A synthetic library for rapid isolation of humanized single-domain antibodies

Relative to conventional full-length immunoglobulin G (IgG) antibodies and antibody fragments, single-domain antibodies, derived from the antigen-binding domain of the immunoglobulin of camelid species or cartilaginous fish, hold great potential for many biotechnological applications due to their small size and excellent physicochemical properties. To bypass animal immunization and facilitate the isolation of antigen-specific single-domain antibodies with ease, we have constructed a synthetic single-domain antibody library comprising three diversified synthetic complementarity determining regions (CDRs) grafted into a humanized camelid heavy- chain antibody VH (VHH) framework. Using three types of model antigens, interleukin-1β (IL-1β), amyloid-β, and vascular endothelial growth factor, the constructed single-domain antibody library, which has a vast diversity of approximately 1.8 × 10¹⁰, was evaluated, and single-domain antibody sequences against them were identified.