Novel format single-domain antibodies using human VL domain as scaffold.

Single-domain antibodies (sdAbs) represent a unique class of antibody fragments with distinct advantageous characteristics: small monomeric size, excellent solubility and high thermal stability. However, most sdAbs currently available are derived from immunized animals and require humanization for therapeutic use. Notably, sdAbs feature a longer and variable-length third complementarity-determining region (CDR3)-a key structural element for antigen binding but one that increases the technical complexity of library construction. To address these challenges, this study, for the first time, describes the development of synthetic sdAb libraries using a stability-engineered human light-chain variable domain (VL) as the scaffold. We performed limited randomization on three loops located on the opposite side of the CDRs using degenerate codons, which were designed based on the natural length and amino acid properties of these loops. Eukaryotic expression assays confirmed that sdAbs generated via this randomization strategy exhibit excellent secretory expression efficiency. We constructed two ribosome display libraries using two sets of degenerate codon primers and compared the panning efficiency of two strategies against four model antigens (Jo-1, SSB, HAV, and CRP): standalone ribosome display and a combined approach of ribosome display followed by phage pIX display. Both strategies successfully yielded target-specific binders, with the combined panning method demonstrating superior performance to standalone ribosome display. The results demonstrate that synthetic sdAb libraries based on a stability-optimized human VL scaffold provide a facile and reliable source of functional sdAbs. This innovative design is anticipated to serve as a promising platform for the development of next-generation single-domain bispecific antibodies.