Data Sheet 1_Split intein-mediated reconstitution of regulatable CAR T-cells.pdf

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.</p>Methods<p>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.1^C 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.1^N split intein. </p>Results<p>Upon delivery of the adaptor module, the intein fragments undergo PTS, generating a functional membrane-bound CD1a CAR with splicing efficiencies of 85%.