Antibody format matters: A comparative analysis of VHH and scFv domains reveals superior in vivo CAR T cell function with VHH domains.

Chimeric antigen receptor (CAR) T cells require an extracellular targeting domain for antigen specificity, typically an scFv. VHH antibodies are emerging as alternative CAR binding regions due to their reduced size, improved stability, and CDR3 loop architecture. Herein, we generated and compared VHH and scFv antibodies targeting CD123, via immunized phage display libraries, selecting ten antibody pairs based on comparable domain targeting and kinetic profiles (KD range M 10-9-10-11). The VHH antibody fragments showed improved stability (Tm50 Δ7.85°C), reduced aggregation, and a favorable surface charge. CARs incorporating a VHH demonstrated higher IL2 and IFNγ secretion than scFv-derived CARs during in vitro analysis and substantially enhanced survival and decreased tumor burden in a xenograft murine model of acute myeloid leukemia (AML). This comprehensive comparison of the two most adopted antibody classes provides a rationale for the selection of VHH as preferred CAR binding domains.