Development and Validation of a Novel Single-Cell Functional Assay for GD2 CAR T Cell Activity Against Adherent Ewing Sarcoma Models

Chimeric Antigen Receptor T cell (CAR T) therapies have transformed the treatment landscape of several hematological malignancies and are increasingly being explored for solid tumors. As cell therapy development and manufacturing continue to expand, there is a growing need for advanced analytical platforms capable of rapidly and reproducibly assessing the functional quality and potency of CAR T cell products. Conventional cytotoxicity assays provide bulk measurements of target cell killing but fail to capture the functional heterogeneity, dynamic interactions, and serial killing behavior that characterize immune cell responses at the single-cell level. This thesis contributed to the development of an innovative application of the VivaCyte® platform, an integrated system combining automated cell culture, liquid handling, and high-content imaging to enable high-throughput single-cell functional profiling of immune cell therapies. The project focused on establishing a novel experimental setup for the analysis of CAR T cell activity against adherent tumor cells, expanding the platform's capabilities beyond its existing applications. To achieve this goal, the proprietary co-culture array (CC-array®) microfluidic technology developed by Cellply was optimized using a functionalized substrate and a dedicated surface coating to promote stable tumor cell adhesion while maintaining compatibility with automated imaging and single-cell analysis workflows. The resulting platform enables the generation and monitoring of thousands of miniaturized tumor–immune cells co-cultures in parallel, providing detailed characterization of effector cell behavior across multiple experimental conditions. The performance of the newly developed assay was validated using GD2 CAR T cells and GD2-positive Ewing sarcoma cell lines as a representative solid tumor model. Multiple CAR T cell batches were evaluated and compared with GFP-expressing T cells used as controls. Results obtained with VivaCyte® were benchmarked against established bulk cytotoxicity assays, demonstrating concordant measurements of antitumor activity while providing substantially deeper insights into cell-to-cell interactions and functional heterogeneity. Overall, this work establishes a robust and reproducible single-cell assay for evaluating CAR T cell activity against adherent tumor targets and demonstrates the potential of VivaCyte® as an advanced platform for cell therapy characterization. By enabling high-content functional profiling with increased automation, throughput, and reproducibility, this technology may support the development, optimization, and quality assessment of next-generation cell therapy products.