Sabine Hünecke, Universitätsklinikum Frankfurt, Klinik für Kinder- und Jugendmedizin, Labor für Stammzelltransplantation und Immuntherapien

Flow cytometry enables the analysis of Advanced Therapy Medicinal Products (ATMPs) concerning phenotype, differentiation, and antigen expression intensity. It also facilitates the detection of immune cell functions and the definition of release specifications. Our focus is on five ATMPs: Natural Killer cells, Cytokine Induced Killer cells, Mesenchymal Stroma cells, CAR-T cells, and CAR-NK cells.
For the application of flow cytometry in the quality control of ATMPs, the assay needs to be validated according to the ICH Q2 guideline.

Parameters considered for the validation of CAR T cell monitoring in vivo are addressed. Chimeric Antigen Receptor (CAR) T cell therapy represents a powerful new treatment option for relapsed or refractory hematologic malignancies. Monitoring CAR T cell kinetics offers insights into the therapy's efficacy. In Frankfurt, over 40 patients were treated with Kymriah. Serial dilution experiments have demonstrated precise and linear quantification down to 0.05% of T cells. An inter-method comparison with real-time PCR revealed a significant correlation (r=0.7; p<0.0001). While long-term CAR T cell detectability and B cell aplasia were observed in most patients, some showed signs of B cell recovery. Subsequent infusions of CAR T cells resulted in detectable but limited re-expansions.
In conclusion, flow cytometry is a reliable method for both quality control of ATMPs and in vivo detection and monitoring of CAR T cells.