Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1 therapeutic antibodies

Abstract

Being regarded as the 'cancer panacea', the anti-PD-1/anti-PD-L1 monoclonal antibodies (mAbs) have become the R&D focus of biopharmaceutical industries. Several marketed such mAbs have been proved particularly effective in treating various cancers. However, the cell-based bioassay to measure the biological activities of the anti-PD-1/anti-PD-L1 mAbs as the lot release or stability test has been a great challenge to quality control laboratories due to the immunomodulating nature of the mAbs. Here, we describe the development and validation of a reporter gene assay consisting of two-cell systems to measure the bioactivity of the anti-PD-1/anti-PD-L1 mAbs. We have generated two cell lines, the CHO-PD-L1-CD3L cell line that stably expresses PD-L1 and the membrane-anchored anti-CD3 single chain antibody fragment (scFv) named CD3L and the Jurkat-PD-1-NFAT cell line that stably expresses PD-1 and the luciferase gene under the control of the NFAT response elements from the IL-2 promoter. The results show good dose-dependent responsiveness to the mAbs and excellent performance characteristics including specificity, accuracy and precision. The biological relevance of the assay, the passage stability of the two cell lines, and the capability of measuring various anti-PD-1/anti-PD-L1 mAbs render this assay applicable not only in lot release and stability test but also in characterization and development of new anti-PD1/anti-PD-L1 mAbs.

Keywords: Bioactivity; Monoclonal antibody; PD-1; PD-L1; Reporter gene assay; Validation.