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Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients

Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products...

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Autores principales: Kunz, Alexander, Gern, Ulrike, Schmitt, Anita, Neuber, Brigitte, Wang, Lei, Hückelhoven-Krauss, Angela, Michels, Birgit, Hofmann, Susanne, Müller-Tidow, Carsten, Dreger, Peter, Schmitt, Michael, Schubert, Maria-Luisa
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078460/
https://www.ncbi.nlm.nih.gov/pubmed/32201711
http://dx.doi.org/10.1016/j.omtm.2020.02.003
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author Kunz, Alexander
Gern, Ulrike
Schmitt, Anita
Neuber, Brigitte
Wang, Lei
Hückelhoven-Krauss, Angela
Michels, Birgit
Hofmann, Susanne
Müller-Tidow, Carsten
Dreger, Peter
Schmitt, Michael
Schubert, Maria-Luisa
author_facet Kunz, Alexander
Gern, Ulrike
Schmitt, Anita
Neuber, Brigitte
Wang, Lei
Hückelhoven-Krauss, Angela
Michels, Birgit
Hofmann, Susanne
Müller-Tidow, Carsten
Dreger, Peter
Schmitt, Michael
Schubert, Maria-Luisa
author_sort Kunz, Alexander
collection PubMed
description Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products and monitoring of CAR T cell frequencies in patients are required. Quantitative polymerase chain reaction (qPCR) is the preferred method for VCN assessment. However, no standardized procedure with high reproducibility has been described yet. Here, we report on a single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) to determine VCN in CAR T cell products. SCG-DP-PCR was validated and compared to the absolute standard curve method (ACM) within the framework of a clinical trial treating patients with good manufacturing practice (GMP)-grade CAR T cells at the University Hospital Heidelberg. Methodologically, SCG-DP-PCR displayed technical advantages over ACM and minimized mathematical analysis. SCG-DP-PCR, as a highly reproducible approach, can be used for clinical follow-up of patients treated with CAR T cells or other GMOs and might replace established methods for VCN quantification. This work will enable clinicians to assess VCN, as well as CAR T cell frequencies, in patients as a basis for decisions on subsequent therapies, including repeated CAR T cell administration.
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spelling pubmed-70784602020-03-20 Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients Kunz, Alexander Gern, Ulrike Schmitt, Anita Neuber, Brigitte Wang, Lei Hückelhoven-Krauss, Angela Michels, Birgit Hofmann, Susanne Müller-Tidow, Carsten Dreger, Peter Schmitt, Michael Schubert, Maria-Luisa Mol Ther Methods Clin Dev Article Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products and monitoring of CAR T cell frequencies in patients are required. Quantitative polymerase chain reaction (qPCR) is the preferred method for VCN assessment. However, no standardized procedure with high reproducibility has been described yet. Here, we report on a single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) to determine VCN in CAR T cell products. SCG-DP-PCR was validated and compared to the absolute standard curve method (ACM) within the framework of a clinical trial treating patients with good manufacturing practice (GMP)-grade CAR T cells at the University Hospital Heidelberg. Methodologically, SCG-DP-PCR displayed technical advantages over ACM and minimized mathematical analysis. SCG-DP-PCR, as a highly reproducible approach, can be used for clinical follow-up of patients treated with CAR T cells or other GMOs and might replace established methods for VCN quantification. This work will enable clinicians to assess VCN, as well as CAR T cell frequencies, in patients as a basis for decisions on subsequent therapies, including repeated CAR T cell administration. American Society of Gene & Cell Therapy 2020-02-20 /pmc/articles/PMC7078460/ /pubmed/32201711 http://dx.doi.org/10.1016/j.omtm.2020.02.003 Text en © 2020 The Author(s) http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Kunz, Alexander
Gern, Ulrike
Schmitt, Anita
Neuber, Brigitte
Wang, Lei
Hückelhoven-Krauss, Angela
Michels, Birgit
Hofmann, Susanne
Müller-Tidow, Carsten
Dreger, Peter
Schmitt, Michael
Schubert, Maria-Luisa
Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients
title Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients
title_full Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients
title_fullStr Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients
title_full_unstemmed Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients
title_short Optimized Assessment of qPCR-Based Vector Copy Numbers as a Safety Parameter for GMP-Grade CAR T Cells and Monitoring of Frequency in Patients
title_sort optimized assessment of qpcr-based vector copy numbers as a safety parameter for gmp-grade car t cells and monitoring of frequency in patients
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078460/
https://www.ncbi.nlm.nih.gov/pubmed/32201711
http://dx.doi.org/10.1016/j.omtm.2020.02.003
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