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Development of a robust reporter gene assay for measuring the bioactivity of OX40‐targeted therapeutic antibodies
Luminescence ( IF 3.2 ) Pub Date : 2020-12-31 , DOI: 10.1002/bio.4004
Meng Li 1 , Lan Wang 1 , Chuanfei Yu 1 , Junzhi Wang 1
Luminescence ( IF 3.2 ) Pub Date : 2020-12-31 , DOI: 10.1002/bio.4004
Meng Li 1 , Lan Wang 1 , Chuanfei Yu 1 , Junzhi Wang 1
Affiliation
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OX40 plays a prominent role in the onset and development of solid tumors, and OX40‐targeted monoclonal antibodies (mAbs) have entered clinical trials for various tumors. Bioactivity determination of therapeutic mAbs is of great significance in product quality, however, mechanism of action‐based bioassays to determine the bioactivity of anti‐OX40 mAbs is still lacking. Here, we established a reporter gene assay system based on two cell lines, namely Jurkat‐OX40‐NFκB‐Luc which stably expresses NFκB‐controlled luciferase, and Raji cells which inherently express FcγRs. In the model, FcγRs on Raji cells could crosslink the Fc of anti‐OX40 mAbs, which leads to the further crosslinking between Fab of anti‐OX40 mAbs and OX40 on Jurkat‐OX40‐NFκB‐Luc cells. OX40 crosslinking could activate Jurkat‐OX40‐NFκB‐Luc cells, and induce the expression of NFκB‐controlled luciferase, the extent of which could reflect the bioactivity of anti‐OX40 mAbs in a dose‐dependent manner. After the optimization of various assay conditions, the validation of the cell‐based bioassay showed good assay performance characteristics, including specificity, accuracy, precision, linearity, and stability. This innovative assay that is based on the OX40‐NFκB pathway can be a powerful pool to measure the bioactivity of OX40‐targeted mAbs.
中文翻译：
开发用于测量OX40靶向治疗性抗体生物活性的可靠的报告基因测定法
OX40在实体瘤的发生和发展中起着重要作用，针对OX40的单克隆抗体（mAb）已进入各种肿瘤的临床试验。治疗性mAb的生物活性测定对产品质量至关重要，但是，仍然缺乏基于作用的生物测定机制来确定抗OX40 mAb的生物活性。在这里，我们建立了一个基于两种细胞系的报告基因测定系统，即稳定表达NFκB调控的荧光素酶的Jurkat-OX40-NFκB-Luc和固有表达FcγRs的Raji细胞。在该模型中，Raji细胞上的FcγR可以使抗OX40 mAb的Fc交联，从而导致抗OX40 mAb的Fab与Jurkat-OX40-NFκB-Luc细胞上的OX40进一步交联。OX40交联可以激活Jurkat-OX40-NFκB-Luc细胞，并诱导NFκB控制的萤光素酶的表达，其程度可能以剂量依赖的方式反映抗OX40 mAb的生物活性。在优化各种测定条件之后，基于细胞的生物测定的验证显示出良好的测定性能特征，包括特异性，准确性，精密度，线性和稳定性。这种基于OX40‐NFκB途径的创新检测方法可以作为衡量OX40靶向mAb生物活性的强大库。
更新日期：2020-12-31
中文翻译：
开发用于测量OX40靶向治疗性抗体生物活性的可靠的报告基因测定法
OX40在实体瘤的发生和发展中起着重要作用，针对OX40的单克隆抗体（mAb）已进入各种肿瘤的临床试验。治疗性mAb的生物活性测定对产品质量至关重要，但是，仍然缺乏基于作用的生物测定机制来确定抗OX40 mAb的生物活性。在这里，我们建立了一个基于两种细胞系的报告基因测定系统，即稳定表达NFκB调控的荧光素酶的Jurkat-OX40-NFκB-Luc和固有表达FcγRs的Raji细胞。在该模型中，Raji细胞上的FcγR可以使抗OX40 mAb的Fc交联，从而导致抗OX40 mAb的Fab与Jurkat-OX40-NFκB-Luc细胞上的OX40进一步交联。OX40交联可以激活Jurkat-OX40-NFκB-Luc细胞，并诱导NFκB控制的萤光素酶的表达，其程度可能以剂量依赖的方式反映抗OX40 mAb的生物活性。在优化各种测定条件之后，基于细胞的生物测定的验证显示出良好的测定性能特征，包括特异性，准确性，精密度，线性和稳定性。这种基于OX40‐NFκB途径的创新检测方法可以作为衡量OX40靶向mAb生物活性的强大库。
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