(726) Development of in vitro models to study CD137 agonist antibody mediated hepatotoxicity

Introduction/Rationale: Immune-mediated hepatotoxicity from biotherapeutics is a growing concern in drug development due to the poor translatability of preclinical safety findings and the lack of suitable in vitro models to predict in vivo risks. Urelumab, a CD137 agonist antibody, presented with severe liver toxicity in clinical trials. In contrast utomilumab, demonstrated a clean safety profile. This difference offers a unique compound set to investigate mechanisms contributing to immune-mediated hepatotoxicity and to develop in vitro models to forecast human risks.

Methods: In house versions of urelumab and utomilumab were produced with their original and altered Fc backbones. Effector CD8+ T cells were cultured with FcγRIIb expressing CHO cells and CD137 agonist antibodies with or without anti-CD3. Effector functions were quantified via IFN-γ, TNF-α, Granzyme B, and Perforin measurements by MSD or ELISA. Incucyte Sx5 was utilized to quantify hepatoxicity (caspase 3/7) induced from urelumab and utomilumab using a co-culture of labeled HepG2 cells (WT or β2M KO) and human effector CD8+ T cells.

Results: Urelumab induced cytokine release from effector CD8+ T cells was enhanced by FcγRIIb crosslinking and T cell receptor (TCR) engagement but was not dependent on these factors, like utomilumab. Additionally, urelumab mediated cytotoxic granule release required parallel TCR engagement and was enhanced by FcγRIIb crosslinking. Using an in vitro co-culture of human effector CD8+ T cells and HepG2 cells, we modeled urelumab mediated hepatotoxicity, demonstrating a lack of dependence on TCR engagement and a correlation between cytokine production and hepatocyte death, which was absent with non-toxic utomilumab.

Conclusion: This fully human in vitro model is a sensitive tool for evaluating immune mediated hepatotoxicity risk for immunostimulatory biotherapeutics.