Structure-based design and biophysical characterization of the linker sequence that associate to the VH-VL domain interface of the single-chain Fv antibody to suppress oligomerization propensity

A single-chain antibody is an artificial small size antibody fragment whose variable regions are connected by a flexible short peptide linker. The scFv antibody is considered an attractive molecule for constructing advanced therapeutic antibodies, such as CAR-T cells and multi-specific 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-chain VH-VL interactions that form dimers, trimers, and aggregates. In this study, we designed a novel peptide linker that interacts with the VH-VL 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-exclusion chromatography demonstrated that the novel linker suppressed dimer formation. Analysis after 7 days of storage in aqueous solution revealed that the designed linker also suppressed oligomer formation. Surface plasmon resonance experiments revealed that antigen-binding affinity was not compromised by the designed linker. The new linker will facilitate the application study of scFv antibodies.