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93 PCR assay to identify woodchuck hepatitis virus posttranscriptional regulatory element in CAR-T and TCR-T cells for fresh and formalin-fixed samples
  1. Shalini Pullarkat1,
  2. Jocelyn Wright1,
  3. David Woolston1,
  4. Erik Kimble1,
  5. Bo Lee1,
  6. Alexandre Hirayama1,
  7. Aude Chapuis1,
  8. Denise Buenrostro1,
  9. Graeme Black1,
  10. Marie Bleakley1,
  11. Brandon Seaton1,
  12. Jennifer Specht2,
  13. Cameron Turtle1 and
  14. Cecilia Yeung1
  1. 1Fred Hutchinson Cancer Center, Seattle, WA, USA
  2. 2University of Washington, Seattle, WA, USA


Background Chimeric antigen receptor T-cell (CART) and engineered T-cell receptor cytotoxic T cell (TCRT) therapy are adoptive cellular therapies developed by modifying T-cells to target surface antigens of cancer cells in CART, and both cell surface and intracellular antigens in the case of TCRT.1 Producing CART and TCRT cells involves transduction with viral vectors that usually contain the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) to enhance transcription.2 As adoptive cellular therapies become more widely used in cancer care, adverse events (AE) and safety monitoring questions have been raised requiring methods to quantify the amount of CART and TCRTs in patient tissues. We developed a PCR assay to target WPRE for use on archival formalin-fixed, paraffin embedded (FFPE) tissues with the goal of providing valuable information regarding CART/TCRT localization and AEs as most engineered cell products used at our institution are transduced with a viral vector containing WPRE.

Methods DNA from 4 pre-infusion CART (JCAR14 and Kymriah anti-CD19 CARTs) and TCRT (HA-1 TCRT, and MAGEA1 TCRT) products along with unmodified peripheral blood mononuclear cells (PBMC) were extracted.3,4,5 WPRE primers were designed to generate short amplicon of 159 base pairs for use on FFPE tissues. Using Phusion High-Fidelity PCR Kit (ThermoFisher Scientific) per manufacture protocol, we verified the assay with fresh DNA as well as FFPE samples. FFPE DNA was extracted from curls cut from paraffin blocks containing cell pellet admixtures of CART or TCRT products and human PBMCs resuspended in Histogel and embedded to mimic cells in fixed tissues.

Results Our PCR protocol detects CART/TCRT constructs in fresh and FFPE samples for JCAR14 CART, Novartis (Kymriah) CART, HA-1 TCRT, MAGEA1 TCRT products. Through limit-of-detection experiments, we detected fresh CART DNA in dilution up to 0.1% (figure 1a) in JCAR14 products. Using fresh TCR-T samples, we detected TCR DNA up to 0.1% (figure 1b) in HA-1 TCRT products. For FFPE-derived DNA, we detected WPRE sequences (figure 1c, d) in four samples with an input amount of 25ng CART/TCRT DNA and various CART/TCRT admixtures confirmed by WPRE-ISH on RNAscope (table 1).

Conclusions Detecting CART/TCRT using a WPRE PCR assay optimized for use on FFPE is a novel yet simple technique suitable for clinical application in understanding AEs and tumor localization of the adoptive cellular therapy. In detecting CART and TCRT in FFPE, we are now able to study trafficking of these immune effector cells when AE occurs or when post-treatment biopsies are available.


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  2. Wright JH, Huang L-Y, Weaver S, Archila LD, McAfee MS, Hirayama AV, et al. Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and Immunohistochemistry. Journal of Immunological Methods. 2021;492:112955.

  3. Hirayama AV, Gauthier J, Hay KA, Sheih A, Cherian S, Chen X, et al. Efficacy and toxicity of JCAR014 in combination with durvalumab for the treatment of patients with relapsed/refractory aggressive B-cell Non-Hodgkin lymphoma. Blood. 2018;132(Supplement 1):1680.

  4. Schuster SJ, Tam CS, Borchmann P, Worel N, McGuirk JP, Holte H, et al. Long-term clinical outcomes of Tisagenlecleucel in patients with relapsed or refractory aggressive B-cell lymphomas (Juliet): A Multicentre, open-label, single-ARM, phase 2 study. The Lancet Oncology. 2021;22(10):1403–15.

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Ethics Approval This study was approved by the University of Washington ethics review board. This study does not involve the use of animal or human subjects.

Abstract 93 Figure 1

(a) Limit of detection dilution series on fresh CART (JCAR14 product). Detection occurred to 0.1% CART , (b) Limit of detection dilution series on fresh TCR (HA-1). Detection occurred to 0.1% TCRT , (c) WPRE assay on FFPE tissues. Tested on 100% CART (+ control), JCAR14, and Novartis (Kymriah) CART products , (d) WPRE assay on FFPE tissues. Tested on 100% CART (+ control), Muc1*, Novartis (Kymriah), JCAR14, CD20 CART, and MAGEA1 TCRT

Abstract 93 Table 1

CART/TCRT products extracted from FFPE, with configuration of block as well as percentage of curl DNA made up of CART/TCRT DNA

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