Refactored intracellular domains enhance CAR expression and function

Chimeric antigen receptor (CAR) intracellular domains (ICDs) couple antigen recognition to T-cell activation. Here we worked with AI agents to refactor CAR ICDs, proposing designs and refining them as measurements accumulated over three rounds. This approach yielded KRGR-ζ1-40, a 45-amino-acid ICD less than a third the length of the clinical 4-1BB-CD3ζ (BBζ) ICD. Compared with BBζ, KRGR-ζ1-40 increased CAR surface expression and antigen-driven IFN-γ release, without detectable antigen-independent IFN-γ release. KRGR-ζ1-40 extended CAR expression and target-cell killing after transient mRNA delivery, supported the expression of four CARs from a shared mRNA dose, and enhanced B-cell depletion after lentiviral and lipid-nanoparticle delivery. These results show that iterative agent-guided design with experimental feedback can yield compact ICDs that enhance CAR expression and sustain CAR T-cell function.