FAS Ablation Confers Resistance to Allogeneic CAR-T Rejection By T Cells in Absence of NK Cell Sensitization

Allogeneic chimeric antigen receptor T cells (allo-CAR-T) derived from healthy donors hold potential to overcome limitations associated with autologous cancer therapies, providing immediate access to standardized, affordable batches of CAR-T with improved efficacy. Concomitant to immune reconstitution, however, allogeneic CAR-T are rejected by the host’s immune system, resulting in reduced therapeutic efficacy and early relapses. To systematically identify targets overcoming allo-CAR-T rejection, we developed in vivo genome-wide CRISPR KO screens based on allogeneic T cell positive selection (screening for KO genes that promote resistance in fully mismatched immunocompetent mice). We show that Fas or B2m deletion increases the survival of allogeneic T cells in vitro and in vivo. Consequently, FAS or B2M silencing in anti-CD19 allo-CAR-T enhances their anti-leukemic activity and promotes mice survival. Nevertheless, while B2M KO allo-CAR-T become highly sensitive to NK cell-mediated rejection, FAS KO CAR-T, like control CAR-T, express normal levels of HLA-I and remain resistant to NK cell killing. We validated the results in a clinically relevant context using base-editing for multiplexing gene-KO in human CAR-T cells. CD3-FAS KO outperforms CD3-B2M KO CAR-T in the control of leukemia growth under allogeneic pressure by both T and NK cells. We conclude that CD3-FAS double-KO allogeneic CAR-T are partially protected from both T and NK cell-mediated allo-rejection, an approach with the potential to improve the efficacy of allogeneic cellular therapies in patients with B-cells neoplasms and more generally in cancer patients.