Abstract
PCSK9 is an effective target for lowering LDL-c. Previously, a camelid-human chimeric heavy chain antibody VHH-B11-Fc targeting human PCSK9 was designed. It had a potent hypolipidemic effect. However, the nanobody VHH-B11 interacts with PCSK9 at low affinity, while camelid VHH exhibits some immunogenicity. Moreover, the interacting epitope is yet to be identified, although VHH-B11 was shown to have distinct hPCSK9-binding epitopes for Evolocumab. This might impede the molecule's progress from bench to bedside. In the present study, we designed various configurations to improve the affinity of VHH-B11 with hPCSK9 (< 10 nM) that in turn enhanced the druggability of VHH-B11-Fc. Then, 17 amino acids were specifically mutated to increase the degree of humanization of the nanobody VHH-B11. Using phage display and sequencing technology, the linear epitope "STHGAGW" (amino acids 447-452) was identified in the hinge region of PCSK9 as the interacting site between VHH-B11-Fc and hPCSK9. Unlike the interaction epitope of Evolocumab, located in the catalytic region of PCSK9, the binding epitope of VHH-B11 is located in the hinge region of PCSK9, which is rarely reported. These findings indicated that a specific mechanism underlying this interaction needs to be explored.