Development and validation of a reporter gene assay for the bioactivity determination of anti-TSLP monoclonal antibodies

Thymic stromal lymphopoietin (TSLP) promotes Th2-mediated inflammation via dendritic cell activation and STAT5 signaling and plays critical roles in inflammatory disorders and allergic diseases. While tezepelumab stands as the first and only approved anti-TSLP monoclonal antibody (mAb) with over 10 anti-TSLP mAbs in clinical development, no validated anti-TSLP mAb bioassay was reported yet. Bioactivity determination is essential for ensuring mAb quality. To bridge this gap, we generated a novel HuT78-STAT5-luc reporter cell line through lentiviral transduction of STAT5 response element-driven luciferase into HuT78 cells. This stable cell line expressed luciferase in a TSLP dose-responsive manner. After systematic optimization of cell density, incubation time, TSLP concentration and mAb concentration, we established a reporter gene assay (RGA) with four-parameter regression compliance for the anti-TSLP mAb bioactivity determination. The RGA underwent full validation according to the International Council for Harmonization (ICH) Q2(R2) guideline, namely specificity, linearity, accuracy, precision and robustness. In conclusion, we established a robust and user-friendly RGA that can be applied for the quality control of anti-TSLP mAbs.