AUTHOR=Peinelt Andreas , Bremm Melanie , Kreyenberg Hermann , Cappel Claudia , Banisharif-Dehkordi Julia , Erben Stephanie , Rettinger Eva , Jarisch Andrea , Meisel Roland , Schlegel Paul-Gerhardt , Beck Olaf , Bug Gesine , Klusmann Jan-Henning , Klingebiel Thomas , Huenecke Sabine , Bader Peter TITLE=Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel JOURNAL=Frontiers in Immunology VOLUME=13 YEAR=2022 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.830773 DOI=10.3389/fimmu.2022.830773 ISSN=1664-3224 ABSTRACT=

Chimeric antigen receptor (CAR) T cell therapy is a potent new treatment option for relapsed or refractory hematologic malignancies. As the monitoring of CAR T cell kinetics can provide insights into the activity of the therapy, appropriate CAR T cell detection methods are essential. Here, we report on the comprehensive validation of a flow cytometric assay for peripheral blood CD19 CAR T cell detection. Further, a retrospective analysis (n = 30) of CAR T cell and B cell levels over time has been performed, and CAR T cell phenotypes have been characterized. Serial dilution experiments demonstrated precise and linear quantification down to 0.05% of T cells or 22 CAR T cell events. The calculated detection limit at 13 events was confirmed with CAR T cell negative control samples. Inter-method comparison with real-time PCR showed appreciable correlation. Stability testing revealed diminished CAR T cell values already one day after sample collection. While we found long-term CAR T cell detectability and B cell aplasia in most patients (12/17), some patients (5/17) experienced B cell recovery. In three of these patients the coexistence of CAR T cells and regenerating B cells was observed. Repeat CAR T cell infusions led to detectable but limited re-expansions. Comparison of CAR T cell subsets with their counterparts among all T cells showed a significantly higher percentage of effector memory T cells and a significantly lower percentage of naïve T cells and T EMRA cells among CAR T cells. In conclusion, flow cytometric CAR T cell detection is a reliable method to monitor CAR T cells if measurements start without delay and sufficient T cell counts are given.