Enhanced anti-tumor activity by zinc finger repressor-driven epigenetic silencing of immune checkpoints and TGFBR2 in CAR-T cells and TILs

Chimeric antigen receptor T (CAR-T) therapies have shown remarkable success in treating hematological malignancies. However, effectiveness against solid tumors remains limited due to the immunosuppressive tumor microenvironment (TME), such as transforming growth factor β (TGF-β) signaling and upregulated immune checkpoints (ICs). Furthermore, identifying universal, tumor-specific targets for CAR-T cells in solid tumors is challenging, but using reinvigorated, immunosuppressive-resistant tumor-infiltrating lymphocytes (TILs) could be a promising alternative approach. Unlike nucleases, which may induce genotoxic DNA double-strand breaks, multiplexed zinc finger repressors (ZFRs) offer a safer alternative for knocking out TME-related immunosuppressive factors. We epigenetically repressed PD-1 expression both in CAR-T cells and TILs from colorectal liver metastases. PD-1 repression did not affect T cell viability, proliferation, or functionality. In a murine B cell lymphoma model, PD-1-repressed CD19-CAR-T cells exhibited enhanced anti-tumor activity and improved survival. Notably, PD-1 repression alone did not increase cytotoxicity against a PD-L1-positive colorectal cell line <i>in vitro</i>. To further increase anti-tumor potency in this context, ZFR-expressing lentiviral vectors (LVs) targeting PD-1 and other ICs (LAG-3, TIM-3, and TIGIT) or TGFBR2 were developed, improving significantly the cytotoxic activity in TILs. This strategy highlights the potential to enhance tumor-reactive T cells and improve anti-cancer immunotherapies by epigenetically repressing immunosuppressive factors in the TME using multiplexed ZFRs.