Designed Ankyrin Repeat Proteins as Her2 Targeting Domains in Chimeric Antigen Receptor-Engineered T Cells

Chimeric antigen receptor (CAR) engineering is a branch of cancer immunotherapy that equips immune cells to target tumor antigens expressed on the cell surface using antibody-derived single-chain variable fragments (scFvs). However, other antibody mimetics, such as designed ankyrin repeat proteins (DARPins), can also serve as antigen-binding domains in CARs. This study shows that CAR-engineered T (CAR-T) cells utilizing Her2-targeting DARPins G3 and 929 can target human epidermal growth factor receptor 2 (Her2)-overexpressing cancer cells as effectively as CAR-T cells with the scFv 4D5 in vitro, and G3 CAR-T cells can slow or eliminate tumor growth in vivo as effectively as 4D5 CAR-T cells. Some DARPins may offer an attractive alternative to scFv usage in CARs, as they are smaller, thermodynamically stable, poorly immunogenic, and can be generated with different binding properties from DARPin libraries.