Intein‐based modular chimeric antigen receptor platform for specific <scp>CD19</scp> / <scp>CD20</scp> co‐targeting

Development of chimeric antigen receptor T-cell therapy has revolutionized the treatment of B-cell malignancies, although challenges such as antigen escape and tumor heterogeneity often decrease treatment success. Modular CARs targeting multiple antigens have been proposed as an interesting solution to address these challenges by reducing the likelihood of tumor cells evading treatment through the loss of a single antigen. In this study, we present a new modular CAR platform, termed CARtein, which takes advantage of intein interactions to jointly target CD19 and CD20 antigens. We demonstrate that the CARtein system, which features a universal CAR signaling backbone that covalently binds to specific scFv-intein recognition partners, generates fully active CARs. Functionality was validated using Raji cells and K562 cells expressing CD19 and/or CD20, observing significant T cell activation through NFAT and NFκB promoter activity and CD69 upregulation. Overall, our study lays the foundation for the establishment of a new way to target multiple antigens through a universal and inert CAR backbone with highly specific activation.