Development of novel humanized VHH synthetic libraries based on physicochemical analyses

Due to the high affinity and specificity of antibodies toward antigens, various antibody-based applications have been developed. Recently, variable antigen-binding domains of heavy-chain antibodies (VHH) have become an attractive alternative to conventional fragment antibodies due to their unique molecular characteristics. As an antibody-generating strategy, synthetic VHH libraries (including humanized VHH libraries) have been developed using distinct strategies to constrain the diversity of amino acid sequences. In this study, we designed and constructed several novel synthetic humanized VHH libraries based on biophysical analyses conducted using the complementarity determining region-grafting method and comprehensive sequence analyses of VHHs deposited in the protein data bank. We obtained VHHs from the libraries, and hit clones exhibited considerable thermal stability. We also found that VHHs from distinct libraries tended to have different epitopes. Based on our results, we propose a strategy for generating humanized VHHs with distinct epitopes toward various antigens by utilizing our library combinations.