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The scFv antibody is considered an attractive molecule for constructing advanced therapeutic antibodies, such as CAR\u2010T cells and multi\u2010specific antibodies. However, the application of scFv antibodies in advanced drugs is limited by their propensity to oligomerize. Due to weak interactions between the VH and VL domains, scFvs exist in an equilibrium between open and closed states, leading to inter\u2010chain VH\u2013VL interactions that form dimers, trimers, and aggregates. In this study, we designed a novel peptide linker that interacts with the VH\u2013VL domain interface to shift this equilibrium toward the closed state. By comparing the structures of various antibodies, we identified a conserved groove in the interface region between the VH and VL domains, whose structure and sequence were conserved among antibodies. Utilizing various computational structural biological techniques, we designed a peptide fragment that binds to this groove effectively assembles the VH and VL domains together. The designed linker was introduced into scFv proteins with human and mouse frameworks and produced using a bacterial expression system. Size\u2010exclusion chromatography demonstrated that the novel linker suppressed dimer formation. Analysis after 7\u2009days of storage in aqueous solution revealed that the designed linker also suppressed oligomer formation. Surface plasmon resonance experiments revealed that antigen\u2010binding affinity was not compromised by the designed linker. The new linker will facilitate the application study of scFv antibodies.<\/jats:p>","DOI":"10.1002\/pro.70450","type":"journal-article","created":{"date-parts":[[2026,1,20]],"date-time":"2026-01-20T12:51:57Z","timestamp":1768913517000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Structure\u2010based design and biophysical characterization of the linker sequence that associate to the\n                    <scp>VH<\/scp>\n                    \u2013\n                    <scp>VL<\/scp>\n                    domain interface of the single\u2010chain Fv antibody to suppress oligomerization propensity"],"prefix":"10.1002","volume":"35","author":[{"given":"Kyo","family":"Okazaki","sequence":"first","affiliation":[{"name":"Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences Kumamoto University  Kumamoto 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