A Comprehensive ddPCR Strategy for Sensitive and Reliable Monitoring of CAR-T Cell Kinetics in Clinical Applications.

Details

Serval ID
serval:BIB_CAF87A80906C
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
A Comprehensive ddPCR Strategy for Sensitive and Reliable Monitoring of CAR-T Cell Kinetics in Clinical Applications.
Journal
International journal of molecular sciences
Author(s)
Wiedemann G., Bacher U., Joncourt R., Solly F., Widmer C.C., Zeerleder S., Novak U., Pabst T., Porret N.A.
ISSN
1422-0067 (Electronic)
ISSN-L
1422-0067
Publication state
Published
Issued date
06/08/2024
Peer-reviewed
Oui
Volume
25
Number
16
Language
english
Notes
Publication types: Journal Article
Publication Status: epublish
Abstract
In this study, we present the design, implementation, and successful use of digital droplet PCR (ddPCR) for the monitoring of chimeric antigen receptor T-cell (CAR-T) expansion in patients with B-cell malignancies treated with different CAR-T products at our clinical center. Initially, we designed a specific and highly sensitive ddPCR assay targeting the junction between the 4-1BB and CD3ζ domains of tisa-cel, normalized with RPP30, and validated it using blood samples from the first tisa-cel-treated patient in Switzerland. We further compared this assay with a published qPCR (quantitative real-time PCR) design. Both assays showed reliable quantification of CAR-T copies down to 20 copies/µg DNA. The reproducibility and precision were confirmed through extensive testing and inter-laboratory comparisons. With the introduction of other CAR-T products, we also developed a corresponding ddPCR assay targeting axi-cel and brexu-cel, demonstrating high specificity and sensitivity with a limit of detection of 20 copies/µg DNA. These assays are suitable for CAR-T copy number quantification across multiple sample types, including peripheral blood, bone marrow, and lymph node biopsy material, showing robust performance and indicating the presence of CAR-T cells not only in the blood but also in target tissues. Longitudinal monitoring of CAR-T cell kinetics in 141 patients treated with tisa-cel, axi-cel, or brexu-cel revealed significant expansion and long-term persistence. Peak expansion correlated with clinical outcomes and adverse effects, as is now well known. Additionally, we quantified the CAR-T mRNA expression, showing a high correlation with DNA copy numbers and confirming active transgene expression. Our results highlight the quality of ddPCR for CAR-T monitoring, providing a sensitive, precise, and reproducible method suitable for clinical applications. This approach can be adapted for future CAR-T products and will support the monitoring and the management of CAR-T cell therapies.
Keywords
Humans, Receptors, Chimeric Antigen/genetics, Receptors, Chimeric Antigen/immunology, Receptors, Chimeric Antigen/metabolism, Immunotherapy, Adoptive/methods, T-Lymphocytes/metabolism, T-Lymphocytes/immunology, Kinetics, Reproducibility of Results, Polymerase Chain Reaction/methods, Sensitivity and Specificity, B-cell lymphoma, CAR-T cell therapies, acute lymphatic leukemia (ALL), droplet digital PCR (ddPCR), molecular monitoring
Pubmed
Open Access
Yes
Create date
09/09/2024 14:34
Last modification date
10/09/2024 7:17
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