Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation

J Biol Chem. 2016 Aug 5;291(32):16659-71. doi: 10.1074/jbc.M116.717736. Epub 2016 Jun 8.

Abstract

Single domain antibodies (sdAbs) correspond to the antigen-binding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ∼300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ∼1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.

Keywords: Familial Hypercholesterolemia; LDL Receptor; cholesterol regulation; cysteine-histidine-rich-domain (CHRD); inhibitor; low-density lipoprotein (LDL); proprotein convertase subtilisin/kexin type 9 (PCSK9); single-domain antibody (sdAb,nanobody).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cholesterol, LDL / genetics
  • Cholesterol, LDL / metabolism*
  • HEK293 Cells
  • Humans
  • Mice
  • Mice, Knockout
  • Proprotein Convertase 9 / antagonists & inhibitors
  • Proprotein Convertase 9 / genetics
  • Proprotein Convertase 9 / metabolism*
  • Proteolysis / drug effects*
  • Receptors, LDL / genetics
  • Receptors, LDL / metabolism*
  • Single-Domain Antibodies / pharmacology*

Substances

  • Cholesterol, LDL
  • LDLR protein, human
  • Receptors, LDL
  • Single-Domain Antibodies
  • PCSK9 protein, human
  • Pcsk9 protein, mouse
  • Proprotein Convertase 9