Optimization of a U937 cell-based reporter system to assess ADCC activity of ustekinumab versus trastuzumab

In the present study, the human monocytic U937 cell line, which intrinsically expresses low levels of membrane-bound IL-12, was systematically optimized to establish a robust target cell system for a surrogate ADCC reporter assay aimed at evaluating the effector function of ustekinumab. ADCC activity was assessed in parallel with trastuzumab, employed as a well-characterized positive control, using a bioluminescent reporter-based platform. To induce surface IL-12 overexpression, U937 cells were stimulated with varying concentrations of IFN-γ and lipopolysaccharide (LPS), and expression levels were quantitatively assessed by flow cytometry using FITC-labelled antibody staining, alongside cell viability evaluation via trypan blue exclusion. An optimized effector function assay was established, demonstrating that stimulation with 25 ng/mL IFN-γ alone was sufficient to achieve consistent and reproducible IL-12 surface overexpression. These findings confirm that stimulated U937 cells represent a reliable target cell model for assessing ADCC-related mechanisms of monoclonal antibodies. Using this optimized system in conjunction with Jurkat-derived reporter effector cells engineered to express FcγRIIIa and an NFAT-responsive luciferase construct, ustekinumab failed to induce measurable ADCC activity. Collectively, these results support the conclusion that ustekinumab primarily functions through neutralization of soluble IL-12 and IL-23 cytokines rather than FcγRIIIa-mediated cytotoxic effector mechanisms.