College of Life Science and Engineering, Henan University of Urban Construction, Pingdingshan, 467036, China.
BGI-Shenzhen, Shenzhen, 518083, China.
Sci Rep. 2022 Jul 16;12(1):12211. doi: 10.1038/s41598-022-16453-3.
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.
PCSK9 是降低 LDL-c 的有效靶点。先前设计了一种针对人 PCSK9 的骆驼科与人嵌合重链抗体 VHH-B11-Fc。它具有强大的降血脂作用。然而，纳米抗体 VHH-B11 与人 PCSK9 的亲和力较低，而骆驼科 VHH 表现出一定的免疫原性。此外，尽管 VHH-B11 与人 Evolocumab 具有独特的 hPCSK9 结合表位，但相互作用的表位尚未确定。这可能会阻碍该分子从实验室走向临床。在本研究中，我们设计了各种构型来提高 VHH-B11 与人 PCSK9 的亲和力（<10 nM），进而增强了 VHH-B11-Fc 的成药性。然后，特异性突变了 17 个氨基酸以增加纳米抗体 VHH-B11 的人源化程度。通过噬菌体展示和测序技术，鉴定了 PCSK9 铰链区中的线性表位“STHGAGW”（氨基酸 447-452）是 VHH-B11-Fc 与人 PCSK9 相互作用的位点。与位于 PCSK9 催化区的 Evolocumab 相互作用表位不同，VHH-B11 的结合表位位于 PCSK9 的铰链区，这很少有报道。这些发现表明，需要探索这种相互作用的特定机制。
