Abstract
Single-chain variable fragments (scFvs) have been recognized as promising agents in cancer therapy. However, short serum half-life of scFvs often limits clinical application. Fusion to albumin affibody (ABD) is an effective and convenient half-life extension strategy. Although one terminus of scFv is available for fusion of ABD, it is also frequently used for fusion of useful moieties such as small functional proteins, cytokines, or antibodies. Herein, we investigated the internal linker region for ABD fusion instead of terminal region, which was rarely explored before. We constructed two internally ABD-inserted anti-HER2 4D5scFv (4D5-ABD) variants, which have short (4D5-S-ABD) and long (4D5-L-ABD) linker length respectively. The model structures of these 4D5scFv and 4D5-ABD variants predicted using the deep learning-based protein structure prediction program (AlphaFold2) revealed high similarity to either the original 4D5scFv or the ABD structure, implying that the functionality would be retained. Designed 4D5-ABD variants were expressed in the bacterial expression system and characterized. Both 4D5-ABD variants showed anti-HER2 binding affinity comparable with 4D5scFv. Binding affinity of both 4D5-ABD variants against albumin was also comparable. In a pharmacokinetic study in mice, the 4D5-ABD variants showed a significantly prolonged half-life of 34 h, 114 times longer than that of 4D5scFv. In conclusion, we have developed a versatile scFv platform with enhanced pharmacokinetic profiles with an aid of deep learning-based structure prediction.