A bifunctional anti-PCSK9 scFv/Exendin-4 fusion protein exhibits enhanced lipid-lowering effects via targeting multiple signaling pathways in HFD-fed mice.

Fusion protein which encompasses more than one functional component, has become one of the most important representatives of macromolecular drugs for disease treatment since that monotherapy itself might not be effective enough to eradicate the disease. In this study, we sought to construct a bifunctional antibody fusion protein by fusing anti-PCSK9 scFv with Exendin-4 for simultaneously lowering both LDL-C and TG. Firstly, three Ex4-anti-PCSK9 scFv fusion proteins were constructed by genetically connecting the C-terminal of Exendin-4 to the N-terminal of anti-PCSK9 scFv through various flexible linker peptides (G4S)n (n = 2, 3, 4). After soluble expression in E. coli, the most potent Ex4-(G4S)4-anti-PCSK9 scFv fusion protein was selected based on in vitro activity assays. Then, we investigated the in vivo therapeutic effects of Ex4-(G4S)4-anti-PCSK9 scFv on the serum lipid profile and bodyweight changes as well as underlying molecular mechanism in HFD-fed C57BL/6 mice. The data showed that Ex4-(G4S)4-anti-PCSK9 scFv exhibits enhanced effects of lowering both LDL-C and TG in serum, reducing food intake and body weight via blocking PCSK9/LDLR, activating AMPK/SREBP-1 pathways, and up-regulating sirt6. Conclusively, Ex4-(G4S)4-anti-PCSK9 has the potential to serve as a promising therapeutic agent for effectively treating dyslipidemia with high levels of both LDL-C and TG.