Nanobodies: Robust miniprotein binders in biomedicine

Variable domains of heavy chain-only antibodies (V_HH), also known as nanobodies (Nbs), are monomeric antigen-binding domains derived from the camelid heavy chain-only antibodies. Nbs are characterized by small size, high target selectivity, and marked solubility and stability, which collectively facilitate high-quality drug development. In addition, Nbs are readily expressed from various expression systems, including E. coli and yeast cells. For these reasons, Nbs have emerged as preferred antibody fragments for protein engineering, disease diagnosis, and treatment. To date, two Nb-based therapies have been approved by the U.S. Food and Drug Administration (FDA). Numerous candidates spanning a wide spectrum of diseases such as cancer, immune disorders, infectious diseases, and neurodegenerative disorders are under preclinical and clinical investigation. Here, we discuss the structural features of Nbs that allow for specific, versatile, and strong target binding. We also summarize emerging technologies for identification, structural analysis, and humanization of Nbs. Our main focus is to review recent advances in using Nbs as a modular scaffold to facilitate the engineering of multivalent polymers for cutting-edge applications. Finally, we discuss remaining challenges for Nb development and envision new opportunities in Nb-based research.