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Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel

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Title: Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel
Authors: Andreas Peinelt, Melanie Bremm, Hermann Kreyenberg, Claudia Cappel, Julia Banisharif-Dehkordi, Stephanie Erben, Eva Rettinger, Andrea Jarisch, Roland Meisel, Paul-Gerhardt Schlegel, Olaf Beck, Gesine Bug, Jan-Henning Klusmann, Thomas Klingebiel, Sabine Huenecke, Peter Bader
Source: Frontiers in Immunology, Vol 13 (2022)
Publisher Information: Frontiers Media S.A., 2022.
Publication Year: 2022
Collection: LCC:Immunologic diseases. Allergy
Subject Terms: acute lymphoblastic leukemia, chimeric antigen receptor (CAR), immunotherapy, flow cytometry, immune monitoring, Immunologic diseases. Allergy, RC581-607
More Details: 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.
Document Type: article
File Description: electronic resource
Language: English
ISSN: 1664-3224
Relation: https://www.frontiersin.org/articles/10.3389/fimmu.2022.830773/full; https://doaj.org/toc/1664-3224
DOI: 10.3389/fimmu.2022.830773
Access URL: https://doaj.org/article/79daa92e8f4244719dcde74aa8452d48
Accession Number: edsdoj.79daa92e8f4244719dcde74aa8452d48
Database: Directory of Open Access Journals
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  Data: Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel
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  Data: 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.
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      – SubjectFull: chimeric antigen receptor (CAR)
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