Abstract 2747: Combined investigation of anti-tumor efficacy and liver safety of bispecific T cell engagers in immune-competent high-throughput co-culturing platform
Authors: Rudnik Michal , Grining Ozlem Yavaş , Hutter Simon , Greve Frauke , Franyuti Daniela Ortiz , Matheis Ramona , Breous-Nystrom Ekaterina , Frey Olivier 
Published: April 2023

Abstract
The superior clinical therapeutic effects and broad applications of bispecific T cell engagers (BiTEs) has transformed treatment strategies in oncology. While benefits of the novel immunotherapies are indisputable, the safety aspects require to be intensively investigated. Considering challenges of animal models for accessing efficacy and safety of biologics leading to failed clinical trials, there is unmet need for human disease relevant systems, which facilitate development of new clinical candidates. We developed a high-throughput, 384-well-format co-culturing platform for combined assessment of anti-tumor efficacy and liver safety of immunotherapeutics. As a model of liver we employed 3D spheroids composed of primary human hepatocytes and Kupffer cells with preserved metabolic and inflammatory functions. In parallel, the solid tumor model was aggerated of human cancer cell lines (HCT116-GFP) and primary cancer associated fibroblasts, mimicking the tumor microenvironment. Both models were cultured in automation-friendly wells pairwise interconnected with microfluidic channels. Gravity-driven tubeless flow ensured tissue-tissue interaction. To evaluate our system, we treated 3D spheroid models with runimotamab (HER2xCD3 BiTE) in the presence of peripheral blood mononuclear cells (PBMCs). Tumor viability and growth was assessed by fluorescence measurements, while liver toxicity and function were monitored by release of liver aminotransferases ALT/AST, albumin secretion and live confocal microscopy. Immune cell activation was assessed by a cytokine bead array. The treatment with runimotamab resulted in a significant decrease of fluorescence and size of tumor spheroids. At the same time, we observed an induced secretion of cytokines with a peak of expression of IL-2, TNFα after 24h and INFγ, IL-6 and IL-17A after 48h. Cytokine release was coupled with elevated levels of clinically relevant liver damage biomarkers ALT and AST with peak at 72h timepoint. This data suggests potential risk of cytokine release syndrome (CRS) followed by liver damage. Interestingly, redosing of the antibody was not followed by another release of liver enzymes, similarly to clinical observations. In summary, we have developed a human disease relevant, high-throughput platform for the evaluation of novel immunotherapies closely emulating clinical results. Automation-compatible with up to 192 co-culture conditions per plate it represents a powerful tool for clinical candidate development.
Citation Format: Michal Rudnik, Ozlem Yavaş Grining, Simon Hutter, Frauke Greve, Daniela Ortiz Franyuti, Ramona Matheis, Ekaterina Breous-Nystrom, Olivier Frey. Combined investigation of anti-tumor efficacy and liver safety of bispecific T cell engagers in immune-competent high-throughput co-culturing platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2747.