Epigenetic Suppression of Transgenic T-cell Receptor Expression via Gamma-Retroviral Vector Methylation in Adoptive Cell Transfer Therapy
Transgenic T-cell receptor (TCR) adoptive cell therapies recognizing tumor antigens are 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 using murine stem cell virus (MSCV) ?-retroviral vectors.  Analysis  of  clinical transgenic adoptive  cell  therapy  products in  vivo revealed  that  despite  strong persistence  of  the  transgenic  TCR  DNA  sequence  over  time,  its  expression  was  profoundly  decreased  over time  at  the  RNA  and  protein  levels.  Patients  with  the  greatest  degrees  of  expression  suppression  displayed significant  increases  in  DNA  methylation  over time  within  the  MSCV  promoter  region,  as  well  as  progressive increases in DNA methylation within the entire MSCV vector over time. These increases in vector methylation occurred  independently of  its  integration  site  within  the  host  genomes.  These  results  have  significant implications for the design of future viral-vector gene engineered adoptive cell transfer therapies. Overall design: 16 patients from our previous transgenic TCR ACT trials directed against MART-1 (n=4) and NY-ESO-1 (n=5) were selected for analysis. DNA library were prepared from PBMC samples at day 0 (infusion), day +30, and day +70. We then carried out target enrichment using RNA probes to capture the MSCV vector, followed by bisulfite conversion.