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In this study, the human IGHV3\u201023*04 germline gene was chosen as the scaffold with a high expression level and favorable thermal stability. Amino acid diversity was introduced into the complementarity determining region 3 (CDR3) to exclude potential sequence liabilities. A library containing 2.6\u2009\u00d7\u200910<jats:sup>11<\/jats:sup> independent clones was successfully constructed. The receptor\u2010binding domain (RBD) of the SARS\u2010CoV\u20102 spike protein, interleukin\u201017A (IL17A), B\u2010cell maturation antigen (BCMA), and G\u2010protein coupled receptor family C group 5 member D (GPRC5D) were used as target antigens to screen and identify VHs. In each case, Thirty\u2010one to fifty\u2010five VHs were screened out. The VH\u2010Fc antibodies showed superior affinities (as high as 4.6\u2009nM) to the corresponding antigens but did not bind to antigen\u2010irrelevant cell CHO\u2010S. Furthermore, the anti\u2010RBD and anti\u2010IL17A VH\u2010Fc antibodies showed strong functional activity in the receptor\u2010blocking assays. The VH\u2010Fc antibodies from the synthetic library exhibited favorable developability (thermal stability, colloidal stability, hydrophilicity, anti\u2010aggregation ability, and no interaction with human IgGs). We demonstrated that high\u2010affinity and highly functional VH domain antibodies were generated from the rationally designed library with desired physicochemical properties. This approach is generally universal to target any antigen and has significant potential to accelerate candidate selection.<\/jats:p>","DOI":"10.1002\/pro.70090","type":"journal-article","created":{"date-parts":[[2025,3,18]],"date-time":"2025-03-18T15:15:04Z","timestamp":1742310904000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A synthetic heavy chain variable domain antibody library (<scp>VHL<\/scp>) provides highly functional antibodies with favorable developability"],"prefix":"10.1002","volume":"34","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-8908-9760","authenticated-orcid":false,"given":"Guiying","family":"Pang","sequence":"first","affiliation":[{"name":"College of Pharmacy Anhui University of Traditional Chinese Medicine  Hefei People's Republic of China"},{"name":"Institute of Innovative Medicine, Biocytogen Pharmaceuticals (Beijing) Co, Ltd.  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