Development and characterization of a camelid derived antibody targeting a linear epitope in the hinge domain of human PCSK9 protein

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.

Biparatopic anti-PCSK9 antibody enhances the LDL-uptake in HepG2 cells

“…In addition, we used SPR to determine the avidity of the Ab for hPCSK9, resulting in an avidity value of approximately 0.036 nM (Fig. 3 D), which represents a considerable improvement compared to B11-Fc antibody (~ 0.69 nM) 19 , 20 .…”

“…During PCSK9 Ab drug development, it is important to detect epitope differences in the interaction of the target antigen with Ab 19 . Specifically, epitope binning assay is required to avoid patent disputes and has been particularly relevant in recent long-term patent infringement litigation involving companies such as Sanofi and Amgen 25 .…”

“…Previously, we reported the development of a camelid-human chimeric HcAb, VHH-B11-Fc, developed based on the llama Ab library using phage display technology 19 , 20 . This Ab exhibited 100% inhibition of PCSK9 in hepatocellular carcinoma models at a specific concentration (1.5 µM).…”

“…This Ab exhibited 100% inhibition of PCSK9 in hepatocellular carcinoma models at a specific concentration (1.5 µM). Moreover, we characterized the interaction epitopes of VHH-B11 in the hinge region of PCSK9 as described previously 19 . In this study, we aimed to enhance the druggability of VHH-B11-Fc in lipid-lowering by designing a novel antibody 20 – 23 .…”

Identification of a Linear B Cell Epitope on p54 of African Swine Fever Virus Using Nanobodies as a Novel Tool

“…In fact, some research uses Nbs to identify special epitopes. For instance, Nbs against human proprotein convertase subtilisin/kexin 9 can recognize a linear epitope in the hinge domain that cannot be recognized by traditional MAbs ( 21 ). Due to the unique advantages of Nbs in recognizing antigenic epitopes, Nbs were used to identify epitope(s) of the p54 protein that were difficult to recognize by conventional antibodies in this study.…”