Development of a ready-to-use cell-based functional neutralizing antibody assay for MK-1484, a PEGylated IL-2 mutein that preferentially activates effector T-cells

BACKGROUND: MK-1484 is an engineered, PEGylated interleukin-2 (IL-2) mutein that preferentially binds the βγ subunits of the IL-2 receptor to activate effector T-cells and natural killer (NK) cells. Owing to high sequence homology, anti-drug antibodies (ADAs), including neutralizing antibodies (NAbs), may cross-react with and neutralize endogenous IL-2; MK-1484 is therefore classified as having a high immunogenicity risk, requiring an NAb assay to be ready for early-phase clinical studies. RESEARCH DESIGN AND METHODS: A highly sensitive competitive ligand-binding assay (cLBA) using mixed recombinant β- and γ-IL-2 receptor subunits was considered impractical, so plate- and flow cytometry-based cell-binding assays were attempted using the IL-2-dependent KIT225 cell line. A cell-based functional luciferase reporter assay (KIT225-STAT5-Luc#8) was then developed and qualified. RESULTS: Both cell-binding assays produced only weak signal. The functional reporter assay was successfully developed and qualified as a ready-to-use format for MK-1484 (cut point S/N 0.62; sensitivity 4.0 µg/mL), overcoming cell-line instability, lengthy recovery, and IL-2 starvation by using MK-1484 itself for post-thaw recovery and reducing the workflow from ~6 to 2 days. CONCLUSIONS: The ready-to-use functional NAb assay is fit-for-purpose for early-phase testing when combined with a more sensitive wild-type IL-2 NAb assay as the primary safety screen, and may be adaptable to other IL-2-based biotherapeutics, subject to case-by-case evaluation.