Synthetic nanobodies for drug discovery—Design, construction, optimization, and applications

Nanobodies (Nbs) are single‐domain antibody fragments derived from camelid heavy‐chain‐only antibodies (HCAbs). Nbs can be readily produced in microbial expression systems and are widely used as research tools while also being actively explored for diagnostic and therapeutic applications. Nbs are traditionally isolated from either immune libraries generated through camelid immunization or naïve libraries constructed from nonimmunized donors. The development of synthetic Nb libraries has transformed this paradigm by eliminating the need for animal immunization and accelerating every stage of Nb discovery—including library design, construction, selection, and optimization—through integrated in vitro and computational approaches. Compared with conventional immune libraries, synthetic libraries provide greater experimental control, broader target coverage, and increased flexibility for generating binders against challenging or predefined targets. This review summarizes current strategies for the design, construction, optimization, and application of synthetic Nbs, with particular emphasis on advances in library engineering, computational design, and their emerging roles in biomedical research and drug discovery.