Retargeting CD19 Chimeric Antigen Receptor T Cells via Engineered CD19-Fusion Proteins

tumor cells. To enhance the modularity of this technology, we engineered a more robust CD19 ECD via deep mutational scanning with yeast display and flow cytometric selections for improved protease resistance and anti-CD19 antibody binding. These enhanced CD19 ECDs significantly increase, and in some cases recover, fusion protein expression while maintaining target antigen affinity. Importantly, CD19-FPs retarget CAR19s to kill tumor cells expressing multiple distinct antigens, including HER2, CD20, EGFR, BCMA, and Clec12A as N- or C-terminal fusions and linked to both antibody fragments and fibronectin ligands. This study provides fundamental insights into CD19 sequence-function relationships and defines a flexible and modular platform to retarget CAR19s to any tumor antigen.