A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo1[S]

Stefania Di Marco,Carl P. Sparrow,Shilpa Pandit,Laurence B. Peterson,Ray Rosa,Ayesha Sitlani,Jon H. Condra,Jing Zhao ,Peter Haytko,Richard Cummings ,Cinzia Volpari,Holly A. Hammond,Lora Hamuro,Salvatore Vitelli,Lingyi Huang,Xiaomei Liu,Dana D. Wood,Rose M. Cubbon,Shengping Wang ,Andrea Carfı́ ,Brian K. Hubbard ,Lyndon J. Mitnaul,Douglas Wisniewski,Fubao Wang,Ni Yan ,M.j. Bottomley ,Douglas G. Johns,Jayne Chin,Xun Shen,Weirong Wang

doi:10.1194/jlr.m011445

Abstract

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

Highlights

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake
To determine whether the binding of 1D05-IgG2 affects PCSK9 function, we studied its effect on the PCSK9/LDLr interaction
Several anti-PCSK9 antibodies have been reported recently. These include monoclonal or polyclonal antibodies directed against the catalytic domain of PCSK9

Summary

Current address

There is extensive evidence that plasma PCSK9 raises LDLc levels by binding to cell surface LDL receptor (LDLr) protein and directing LDLr to lysosomes for degradation [7,8,9,10,11] Consistent with this mechanism, inhibition of PCSK9 by recombinant LDLr fragments [12,13,14] or by mono- or polyclonal antibodies [15, 16] restored LDLc uptake in cells. Intravenous (iv) injection of a monoclonal antibody (MAb) that disrupted the PCSK9/ LDLr interaction [15] or small interfering RNAs targeting liver PCSK9 [17] was found to reduce plasma LDLc in mice and nonhuman primates. By using highly sensitive dissociation-enhanced lanthanide fluorescence immunoassays (DELFIAs), which selectively detect total or antibody-free PCSK9, we demonstrate that the LDL-lowering effect of the 1D05-IgG2 antibody closely follows the reduction in antibody-free-PCSK9 levels and the increase in percentage of PCSK9 bound to 1D05-IgG2

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION