Abstract
Chimeric antigen receptor T cell (CAR-T) therapies have revolutionized cancer immunotherapy. Traditional single-chain variable fragments (ScFvs) used as CAR recognition moieties face challenges such as high tonic signaling, compromised binding epitopes, and suboptimal affinity. Single-domain antibodies (SdAbs) offer an attractive alternative due to their smaller size, stability, and reduced immunogenicity. In this work, we developed an SdAb-CAR-T cell discovery platform integrating generation, characterization, and selection of SdAbs based on various properties. This approach was demonstrated by developing CAR-T cells with SdAbs against CD33, a target for acute myeloid leukemia (AML). We identified diverse SdAbs against CD33, with affinities ranging from 3.9-115 nM, and characterized their binding kinetics and epitope recognition. Using SdAb-based second-generation CARs, we assessed tonic signaling, T cell phenotypes, cytotoxicity and cytokine release in vitro, resulting in reduced tonic signaling and increased cytokine production. In vivo, SdAb-based CAR-T cells exhibited enhanced efficacy at lower doses, in a xenograft AML mouse model, demonstrating advantages over ScFv-based CD33 CAR-T cells.