Epigenetically programmed 'stem-like' tissue resident memory CAR T-cells exhibit robust activity against solid and liquid tumors [bulk_RNA]

Chimeric antigen receptor (CAR) T-cells have shown remarkable success in the therapy of hematological malignancies, but they have not yet proven nearly as effective in treating non-hematopoietic cancers. This study proposes augmentation of CAR T-cell function and accumulation in solid tumors by modifying the epigenetic landscape that governs early memory differentiation and adaptation to tissue residency. We identified that a key factor in human tissue-resident memory CAR T-cell (CAR-TRM) formation is activation in the presence of the pleotropic cytokine, TGF-ß, which epigenetically enforces a core transcriptional program of both 'stemness' and sustained tissue residency. This strategy results in a readily actionable in vitro production method for engineering peripheral blood T-cells into large numbers of 'stem-like' CAR-TRM cells resistant to epigenetically-imposed exhaustion that possess enhanced ability to accumulate in situ and rapidly kill cancer cells for more effective immunotherapy. Overall design: Anti-mesothelin M5 CAR-TCONV and CAR-TRM cells were harvested after nine days of in vitro expansion. CD8+ CAR T-cells were magnetically isolated using anti-mesothelin-coated beads (ACROBiosystems) and anti-CD8 microbeads (Miltenyi Biotec), followed by resuspension in 500µL of TRIzol (Invitrogen). Total RNA was prepared using RNA Clean & Concentrator™ kits (Zymo Research) following the manufacturer's instructions. Transcriptome sequencing was performed by Novogene using the NovaSeq 6000 system (Illumina, 150bp paired end reads).