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.

Autor: Pullarkat S; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America., Black G; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America., Bleakley M; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Department of Pediatrics, University of Washington, Seattle, Washington, United States of America.; Program in Immunology, University of Washington, Seattle, Washington, United States of America., Buenrostro D; Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Program in Immunology, University of Washington, Seattle, Washington, United States of America., Chapuis AG; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Program in Immunology, University of Washington, Seattle, Washington, United States of America.; Department of Medicine, Division of Hematology and Oncology, University of Washington, Seattle, Washington, United States of America., Hirayama AV; Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Department of Medicine, Division of Hematology and Oncology, University of Washington, Seattle, Washington, United States of America., Jaeger-Ruckstuhl CA; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America., Kimble EL; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Department of Medicine, Division of Hematology and Oncology, University of Washington, Seattle, Washington, United States of America., Lee BM; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America., Maloney DG; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Department of Medicine, Division of Hematology and Oncology, University of Washington, Seattle, Washington, United States of America., Radich J; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America., Seaton BW; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America., Specht JM; Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Department of Medicine, Division of Hematology and Oncology, University of Washington, Seattle, Washington, United States of America., Turtle CJ; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Department of Medicine, Division of Hematology and Oncology, University of Washington, Seattle, Washington, United States of America.; Integrated Immunotherapy Research Center, Fred Hutchinson Cancer Center, Seattle, WA, United States of America., Woolston DW; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America., Wright JH; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Program in Immunology, University of Washington, Seattle, Washington, United States of America., Yeung CCS; Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, Washington, United States of America.; Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, Washington, United States of America.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2024 Jun 06; Vol. 19 (6), pp. e0303057. Date of Electronic Publication: 2024 Jun 06 (Print Publication: 2024).
DOI: 10.1371/journal.pone.0303057
Abstrakt: 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.
Competing Interests: All authors have read and understand PLOS ONE’s policies. The below commercial interests did not fund and have no association with the work performed in this study; therefore, this does not alter our adherence to PLOS ONE’s policies on sharing data and materials. The authors of this manuscript have the following competing interests: A.G.C. received research funding from Juno Therapeutics, a Bristol Myers Squibb Company. E.K. received research funding from Juno Therapeutics, a Bristol Myers Squibb Company. A.V.H. has received research funding from Juno Therapeutics, a Bristol Myers Squibb Company, and Nektar Therapeutics; has received honoraria from Bristol Myers Squibb. C.J.T. has received research funding from Juno Therapeutics/BMS, Nektar Therapeutics. C.J.T. is on the scientific advisory board for Caribou Biosciences, T-CURX, Myeloid Therapeutics, ArsenalBio, Cargo Therapeutics, Celgene/BMS Cell Therapy. C.J.T. is a DSMB member for Kyverna. C.J.T. ad hoc advisory boards/consulting (last 12 months) include: Nektar Therapeutics, Century Therapeutics, Legend Biotech, Allogene, Sobi, Syncopation Life Sciences, Prescient Therapeutics, Orna Therapeutics, IGM Biosciences, Abbvie. C.J.T. stock options: Eureka Therapeutics, Caribou Biosciences, Myeloid Therapeutics, ArsenalBio, Cargo Therapeutics. C.J.T. has the right to receive payment from Fred Hutch as an inventor on patents related to CAR T-cell therapy C.Y. has received research funding from Minerva, Lonza. C.Y. consults for TwinStrand Biosciences.
(Copyright: © 2024 Pullarkat et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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