Abstract 735: Mitigation of liver toxicity effects of bispecific T cell engagers in immune-competent liver-tumor co-culturing high-throughput platform
Authors: Rudnik Michal , Hölting Lisa , Häfeli Tamara , Grining Özlem Yavaş , Greve Frauke , Franyuti Daniela Ortiz , Matheis Ramona , Ligeon Laure-Anne , Breous-Nystrom Ekaterina , Frey Olivier 
Published: March 2024

Abstract
The remarkable clinical therapeutic effects and wide-ranging applications of bispecific T cell engagers (BiTEs) have revolutionized the treatment approaches in the field of oncology. While the advantages of these innovative immunotherapies are undeniable, it is imperative to thoroughly investigate their safety aspects. With increasing use of biologics for treatment of various cancers, the incidence of immune-related adverse events (irAEs) will undoubtedly increase. Therefore, there is a compelling need to develop measures to effectively prevent or manage irARs already on the stage of the development of new clinical candidates.
To address this need, we have created a high-throughput, automation friendly, 384-well-format platform in which 3D spheroid models are co-cultured in units interconnected by microfluidic channel. The tissue-tissue interaction was facilitated by a gravity-driven, tubeless flow system. The design allows for the simultaneous evaluation of the anti-tumor efficacy and liver safety of immunotherapeutics. In our system, we employed 3D spheroids comprised of primary human hepatocytes and Kupffer cells to model the liver, ensuring the preservation of their metabolic and inflammatory functions. Additionally, we established a solid tumor model using human cancer cell lines (HCT116-GFP) and primary cancer-associated fibroblasts, effectively mimicking the complex tumor microenvironment We treated 3D spheroid models with runimotamab (HER2xCD3 BiTE), catumaxomab (EPCAMxCD3), nivatrotamab (GD2xCD3) and control bispecific antibodies 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 and microscopy. To mitigate potential liver toxicity, we co-treated spheroid co-cultures with adalimumab (anti-TNF), tocilizumab (anti-IL-6R), raleukin (IL-1R antagonist) (each 100ug/ml), desatanib, dexamethasone or ruxolitinib (each 100nM). The treatment with all bispecific antibodies resulted in a significant decrease of fluorescence and size of tumor spheroids in comparison to control antibody. At the same time increased ALT concentrations were observed after the treatment with BiTEs. The application of small molecular drugs protected against liver damage, but also severely disrupted tumor killing. Interestingly, adalimumab revealed liver protective actions without affecting tumor killing, suggesting TNF-mediated mechanism of toxicity. In summary, we have developed a relevant, high-throughput platform for the evaluation of novel immunotherapies closely reflecting clinical scenarios. High throughput of the assay represents a powerful screening tool for clinical candidate development.
Citation Format: Michal Rudnik, Lisa Hölting, Tamara Häfeli, Özlem Yavaş Grining, Frauke Greve, Daniela Ortiz Franyuti, Ramona Matheis, Laure-Anne Ligeon, Ekaterina Breous-Nystrom, Olivier Frey. Mitigation of liver toxicity effects of bispecific T cell engagers in immune-competent liver-tumor co-culturing high-throughput platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 735.