A Tetrameric Biotin–Avidin Scaffold Using Anti-CD19 (FMC63) Traptavidin Fusion Enables High-Avidity CAR-T Cells for Leukemia Therapy

Chimeric antigen receptor-T cell (CAR-T) therapy shows promise for treating hematologic malignancies but faces limitations including insufficient CAR-T cell activation. We developed a novel tetramer CAR-T (tCAR-T) system targeting CD19, which is overexpressed in hematologic malignancies. We created a tetrameric antibody scaffold by linking anti-CD19 scFv to traptavidin, then engineered T cells expressing CD8 hinge, CD8 transmembrane, costimulatory domains (4-1BB, CD3ζ), and YFP, followed by biotinylation using biotin ligase. The tCAR-T system was assembled via biotin-avidin interaction. This tetrameric arrangement significantly enhanced antibody avidity, promoting stronger antigen engagement and demonstrating potent cytotoxicity against CD19⁺ B-cell lymphoblast lines including Raji, Nalm-6, and Ramos. Notably, tCAR-T exhibited superior antitumor activity compared to Kymriah, with enhanced cytokine release and improved target cell elimination. This innovative approach improves cancer treatment efficacy through enhanced binding avidity and modular targeting flexibility, demonstrating that tCAR-T can overcome limitations of conventional CAR-T therapy and highlighting its potential to improve therapeutic outcomes for cancer patients.