基于Wnt/β-catenin信号通路的DKK-1抗体药物生物活性报告基因法开发与评估

Development and Evaluation of a Reporter Gene Assay for DKK-1 Antibody Drugs Based on the Wnt/β-Catenin Pathway

生物活性检测对于确保治疗性抗体的安全性和有效性至关重要,需要准确反映药物的作用机制(MOA)。作为一类新兴靶点药物,DKK-1抗体在骨质疏松和肿瘤治疗领域展现出巨大潜力。然而,当前针对DKK-1抗体的生物活性评价主要基于碱性磷酸酶活性测定,该方法存在操作复杂、耗时长及结果变异性大等问题,在一定程度上限制了此类抗体的研发及质控。为了建立一种DKK-1抗体高性能生物活性检测方法,本研究引入了报告基因法(RGA),通过将TCF/LEF反应元件控制的稳定表达荧光素酶的报告基因质粒转染HEK 293细胞,成功建立了一种基于DKK-1抗体MOA的Wnt/β-catenin信号通路的报告基因检测方法。该方法经过优化及模拟实验评估,显示出在实际应用中的潜力和可靠性。该新方法的建立预计将进一步促进DKK-1抗体类药物的筛选评估及其在后续开发过程中的质量控制。

The determination of bioactivity is crucial for ensuring the safety and efficacy of therapeutic antibodies,necessitating an accurate reflection of the drug’s mechanism of action(MOA).DKK-1 antibodies,as novel targeted therapies,have shown significant potential in the treatment of osteoporosis and cancer.However,the current bioactivity evaluation for DKK-1 antibodies primarily relies on the detection of alkaline phosphatase activity.This process is complex,time-consuming,and highly variable,which limits the development and quality control of DKK-1 antibodies.To establish a high-performance bioactivity assay for DKK-1 antibodies,this study introduced a reporter gene assay(RGA).By transfecting HEK 293 cells with a reporter gene plasmid stably expressing luciferase reporter under the control of TCF/LEF response elements,we successfully established a reporter gene assay based on the DKK-1 antibody MOA—the Wnt/β-catenin pathway.Through optimization and simulation experiment evaluation,the method has shown promising potential and reliability in practical application.The development of this innovative approach is expected to enhance the screening,assessment,and quality control of DKK-1 antibody drugs in future development.