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.

超过 100 种单克隆抗体 (mAb) 已获得 FDA 批准。作用机制 (MoA) 涉及通过 Fab 区中和特定靶标和通过 Fc 区发挥 Fc 效应器功能，而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）。ADCP 已被公认为最重要的 MoA 之一，尤其是近年来的抗癌 mAb。然而，测量 ADCP 的传统生物测定法总是引入原代巨噬细胞和流式细胞仪，它们难以处理且变化很大。在这项研究中，我们设计了一种单克隆 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 可以通过其 Fab 片段与靶细胞上的膜抗原结合，并通过其 Fc 片段与效应细胞上的 CD32a-FcεRIγ 结合，导致 CD32a-FcεRIγ 的交联和随后 NFAT 控制的荧光素酶的表达，其代表基于 mAb 的 MoA 的 ADCP 的生物活性。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，采用实验设计（DoE）策略优化生物测定。然后我们根据ICH-Q2(R1)对所建立的生物测定法进行了充分验证，证明了该生物测定法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 可用于评估抗 CD20 mAb 在批次放行、稳定性测试以及生物类似物可比性中的 -ADCP 生物活性。工程细胞也可能用于评估 mAb 与其他靶标的 ADCP 生物活性。