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

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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8926389/
https://www.ncbi.nlm.nih.gov/pubmed/35309367
http://dx.doi.org/10.3389/fimmu.2022.830773
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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
author_facet 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
author_sort Peinelt, Andreas
collection PubMed
description 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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spelling pubmed-89263892022-03-17 Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel 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 Front Immunol Immunology 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. Frontiers Media S.A. 2022-03-02 /pmc/articles/PMC8926389/ /pubmed/35309367 http://dx.doi.org/10.3389/fimmu.2022.830773 Text en Copyright © 2022 Peinelt, Bremm, Kreyenberg, Cappel, Banisharif-Dehkordi, Erben, Rettinger, Jarisch, Meisel, Schlegel, Beck, Bug, Klusmann, Klingebiel, Huenecke and Bader https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Immunology
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
Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel
title Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel
title_full Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel
title_fullStr Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel
title_full_unstemmed Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel
title_short Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel
title_sort monitoring of circulating car t cells: validation of a flow cytometric assay, cellular kinetics, and phenotype analysis following tisagenlecleucel
topic Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8926389/
https://www.ncbi.nlm.nih.gov/pubmed/35309367
http://dx.doi.org/10.3389/fimmu.2022.830773
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