Abstract 2303: In vitro evaluation of TCR efficacy and toxicity using 3D spheroid models

Abstract

Abstract Clinical studies using TCR-transgenic T cells for adoptive T cell therapy revealed the efficacy of this therapeutic approach but also uncovered possible toxic effects against healthy tissues, such as neuronal or cardiac cells, caused by on-target/off-tumor or off-target toxicity (Morgan et al., J Immunother. 2013; Linette et al., Blood 2013). Due to the lack of adequate in vivo models for prediction of TCR efficacy or potential TCR-mediated toxicity against healthy tissues, physiologically relevant in vitro models need to be developed. The usage of 3D spheroids as targets for TCR-transduced T cells represents a powerful strategy to test killing capacity or possible safety issues of TCRs, which could fill the gap between 2D co-cultures and animal models. Compared to 2D cell layers, cells within a 3D spheroid structure more closely resemble the physiological in vivo situation regarding cell-cell-interactions, proliferation rates, hypoxia, and gene expression. Here we describe the use of 3D spheroids generated from tumor cell lines for assessment of TCR efficacy or healthy primary cells/iPSC-derived cells for evaluation of potential TCR-derived toxicity. HLA-A2-positive cells served as target cells in functional co-culture assays with CD8+ T cells transduced with an HLA-A2-restricted TCR. 3D spheroids have been generated using ULA (ultra-low attachment) plates or via the hanging-drop culture method. Spheroids were co-cultured with TCR-transduced T cells and killing of spheroids was analyzed in real-time using the IncuCyte live cell imaging system. For better visualization of spheroid killing, tumor cell lines were transduced using a lentivirus encoding a nuclear-restricted red fluorescent protein or by adding Annexin V red fluorescent reagent for detection of apoptotic cells. Thereby, spheroid killing could be quantified via decrease or increase in red fluorescence over time. Killing of 3D spheroids derived from healthy cells was analyzed via phase contrast and evaluation of spheroid morphology. TCR-transduced T cells showed efficient killing of tumor cell spheroids, confirming the results generated in a 2D system. Furthermore, 3D spheroids generated from healthy cells (e.g. normal human lung fibroblasts) or iPSC-derived cells (e.g. cardiomyocytes or neurons) were not killed via TCR-transduced T cells, whereas exogenous loading of the spheroids with the specific peptide led to efficient killing, showing the general susceptibility to TCR-mediated killing of healthy cell 3D spheroids.In summary, we showed that functional in vitro assays using 3D spheroid structures using tumor cell lines or healthy cells represent an elegant approach to assess both efficacy and potential toxicity of a given TCR in vitro. The analyzed TCR showed potent efficacy as well as a favorable safety pattern in 3D in vitro cultures. Citation Format: Maja Buerdek, Kathrin Mutze, Kai Pinkernell, Dolores J. Schendel. In vitro evaluation of TCR efficacy and toxicity using 3D spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2303.