Dual-inactivation of Regnase-1 and SOCS1 rewires exhausted CD8 + T cell fate to enhance anti-tumor functionality

ABSTRACT The solid tumor microenvironment inhibits the functionality of tumor infiltrating T cells recognizing cognate tumor antigen, driving their differentiation towards terminal exhaustion. Interventions are sought to enhance the anti-tumor functionality of tumor-reactive T cells for clinical benefit. The functional genome regulating CD8 + T cell function against solid tumors was mapped by performing genome-wide, focused, and combination in vivo CRISPR/Cas9 screens using OT1 and PMEL TCR transgenic T cells in B16-OVA, MC38-gp100 and EG7-OVA syngeneic tumor models. The ability of the top single hits and combinations, which include Regnase-1 and SOCS1, to enhance CD8 + T cell anti-tumor function was evaluated in the OT1/B16-OVA model with large and established tumors, the disseminated PMEL/B16F10 tumor model, and in a novel murine TIL syngeneic model. The impact of Regnase-1 and SOCS1 single and dual-inactivation on the differentiation of exhausted CD8 + T cell subsets and on long-term persistent memory following tumor clearance was evaluated in OT1 CD8 + T cells in the B16-OVA model. The impact of single and dual-inactivation of Regnase-1 and SOCS1 on the anti-tumor function of experimental human T cell therapeutics was characterized in CRISPR/Cas9-engineered human TIL derived in vitro and in mesothelin-targeting CAR-Ts in vivo. NF-κB and cytokine signaling were identified as the top pathways regulating CD8 + T cell anti-tumor function, with Regnase-1 and Suppressor of Cytokine Signaling 1 (SOCS1) the top single and combination edits regulating the accumulation of tumor-specific TCR transgenic CD8 + T cells in syngeneic tumor models. Dual-inactivation of Regnase-1 and SOCS1 cooperated through non-redundant mechanisms to strongly expand intermediate (Tex int ) and effector (Tex eff ) exhausted CD8 + T cells within lymphoid tissues and tumor, with CD8 + T cells rewired to display an enhanced effector state and suppressed expression of TOX. Dual-edited persistent T effector memory cells (T em ) were formed following tumor clearance. Lastly, Regnase-1 and SOCS1 inactivation enhanced human Tumor Infiltrating Lymphocyte (TIL) and chimeric antigen receptor T cells (CAR-T) therapy functionality. Collectively, this study systematically mapped pathways regulating CD8 + T cell anti-tumor functionality, with Regnase-1 and SOCS1 dual-inactivation found to maximize anti-tumor function through non-redundant mechanisms.