Abstract 6590: Epigenetic suppression of transgenic T-cell receptor (TCR) expression in adoptive cell transfer (ACT) therapy
Authors: Nowicki Theodore S. , Farrell Colin , Morselli Marco , Rubbi Liudmilla , Campbell Katie , Berent-Maoz Beata , Comin-Anduix Begonya , Pellegrini Matteo , Ribas Antoni 
Published: September 2020

Background: ACT of TCR transgenic T cells recognizing shared tumor antigens is associated with robust initial response rates, but frequent disease relapse. This usually occurs in the setting of poor long-term persistence of cells expressing the transgenic TCR, generated most frequently using a murine stem cell virus (MSCV) gamma retroviral vector.

Patients and Methods: Between 2009 and 2016, 24 patients were treated at UCLA using transgenic TCR ACT directed against the tumor antigens MART-1 or NY-ESO-1 for the treatment of melanoma or sarcoma, respectively. We analyzed 16 of these patients' cell therapy products for in vivo persistence and expression of the transgenic TCR at baseline and over time. Transgenic TCR DNA, RNA, and surface protein expression were assessed over time via TCR sequencing, qRT-PCR, and MHC dextramer analysis via FACS, respectively. DNA methylation status of the MSCV 5'LTR promoter region was determined via targeted bisulfite sequencing. Custom biotinylated RNA probes that target the entire MSCV vector were utilized to assess whole vector methylation over time, as well as integration sites within the host genome.

Results: Despite overall strong persistence of the transgenic TCR at the DNA level in all patients over time, its expression was profoundly decreased over time at the RNA and protein levels. In a subset of these patients (N=6), the degree of TCR surface expression at day +70 was &lt;0.5%, and was significantly lower than the other patients (p&lt;0.0001). These patients displayed significant increases in DNA methylation over time within the MSCV 5'LTR promoter region, as determined via targeted bisulfite sequencing. These increases in MSCV promoter methylation were inversely correlated with the degree of transgenic TCR RNA and protein expression, and the patients with increases in the MSCV 5'LTR promoter region displayed significantly lower expression of the transgenic TCR at the protein and RNA levels (p&lt;0.001 and p&lt;0.01, respectively). Analysis of the entire MSCV vector revealed progressive increases in DNA methylation within the entire MSCV vector over time, which recapitulated the significantly higher degree of vector methylation in patients with the most profound decreases in transgenic TCR expression (p&lt;0.0001). These increases in vector methylation occurred independently of its integration site within the host genome.

Conclusions: Progressive acquisition of DNA methylation within the MSCV retroviral vector is associated with suppression of expression of the encoded transgenic TCR. These results have significant implications for the design of future transgenic cell therapies and their associated viral vectors.

Citation Format: Theodore S. Nowicki, Colin Farrell, Marco Morselli, Liudmilla Rubbi, Katie Campbell, Beata Berent-Maoz, Begonya Comin-Anduix, Matteo Pellegrini, Antoni Ribas. Epigenetic suppression of transgenic T-cell receptor (TCR) expression in adoptive cell transfer (ACT) therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6590.