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

PLoS One. 2024 Jun 6;19(6):e0303057. doi: 10.1371/journal.pone.0303057. eCollection 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.

MeSH terms

  • Formaldehyde*
  • Hepatitis B Virus, Woodchuck* / genetics
  • Humans
  • Immunotherapy, Adoptive / methods
  • Real-Time Polymerase Chain Reaction / methods
  • Receptors, Antigen, T-Cell* / genetics
  • Receptors, Antigen, T-Cell* / immunology
  • Receptors, Antigen, T-Cell* / metabolism
  • Receptors, Chimeric Antigen / genetics
  • Receptors, Chimeric Antigen / immunology
  • Receptors, Chimeric Antigen / metabolism
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism
  • Tissue Fixation / methods

Substances

  • Formaldehyde
  • Receptors, Antigen, T-Cell
  • Receptors, Chimeric Antigen

Grants and funding

Funding for this project was granted by The Judith A. Lese Breast Cancer Foundation. M.B. is supported by NIH/NCI 5 P30 CA015704-48. E.K. received The Paul Calabresi Career Development Award for Clinical Oncology in Pediatric and Medical Hematology/Oncology (NIH/NCI; 5K12CA076930-24). Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.