Strategies for successful monitoring of CAR T-cells by flow cytometry

Background & Aim As the success of Chimeric Antigen Receptor T-cell (CAR T-cell) therapies continues and more cell products enter clinical development, the need for robust measurements of these “living drugs” increases. The flow cytometry platform is ideally suited for characterization of cellular therapies owing to its highly specific multiplexing capabilities. Methods, Results & Conclusion Characterization of cellular therapies includes immunophenotypic characterization of CAR T-cells and other cellular components of the infusion product, as well as cellular tracking and quantification following infusion in treated patients. Nonetheless, flow cytometric analysis of CAR T-cells presents unique challenges in addition to those that are present in all other clinical and non-clinical flow assays. The primary challenges associated with monitoring of CAR T-cells post-infusion include: identifying and/or producing reagents and staining methodology for specific CAR-T cell detection; establishing appropriate assay sensitivity in patients lymphodepleted following treatment; maintaining assay specificity with CAR antigen down-regulation in vivo; establishing the accuracy of cell counting methods; defining an appropriate quality control sample for longitudinal monitoring. In this poster we describe these challenges and propose solutions for mitigation. In addition, we present recommendations for method validation aligned with the Context-of-Use as described in the new Clinical Laboratory and Standards Institute's new guidelines for the Validation of Methods Preformed by Flow Cytometry, CLSI H62.