Abstract 1318: A PCSK9 C-terminal Domain Binding Fab Inhibits PCSK9 Internalization and Restores LDL-uptake
Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) binds to LDL receptor (LDLR) protein, which ultimately leads to LDLR degradation and inhibition of plasma LDL cholesterol (LDL-C) clearance. Consequently, PCSK9’s role in modulating circulating LDL-C makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally-occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a Fab (Fragment antigen binding) phage display library, we have identified an anti-PCSK9 Fab antibody, 1G08, from a phage display library, with sub-nanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, Fab 1G08 partially (~50%) inhibited PCSK9-dependent effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9/LDLR interaction, but inhibited the internalization of PCSK9 in HEK293 cells. Furthermore, limited proteolysis and site-directed mutagenesis studies demonstrated that 1G08 binds the PCSK9 C-terminal domain in a region of β-strands encompassing Arg549, Arg582 and Glu607. Consistent with these results, 1G08 fails to bind PCSK9ΔC, a C-terminal truncated variant of PCSK9. Additional studies revealed that PCSK9ΔC has a reduced ability to inhibit LDL uptake. Together, our results highlight a novel functional site at the C-terminal domain of PCSK9.