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.
Highlights
Despite major improvements in the long-term survival of children and young adults diagnosed with acute lymphoblastic leukemia (ALL) [1, 2], the subset of patients with relapsed or refractory (r/r) disease continues to show poor outcomes [3] and new therapeutic approaches are urgently needed
CD19 Chimeric antigen receptor (CAR) Detection Reagent and Anti-Biotin PE volumes were titrated to determine the reagent volume that results in optimal separation of CAR positive and CAR negative populations. 1 μl, 2.5 μl, and 5μl were considered within the optimal range (Figure 1), and 1 μl was subsequently chosen to be continued with in our CAR T cell monitoring
Many centers administering CAR T cells have not yet established CAR T cell detection methods, and patient specimens have to be shipped to cooperating centers for analysis
Summary
Despite major improvements in the long-term survival of children and young adults diagnosed with acute lymphoblastic leukemia (ALL) [1, 2], the subset of patients with relapsed or refractory (r/r) disease continues to show poor outcomes [3] and new therapeutic approaches are urgently needed. Large clinical studies were able to provide considerable evidence of the advantages of CAR T cell therapy in r/r pediatric andAlthough ? The duration of detectable CAR T cell levels has been found to correlate with event free survival [20]. While the mere monitoring of B cell aplasia (BCA) does indicate continued functional activity of CAR T cells [21, 22], the direct monitoring of CAR T cell levels provides a deeper dimension of information and can contribute to clinical decision making
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