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bioRxiv · 10.64898/2026.10.28.762481

Refactored intracellular domains enhance CAR expression and function

Bai, L.·Kumar, A.·Desai, H. J.·Li, J.·de Alwis, D. P.·Hazuda, D.·Grigoryan, G.·Wang, W.·Yi, J.
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Abstract

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-{zeta}1-40, a 45-amino-acid ICD less than a third the length of the clinical 4-1BB-CD3{zeta} (BB{zeta}) ICD. Compared with BB{zeta}, KRGR-{zeta}1-40 increased CAR surface expression and antigen-driven IFN-{gamma} release, without detectable antigen-independent IFN-{gamma} release. KRGR-{zeta}1-40 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.

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Bai, L., Kumar, A., Desai, H. J., Li, J., de Alwis, D. P., Hazuda, D., Grigoryan, G., Wang, W., Yi, J.. 2026-10-29. Refactored intracellular domains enhance CAR expression and function. https://doi.org/10.64898/2026.10.28.762481

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