Discovery and preclinical development of a SdAb-based CAR-T technology for targeting CD33 in AML

Autores de IIS La Fe

• Nuria Gómez Cebrián

  Autor

• Leonor Puchades Carrasco

  Autor

Participantes ajenos a IIS La Fe

• Bernasconi-Bisio, Franco
• Molina, Eva
• Ibarra, Vianca
• Ibanez-Sala, Ines
• Rochira, Federica
• Jauregui, Patricia
• Rodriguez-Diaz, Saray
• Martinez-Turrillas, Rebeca
• Azagra-Barber, Inigo
• Jose Lasarte, Juan
• Vanrell, Lucia
• Roberto Rodriguez-Madoz, Juan
• Prosper, Felipe
• Pineda-Lucena, Antonio

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.

© 2025 The Authors.

Keywords

• CELLS; AVIDITY