Reporter Gene Method Or The Bioactivity Determination Of Therapeutic Anti-RANKL And Anti-BLyS Monoclonal Antibody

With the rapid development of genetic recombination technology and biomedical engineering,antibody drugs have clearly become an important choice for biotherapies.Therefore,it is necessary to establish a relevant technical system of quality evaluation to ensure the stability,safety and effectiveness of the final products.Bioactivity measurement is one of the most important indicators to ensure the quality of antibody drugs.The most common methods for the bioactivity determination of antibody drugs are developed based on in vitro cell detection,such as cell proliferation,cytotoxicity and antibody-dependent cell-mediated cytotoxicity(ADCC).Nowadays,the emerging reporter gene assay is different from the above methods,the basic principle of which involves the introduction of the reporter gene into the cell based on the transgenic technology,and measure the level of specific product expressed in the cell to determine the bioactivity of the antibody drug.The first fully humanized anti-RANKL mAb,denosumab(trade name as Xgeva),was approved by the FDA in 2010 for the treatment of postmenopausal osteoporosis.However,the bioactivity assay is based on HTRF(Homogeneous Time-Resolved Fluorescence)method,which can only reflect the binding activity of denosumab,but not reflect its real MOA(mechanism of action).In recent years,with the rapid development of the domestic biomedical industry,anti-RANKL mAbs have also emerged,and the corresponding MOA-based bioassay method(enzyme activity analysis based on RAW264.7 cells)has also been developed.However,this method has inherent weakness of complicated operation process,long duration(4-5 days),high result variability and low cell sensitivity.The aim of this study was to establish a simpler and more stable reporter gene assay for the bioactivity determination of anti-RANKL mAbs.Monocyte/macrophage RAW264.7 cells naturally expresses RANKL receptor,RANK,and can be adopted as an effector cell for transgenic cell line construction.The NF-?B-driven luciferase reporter gene vector was transiently transfected into the cell,and after the pressure screening with hygromycin B,single clone derived stable transgenic RAW264.7 cells could express luciferase under the control of RANKL-RANK-NF-?B pathway.After the reporter gene assay method was established to determine the bioactivity of anti-RANKL mAb,several key parameters of the method were optimized,followed by the validation of the method.The research results show that the established reporter gene assay method has the advantages of simple operation,high sensitivity,good accuracy and precision.Therefore,the method can be used as an effective supplement to the enzyme activity analysis method based on RAW264.7 cells,and can also be used as a platform method to evaluate the bioactivities of mAbs with the same target of different pharmaceutical companies.Systemic lupus erythematosus(SLE)is caused by autoimmunity disorder,resulting in the production of autoantibodies in the body and their precipitation in the skin,joints or other parts.Beliumab(trade name as Benlysta)was developed by GlaxoSmithKline to mainly treat systemic lupus erythematosus,which can bind to BLyS involved in the development of the disease and block its binding to cell surface receptors.In recent years,with the gradual increase of the incidence of the disease,many pharmaceutical companies in China have carried out research and development of mAbs with the same target,such as 1213 antibody of TopAlliance Biosciences Inc..However at present,no MOA based bioassay is developed for the bioactivity determination of anti-BLyS mAb,while the reporter gene assay established by this study fills the gap.BLyS receptor(TACI)and NF-?B driven luciferase were stably coexpressed into HEK293 cells,and the following method optimization and validation were performed.The results show that this method could effectively reflect the bioactivity of anti-BLyS mAb with good stability and better specificity,accuracy,and precision.Through the research,we successfully screened and constructed RAW264.7/NF-?B-Luci and TACI/NF-?B-Luci/HEK293 transgenic cell lines,and established reporter gene methods for the determination of anti-RANKL and anti-BLyS mAbs.The establishment of the two methods not only broadens the application of the reporter gene assay methods,but also accelerates the R&D of the corresponding mAbs and is of importance to strengthen their quality control.