Development and validation of a reporter gene assay to determine the bioactivity of anti-CTLA-4 monoclonal antibodies.

CTLA-4 is an important immune checkpoint for the regulation of T cell activation, and anti-CTLA-4 monoclonal antibodies (mAbs) are being developed as mono- or combination therapy for various tumors with reliable clinical efficacy. Ipilimumab is the first approved inhibitor of immune checkpoint, and many other anti-CTLA-4 mAbs, including ipilimumab biosimilars, are in different stages of clinical trials. However, due to the immunomodulating nature of the mAbs targeting CTLA-4, mode of action (MoA) and cell-based bioassay to determine their bioactivities as the lot release or stability test has been a great challenge to quality control laboratories. In this study, we have developed and validated a reporter gene assay (RGA), in which two kinds of cell lines were engineered to measure the bioactivity of anti-CTLA-4 mAbs. Raji cells were stably transfected with the membrane-anchored anti-CD3 single chain antibody fragment (scFv) as antigen-presenting cells (APCs, Raji-CD3scFv cells), while Jurkat cells were stably transfected with CTLA-4 with Y201V mutation and NFAT controlled luciferase as the effector cells (Jurkat-CTLA-4-NFAT-luc cells). The ligation of CD80/CD86 on the APCs with CTLA-4 could reduce the luciferase expression accompanied with the activation of effector cells, while the anti-CTLA-4 mAb could reverse the reduction, which resulted in good dose response curve to determine its bioactivity. After optimizing various assay conditions, we performed full validation according to ICH-Q2 (R1), which demonstrated the excellent specificity, accuracy, precision, linearity, and the cell passage stability. The satisfied performance characteristics render the RGA a good bioassay in the bioactivity determination of anti-CTLA-4 mAbs, as applied in characterization, batch release control, stability study, and biosimilar assessment.