O-13.03 Ablation of FAS confers allogeneic CD3- CAR T cells with resistance to rejection by T cells and natural killer cells

<h3>Background</h3> 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. <h3>Materials and Methods</h3> To systematically identify targets overcoming allo-CAR-T rejection, we developed <i>in vivo</i> genome-wide CRISPR KO screens based on allogeneic T cell positive selection (screening for KO genes that promote resistance in fully mismatched immunocompetent mice). <h3>Results</h3> We show that <i>Fas</i> or <i>B2m</i> deletion increases the survival of allogeneic T cells <i>in vitro</i> and <i>in vivo</i>. Consequently, <i>FAS</i> or <i>B2M</i> silencing in anti-CD19 allo-CAR-T enhances their anti-leukemic activity and promotes mice survival. Nevertheless, while <i>B2M</i> KO allo-CAR-T become highly sensitive to NK cell-mediated rejection, <i>FAS</i> 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. <i>CD3-FAS</i> KO outperforms <i>CD3-B2M</i> KO CAR-T in the control of leukemia growth under allogeneic pressure by both T and NK cells. <h3>Conclusions</h3> We conclude that <i>CD3-FAS</i> 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. S. Menegatti: None. S. Lopez-Cobo: None. A. Sutra Del Galy: None. J. Fuentealba: None. L. Silva: None. L. Perrin: None. S. Heurtebise-Chrétien: None. V. Pottez-Jouatte: None. A. Privat: None. A. Darbois Dalahousse: None. A. Simon: None. M. Laprie-Santenac: None. M. Saitakis: None. B. Wick: None. B. Webber: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Luminary Therapeutics. B. Moriarity: F. Consultant/Advisory Board; Significant; Luminary Therapeutics. O. Lantz: None. S. Amigorena: E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Mnemo Therapeutics. L. Menger: None.