Nanobodies in biomedicine: from molecular characteristics to fabrication and clinical translation.

Nanobodies (Nbs), the antigen-binding single-domain fragments derived from camelid heavy-chain antibodies (Abs), have rapidly become a focus of biomedical research due to their compact size, high stability, strong antigen affinity, and ease of molecular engineering. This review systematically outlines their structural and functional features, current strategies for acquisition, screening, optimization, and large-scale production, and comprehensively discusses their wide-ranging applications in therapeutics, diagnostics, and basic research. Specifically, Nbs have shown outstanding efficacy in tumor, toxin, infectious, and cardiovascular disease treatments, while serving as versatile tools for molecular imaging, biosensing, protein purification, structural analysis, and intracellular regulation. The challenges of immunogenicity, off-target effects, and industrial-scale manufacturing are also critically examined. Furthermore, the integration of artificial intelligence in structure prediction, de novo design, and immunogenicity assessment has opened powerful new avenues for rational Nb engineering. Combined with emerging technologies such as gene therapy, nanomaterial delivery, and multispecific architectures, these advances promise to accelerate clinical translation. Overall, Nb technology is poised to become a cornerstone of next-generation precision medicine and biotechnology, offering innovative solutions for disease diagnosis, targeted therapy, and molecular discovery.