Adoptive cell therapies, also known as cellular immunotherapies, are a personalized treatment strategy that involves engineering patients’ immune cells to recognize and attack their own tumors. This therapy involves extracting T cells, a type of immune cells, from the patient, modifying them in the lab to enable targeting and killing of patient cancer cells, and then re-introducing them into the patient to perform this function. A variety of adoptive cell therapies are available, including engineered T cell receptor (TCR) therapy and chimeric antigen receptor (CAR) T cell therapy. In TCR therapy, T cells are equipped with a new T cell receptor that targets specific cancer antigens–molecules unique to or highly expressed by cancer cells that often exist on the cell surface. Engineered T cells can only recognize antigens presented by antigen presenting cells (APCs) via the major histocompatibility complex (MHC). MHCs are proteins present on the surface of APCs that bind and present these antigens to T cells for recognition. As a result of the T cell receptor meeting an MHC-presented cancer antigen, T cells are activated to seek out and destroy cancerous cells. In contrast, CAR T cell therapy involves extraction of T cells from a patient, they are genetic engineered so that they express an entirely new antigen receptor -called CAR. The CARs can bypass the need for antigen-presenting cells (APCs) to mediate their activation by specifically binding to antigens on the surface of cancer cells. As these therapies become more common in cancer treatment, several questions regarding their ability to traffic to and kill tumors as well as safety arise, since adverse events—although limited in occurrence—have been reported in patients who have received these therapies.

A major question has been are these therapeutic T cells making it into the tumors and will a second dose help? One particular question related to safety is, are there non- tumor sites where these engineered cells congregate, and do they cause problems? A challenge with attempting to answer these questions is the fact that these engineered T cells are in fact made to be similar to the patient’s own T cells so identifying them with routine lab testing is hard. Current clinical techniques such as immunohistochemistry cannot distinguish between normal or diseased T cells and therapy products such as CAR-T cells and TCR-T cells. Additionally, techniques such as flow cytometry require fresh or frozen viable cells and require a product-specific assay to identify the engineered cell. To tackle this problem, a team in Dr. Cecilia Yeung’s lab, comprising of Shalini Pullarkat and Dr. Jocelyn Wright, aimed to “identify CAR-T and TCR-T cells in patient samples in order to assess treatment effectiveness and monitor the trafficking of these cells.” “We aimed to develop a tool for understanding the causes of adverse events associated with these therapies,” Pullarkat added. 

 To answer this question, Pullarkat  took advantage of how these cells are engineered. Let me explain! Once researchers receive T cells, they use lentiviral vectors to deliver genetic material—aka genes—into cells, changing the instructions within the cells. It is important to note that these vectors contain a number of sequences required to enhance the expression of the target gene, which in this case is the TCR or CAR. One of these sequences is the  Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Elements (WPRE) sequence, which flanks the TCR or CAR gene for the genetic material delivered to cells in most all types engineered cells. Thus, this WPRE sequence can be detected by quantitative PCR (qPCR) only in genetically engineered CAR-T and TCR-T cells. Validation of this methodology was recently published in PlosOne.

To do this, the team developed a specific quantitative PCR (qPCR) assay for the detection of the WPRE sequences with high sensitivity that can be used in both fresh samples and FFPE tissues standardly acquired and archived in the course of clinical treatment. The first step was to generate a standard curve to determine the limit of detection of WPRE in patient samples. They found that qPCR can detect less than 100 copies of WPRE in a single reaction with high reliability. In addition, the team validated these results using a variety of FFPE samples, including small needle biopsies with low DNA yield, and demonstrated the ability to detect WPRE sequences in CAR-T and TCR-T cells within these tissues. Excitingly, these finding support that “this assay can be used in ongoing translational research studies to investigate CAR-T cell and TCR-T cell trafficking,” commented the authors. “Insight into whether engineered cells are homing, expanding, and persisting in tumor tissues can inform researchers of immunotherapy efficacy and barriers.” The authors added “this assay provides an avenue for retrospective analysis of past patient cases to assess product infiltration into tumors. Analysis using our assay on patient FFPE samples provided insight as to how these cells disseminated and persisted in vivo.” Furthermore, “assaying WPRE in tissue biopsies from sites of adverse events (AEs) via qPCR could contextualize engineered T cells’ contributions to tissue specific AEs, which can inform clinical decision making in turn,” they added.

Overall, Dr. Yeung and her team “propose a simple, novel, and cost-effective assay with a rapid turnaround time and broad sample type applicability for detecting tissue-specific CAR-T/TCR-T cell presence that is sensitive, specific, and applicable to different cell products.”

"We aim to use this research's findings to further study the adverse events that have occurred after immunotherapy treatment,” Yeung and Pullarkat commented. Now, the team is interested in “examining how CAR-T and TCR-T cells are contributing to the pathology of ICANS-CRS, pancytopenia, colitis, and dermatitis among several other observed adverse events.” In the near future, “our lab would like to establish a CAR-T/TCR-T detection clinical service to enable clinical grade assessment using assays such as this one, " Yeung and Wright concluded.

The spotlighted worked was funded by the Judith A. Lese Breast Cancer Foundation, the National Institute of Health, and the Paul Calabresi Career Development Award for Clinical Oncology in Pediatric and Medical Hematology/Oncology.

Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Drs. Marie Bleakley, Aude Chapuis, Erik Kimble, Alexandre Hirayama, David Maloney, Jerald Radich, Jennifer Specht, Cameron Turtle and Cecilia Yeung contributed to this work.

Pullarkat S, Black G, Bleakley M, Buenrostro D, Chapuis AG, Hirayama AV, Jaeger-Ruckstuhl CA, Kimble EL, Lee BM, Maloney DG, Radich J, Seaton BW, Specht JM, Turtle CJ, Woolston DW, Wright JH, Yeung CCS. 2024. 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.