Design optimization of antibody-ligand motifs to enhance CAR-T redirection activity against solid tumors.

Antigenic heterogeneity and the tumor microenvironment remain major obstacles to effective chimeric antigen receptor T (CAR-T) therapy, but natural ligands engaging multiple antigens within tumors and their milieu offer a promising solution. Here, we present a multitarget switchable CAR-T (sCAR-T) strategy that integrates a universal receptor on T cells with an antibody-ligand motif, combining an anti-Her2 single-chain variable fragment (scFv) (4D5) and spliced VEGF-A (VEGF121) to target Her2, VEGFR1, and VEGFR2. Optimization of the switch and CAR hinge preserved the ligand's native dimeric conformation, enhancing antigen recognition and promoting immunological synapse formation. In syngeneic and xenograft models, sCAR-T achieves superior tumor eradication and vasculature disruption compared with conventional CAR-T, overcoming immune escape driven by antigenic heterogeneity. We further extend the sCAR-T design to target receptor tyrosine kinase-like orphan receptor 1 (ROR1), demonstrating that the antibody-ligand motif-based strategy provides a versatile framework for complex antigen targeting, with potential to improve efficacy and safety and enable broader application in immunotherapies targeting complex antigenic combinations.