (DoE) to optimize the bioassay. Then we fully validated the established bioassay according to ICH-Q2(R1), which proved the good assay performance characteristics of the bioassay, including specificity, accuracy, precision, linearity, stability and robustness. This RGA can be applied to evaluate the -ADCP bioactivity for anti-CD20 mAbs in lot release, stability testing as well as biosimilar comparability. The engineered cells may also potentially be used to evaluate the ADCP bioactivity of mAbs with other targets.
已有超过 100 种单克隆抗体 (mAb) 通过 FDA 批准。其作用机制 (MoA) 通过 Fab 区域中和特定靶点，通过 Fc 区域发挥 Fc 效应功能，后者包括补体依赖性细胞毒性 (CDC)、抗体依赖性细胞介导的细胞毒性 (ADCC) 和抗体依赖性细胞吞噬作用 (ADCP)。近年来，ADCP 已被认为是最重要的 MoA 之一，尤其是针对抗癌 mAb。然而，传统的 ADCP 生物测定方法通常需要使用原代巨噬细胞和流式细胞术，这两种方法都难以操作且高度可变。在这项研究中，我们构建了一个单克隆 Jurkat/NFAT/CD32a-FcεRIγ 效应细胞系，该细胞系稳定表达 CD32a-FcεRIγ 嵌合受体和 NFAT 控制的荧光素酶。相应的 mAb 可以通过 Fab 片段与靶细胞上的膜抗原结合，通过 Fc 片段与效应细胞上的 CD32a-FcεRIγ 结合，导致 CD32a-FcεRIγ 交联，随后表达 NFAT 控制的荧光素酶，这代表了基于 mAb MoA 的 ADCP 生物活性。以利妥昔单抗为模型 mAb，Raji 细胞为靶细胞，Jurkat/NFAT/CD32a-FcεRIγ 细胞为效应细胞，我们采用实验设计 (DoE) 策略优化生物测定。然后，我们根据 ICH-Q2(R1) 充分验证了建立的生物测定，证明了生物测定具有良好的测定性能特征，包括特异性、准确性、精密度、线性、稳定性和稳健性。该 RGA 可用于评估批放行、稳定性测试以及生物类似药可比性中抗 CD20 mAb 的 -ADCP 生物活性。这些工程细胞也可能潜在地用于评估其他靶点的 mAb 的 ADCP 生物活性。
