Rational Engineering of Notch1 Signaling to Boost the Proliferation of CD19-BBζ CAR-T.

CAR T-cells targeting CD19 have revolutionized the treatment of relapsed/refractory B-cell malignancies. However, approximately 50% of patients relapse. Patient-derived data indicates that CD19+ relapse is linked to poor CAR T-cell proliferation and/or persistence. We aimed to improve CD19-CAR T-cell survival by manipulating Notch1, a receptor that is activated following T-cell receptor ligation and is essential for upregulating pro-inflammatory and survival genes in T-cells. However, its role in the context of CAR T-cells remains poorly understood. We found that both lentiviral overexpression of Notch1's intracellular domain (N1ICD) and Notch1 genetic knockout resulted in defective CAR-T cell activation and proliferation. This suggests that CAR T-cells require a precise, balanced level of Notch1 signaling for strong effector function. The PEST domain, located at the most distal C-terminus of the Notch1 intracellular domain, regulates the receptor's half-life through ubiquitylation. To augment the levels of active Notch1 and prolong the receptor's signaling, we genetically deleted this domain in the endogenous N1ICD of CAR T-cells. Notch1 PEST-deleted CAR T-cells showed enhanced proliferation post-activation, leading to increased cytotoxicity against CD19+ tumors in vitro. Transcriptionally, Notch1 PEST deletion resulted in upregulation of interferon response pathways and proliferative genes during CAR activation, consistent with Notch1 activation. In B-ALL xenograft in vivo models, treatment with Notch1 PEST-deleted CAR T-cells resulted in greater tumor reduction and increased CAR-T expansion compared to control CAR T-cells. Our findings demonstrate a critical role of Notch1 in CAR-T effector function and provide a novel strategy to augment endogenous Notch1 activity by leveraging native gene regulation.