Journal of Molecular Biology
Volume 426, Issue 4, 20 February 2014, Pages 843-852
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An Antibody against the C-Terminal Domain of PCSK9 Lowers LDL Cholesterol Levels In Vivo

https://doi.org/10.1016/j.jmb.2013.11.011Get rights and content

Highlights

  • We describe a functional antibody that binds to the CTD of PCSK9.

  • The antibody does not inhibit binding of PCSK9 to the LDLR.

  • Blocking of the CTD is sufficient to lower LDL cholesterol in cynomolgus monkeys.

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.

Introduction

Hypercholesterolemia describes the state of elevated levels of LDL (low-density lipoprotein) cholesterol and is closely linked to coronary heart disease, the major cause of morbidity and death in the world [1]. LDL is cleared from the blood by LDL receptor (LDLR) protein on the cell surface of hepatocytes [2]. The complex of LDL and LDLR is internalized by clathrin-mediated endocytosis. After migration to the endosome and subsequent acidification of the pH, LDL is released from the complex for degradation in the lysosomes [3], whereas LDLR is recycled and returned to the cell surface [4]. Loss-of-function mutations in LDLR or apolipoprotein B (ApoB), the component of LDL that binds to LDLR, result in familial hypercholesterolemia and coronary heart disease [5]. Statins are currently used as first-line drugs to treat hypercholesterolemia. They induce LDLR expression by inhibiting HMG-CoA reductase and cholesterol synthesis [6]. In 2003, proprotein convertase subtilisin/kexin type 9 (PCSK9), also referred to as NARC-1 (neural apoptosis-regulated convertase 1), was discovered as the ninth member of the proprotein convertase family [7]. Gain-of-function mutations in the PCSK9 gene were identified to be linked to autosomal dominant hypercholesterolemia and, therefore, as the third cause of familial hypercholesterolemia, besides mutations in the genes encoding LDLR and ApoB [8]. Subsequent studies demonstrated that PCSK9 overexpression in mice resulted in reduced LDLR protein levels in the liver and thus increased levels of serum cholesterol [9], [10], [11]. Individuals with loss-of-function mutations in PCSK9 display a reduced risk of coronary heart disease [12].

After cleavage of the signal peptide (residues 1–30), PCSK9 consists of three discrete domains, a prodomain (PD, residues 31–152), a catalytic domain (CD, residues 153–451) and a C-terminal domain (CTD, residues 452–692), that is also referred to as cysteine- and histidine-rich domain or cysteine-rich domain in the literature. In contrast to the other proprotein convertases, PCSK9 lacks a classical P domain that is important for folding and regulation of protease activity [13]. During maturation, PCSK9 undergoes autocatalytic cleavage between the prodomain and the catalytic domain in the endoplasmic reticulum [7]. This cleavage is necessary for subsequent secretion from the hepatocytes [7]. Unlike other proprotein convertases, PCSK9 lacks a second cleavage site at its prodomain. The prodomain remains associated with the catalytic domain, blocks access to the catalytic triad and thereby self-inhibits its proteolytic activity, as seen in the crystal structures [14], [15], [16].

Functional studies showed that PCSK9 does not degrade LDLR but binds to it on the surface of hepatocytes. In the presence of the endocytic adaptor protein ARH (autosomal recessive hypercholesterolemia) that binds LDLR in the cytosol, the whole PCSK9-bound receptor complex is internalized and undergoes lysosomal degradation [17], [18]. The main interaction is formed between PCSK9 and the epidermal growth factor-like repeat A (EGF-A) domain of LDLR [19], [20]. In 2011, Surdo et al. reported the crystal structure of PCSK9 in complex with full-length LDLR at neutral pH that provides an explanation for the mode of action of PCSK9 [21]. They identified an additional interaction between the prodomain of PCSK9 and the LDLR β-propeller domain, which keeps the LDLR in an extended conformation. At acidic endosomal pH, this inhibits rearrangements to a closed receptor conformation that is thought to be required for release of LDL and subsequent recycling of LDLR [22]. At the same time, the affinity of PCSK9 for EGF-A is enhanced at low pH [19], [23]. In addition to this extracellular mode of action, there is also an intracellular pathway of PCSK9-induced LDLR degradation [24]. However, the key mediator of LDLR function seems to be secreted PCSK9 as demonstrated by Chan and colleagues. In nonhuman primates, a monoclonal antibody against PCSK9 lowered serum LDL by 80% [25]. Several other groups described antibodies that showed comparable effects, thereby giving more evidence that this is a promising strategy to treat hypercholesterolemia [26], [27], [28]. Notably, statins have been found to up-regulate PCSK9 expression and thereby reducing their own LDL-lowering effects [29]. The activation of PCSK9 expression by statins was further confirmed and it was shown that PCSK9 transcription is also induced by HNF1a [30]. In a cellular assay, a combination of anti-PCSK9 monoclonal antibodies with statins increased LDLR levels more than either treatment alone [25] and was also effective in humans [28]. Such a combined treatment may be beneficial for patients not reaching desired LDL levels by statins alone or hypercholesterolemic patients who do not tolerate high doses of statins.

Here were report data on the generation and biophysical, structural and pharmacological characterization of an antibody named mAb1. Ni et al. previously described an antibody against the CTD of PCSK9 that lowers LDL uptake in a cellular assay [27]. Here we report on mAb1 that binds to the CTD, lowers LDL uptake in a cellular assay and shows LDL-lowering effects in vivo in cynomolgus monkeys.

Section snippets

Biophysical characterization

We determined the affinity of mAb1 to PCSK9 by injecting increasing concentrations of PCSK9 and PCSK9ΔCTD over a surface plasmon resonance (SPR) chip with mAb1 captured (Fig. 1A). PCSK9 binds with a KD of 4.1 nM (ka = 3.45 × 105 M 1 s 1; kd = 1.40 × 10 3 s 1) to mAb1. By comparison, a literature antibody 3H42 (PDB ID 3H42) that binds in an LDLR competitive fashion to the catalytic domain of PCSK9 was reported to have an affinity of 4 pM [25]. For PCSK9ΔCTD, no binding of mAb1 was observed; thus, the SPR

Antibody generation

Antibodies to PCSK9 were generated in mice using purified PCSK9 (see the expression section below) immobilized on polystyrene beads [49]. Hybridoma generation and screening was performed essentially as previously described [50].

Fab preparation

mAb1 was expressed in a stably transfected Chinese hamster ovary cell line. The full-length antibody was purified using MabSelect Xtra (GE Healthcare Life Sciences, New Jersey, USA) as described in the manufacturer's instructions. Purified mAb1 was digested with

Acknowledgements

We gratefully acknowledge the excellent technical assistance of Adelheid Loehle and Carmen Steinmetz. We thank Jessica Grunwald for measuring LDL cholesterol in the monkey serum.

References (62)

  • B. Dong et al.

    Strong induction of PCSK9 gene expression through HNF1α and SREBP2: mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters

    J Lipid Res

    (2010)
  • P. Bork et al.

    The immunoglobulin fold: structural classification, sequence patterns and common core

    J Mol Biol

    (1994)
  • M. Abifadel et al.

    Identification and characterization of new gain-of-function mutations in the PCSK9 gene responsible for autosomal dominant hypercholesterolemia

    Atherosclerosis

    (2012)
  • T. Kosenko et al.

    Low density lipoprotein binds to proprotein convertase subtilisin/kexin type-9 (PCSK9) in human plasma and inhibits PCSK9-mediated low density lipoprotein receptor degradation

    J Biol Chem

    (2013)
  • I.K. Kotowski et al.

    A spectrum of PCSK9 alleles contributes to plasma levels of low-density lipoprotein cholesterol

    Am J Hum Genet

    (2006)
  • L. Pisciotta et al.

    Additive effect of mutations in LDLR and PCSK9 genes on the phenotype of familial hypercholesterolemia

    Atherosclerosis

    (2006)
  • G. Mayer et al.

    Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels

    J Biol Chem

    (2008)
  • R.M. DeVay et al.

    Characterization of proprotein convertase subtilisin/kexin type 9 (PCSK9) trafficking reveals a novel lysosomal targeting mechanism via amyloid precursor-like protein 2 (APLP2)

    J Biol Chem

    (2013)
  • T. Yamamoto et al.

    A two-step binding model of PCSK9 interaction with the low density lipoprotein receptor

    J Biol Chem

    (2011)
  • C. Becker et al.

    Generation of monoclonal antibodies against human regulatory T cells

    J Immunol Methods

    (2010)
  • J.E. Park et al.

    Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR

    J Biol Chem

    (1994)
  • R. Karlsson et al.

    Experimental design for kinetic analysis of protein–protein interactions with surface plasmon resonance biosensors

    J Immunol Methods

    (1997)
  • E. Krissinel et al.

    Inference of macromolecular assemblies from crystalline state

    J Mol Biol

    (2007)
  • V.L. Roger et al.

    Heart Disease and Stroke Statistics—2011 Update 1

    Circulation

    (2011)
  • M.S. Brown et al.

    A receptor-mediated pathway for cholesterol homeostasis

    Science

    (1986)
  • H. Jeon et al.

    Structure and physiologic function of the low-density lipoprotein receptor

    Annu Rev Biochem

    (2005)
  • C.G. Davis et al.

    Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region

    Nature

    (1986)
  • M. Varret et al.

    Genetic heterogeneity of autosomal dominant hypercholesterolemia

    Clin Genet

    (2008)
  • P. Ma et al.

    Mevinolin, an inhibitor of cholesterol synthesis, induces mRNA for low density lipoprotein receptor in livers of hamsters and rabbits

    Proc Natl Acad Sci

    (1986)
  • N.G. Seidah et al.

    The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation

    Proc Natl Acad Sci

    (2003)
  • M. Abifadel et al.

    Mutations in PCSK9 cause autosomal dominant hypercholesterolemia

    Nat Genet

    (2003)
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