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qPCR assay for detection of Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Elements from CAR-T and TCR-T cells in fresh and formalin-fixed tissue.

Authors :
Pullarkat, Shalini
Black, Graeme
Bleakley, Marie
Buenrostro, Denise
Chapuis, Aude G.
Hirayama, Alexandre V.
Jaeger-Ruckstuhl, Carla A.
Kimble, Erik L.
Lee, Bo M.
Maloney, David G.
Radich, Jerald
Seaton, Brandon W.
Specht, Jennifer M.
Turtle, Cameron J.
Woolston, David W.
Wright, Jocelyn H.
Yeung, Cecilia C. S.
Source :
PLoS ONE. 6/6/2024, Vol. 19 Issue 6, p1-13. 13p.
Publication Year :
2024

Abstract

As adoptive cellular therapies become more commonplace in cancer care, there is a growing need to monitor site-specific localization of engineered cells—such as chimeric antigen receptor T (CAR-T) cells and T-cell receptor T (TCR-T) cells—in patients' tissues to understand treatment effectiveness as well as associated adverse events. Manufacturing CAR-T and TCR-T cells involves transduction with viral vectors commonly containing the WPRE gene sequence to enhance gene expression, providing a viable assay target unique to these engineered cells. Quantitative PCR (qPCR) is currently used clinically in fresh patient tissue samples and blood with target sequences specific to each immunotherapy product. Herein, we developed a WPRE-targeted qPCR assay that is broadly applicable for detection of engineered cell products in both fresh and archival formalin-fixed paraffin embedded (FFPE) tissues. Using both traditional PCR and SYBR Green PCR protocols, we demonstrate the use of this WPRE-targeted assay to successfully detect two CAR-T cell and two TCR-T cell products in FFPE tissue. Standard curve analysis reported a reproducible limit of detection at 100 WPRE copies per 20μL PCR reaction. This novel and inexpensive technique could provide better understanding of tissue abundance of engineered therapeutic T cells in both tumor and second-site toxicity tissues and provide quantitative assessment of immune effector cell trafficking in archival tissue. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
19326203
Volume :
19
Issue :
6
Database :
Academic Search Index
Journal :
PLoS ONE
Publication Type :
Academic Journal
Accession number :
177722819
Full Text :
https://doi.org/10.1371/journal.pone.0303057