Reporter Gene Assays For Bioactivity Determination Of Anti-PD-1/PD-L1 And IL-6/IL-6R Antibodies

The rapid development of transgenic technology lays the foundation for the construction of cell lines which stably express certain proteins.Reporter gene assay(RGA)applies this principle to verify whether certain signal pathway is activated upon the interaction between receptors and ligands.Bioactivity could determine the active pharmaceutical ingredient(API),evaluate the strength and potency,thus comes into an important quality control index for antibody based biotherapeutics.As the evaluation technique of bioactivity,RGA has the advantages of simple operation,short experiment period and little variation,and can overcome the lack of sensitive cell lins regarding to checkpoint inhibitors.Anti-PD-1/PD-L1 antibody is one of the most popular checkpoint inhibitors in the filed of tumor immunotherapy.At present,two anti-PD-1 antibodies,pembrolizumab and nivolumab,and two anti-PD-L1 antibodies,atezolizumab and durvalumab,have been approved to treat various cancers,including advanced melanoma,non-small cell lung cancer,renal cell carcinoma and non-hodgkin’s lymphoma.However,there is no cell-based bioassay to evaluate the bioactivity of anti-PD-1/PD-L1 antibody in the lot release test due to the lack of sensitive cell lines,so we decide to use RGA to establish the corresponding bioassay.We first constructed two cell lines,CHO-PD-L1-CD3 L stably expressing PD-L1 and anti-CD3 single chain antibody fragment,and Jurkat-PD-1-NFAT cells stably expressing PD-1 and luciferase gene driven by NFAT response element.Then,we set up the reporter gene bioassay through optimization and validation of performance characteristics,including specificity,accuracy,precision and cell line stability.Different anti-PD-1/PD-L1 antibodies from different manufacturers shows good dose-dependent responsiveness,proving that this method can be a platform approach to evaluate the biological activity of anti-PD-1/PD-L1 antibody.Another important indication for antibody drugs is autoimmune disease.Tocilizumab(trade name: Actemra)specifically targets to IL-6 receptor and blocks the binding between IL-6 and its receptor IL-6R,relieving symptoms of rheumatoid arthritis(RA).However,the current bioassay for Tocilizumab in the lot release test is the anti-proliferation assay which requires at least 72 hours,so we generated a transgenic IL-6-dependent DS-1 cell line expressing the SIE luciferase gene to evaluate the blocking effect of Tocilizumab.After a serials of optimization,including initial concentration and incubating time of IL-6 and initial concentration and dilution fold of Tocilizumab,we obtained a good dose-response curve.The two reporter gene bioassays evaluating the biological activities of anti-PD-1/PD-L1 and IL-6/IL-6R antibodies have been established in this study.It will not only make the bioactivity evaluation of anti-IL-6/IL-6R antibody simple and convenient and improve the quality control of anti-PD-1/PD-L1 antibody,but also promote the application of RGA in drug quality control and provide technical insight for the bioassay establishment of other antibodies.