Comprehensive genome editing confers ‘off-the-shelf’ CAR-T cells superior efficacy against solid tumors

Abstract Biochemical and immunological negative regulators converge to inhibit tumor-reactive Chimeric Antigen Receptor T (CAR-T) cells, which may explain clinical failures of CAR-T cell therapies against solid tumors. Here, we developed a multifaceted approach to genetically engineer allogeneic (‘off -the-shelf’) CAR-T cells resistant to both biochemical (adenosine) and immunological (PD-L1 and TGF-β) inhibitory signaling. We multiplexed an adenine base editor with a CRISPR-Cas12b nuclease to manufacture a CAR-T cell product comprising six gene edits to evade allorejection ( B2M, CIITA ), prevent graft-versus-host disease ( CD3E ) and resist major biochemical ( ADORA2A ) and immunological ( PDCD1 , TGFBR2 ) immunosuppressive barriers in solid tumors. Combinatorial genetic disruption in CAR-T cells enabled superior anti-tumor efficacy leading to improved tumor elimination and survival in humanized mouse models that recapitulated the suppressive features of a human tumor microenvironment (TME). This novel engineering strategy conferred CAR-T cells resistance to a diverse TME, which may unlock the therapeutic potential of CAR-T cells against solid tumors. One Sentence Summary Multiplex genome engineered CAR-T cells resistant to allorejection and the convergence of biochemical and immunological negative regulators within the tumor microenvironment exhibit superior efficacy against solid tumors.