Split intein-mediated reconstitution of regulatable CAR T-cells.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of B-cell malignancies. However, strategies that improve the modularity and controllability of CAR signaling could broaden its applicability and safety across additional clinical contexts. Here, we describe a regulable CAR T-cell platform based on split intein-mediated protein trans-splicing (PTS) to conditionally reconstitute functional receptors in primary T-cells.

METHODS: As a proof of concept, we engineered a split CAR targeting CD1a (NCT05679895), a surface antigen expressed in T-cell acute lymphoblastic leukemia (T-ALL). The system consists of a membrane-anchored C-terminal scaffold containing the IMPDH.1C split intein fused to 4-1BB and CD3ζ signaling domains, and a soluble N-terminal adaptor composed of an anti-CD1a single-chain variable fragment (scFv) fused to the complementary IMPDH.1N split intein.

RESULTS: Upon delivery of the adaptor module, the intein fragments undergo PTS, generating a functional membrane-bound CD1a CAR with splicing efficiencies of 85%. Reconstituted CAR T-cells exhibited antigen-specific cytotoxicity against CD1a+ T-ALL cells coupled to high levels of interferon-γ secretion.

DISCUSSION: These findings demonstrate that split intein-mediated CAR reconstitution enables a modular platform for conditional CAR reconstitution and transient control of receptor availability.