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

Abstract

Single domain antibodies (sdAbs) correspond to the antigenbinding 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 ϳ300 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 ϳ1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ϳ2.3-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.For over 30 years, a large number of clinical trials have firmly consolidated the importance of lowering LDL-cholesterol (LDLc) 3 in the prevention of cardiovascular diseases (CVD) and its associated devastating sequelae (1). Healthy diets and exercise are highly recommended to lower LDLc in patients with high baseline levels. However, many individuals, including those suffering from familial hypercholesterolemia (FH), cannot reach the recommended LDLc levels to prevent cardiovascular complications. With an overall incidence of ϳ1:200, FH is a common inherited disease that affects at least 30 million people, of whom Յ1% have been diagnosed. It is characterized by plasma LDLc levels greater that the 95th percentile, which result in tendon xanthomas, xanthelasmas, corneal arcus, and premature atherosclerosis, leading to premature ischemic vascular disease and mortality if left untreated. In most cases, FH subjects exhibit mutations in the LDL receptor (LDLR; 67%) and its ligand apolipoprotein B (apoB; 14%), hampering LDL clearance from the circulation (2). In 2003, merging biological studies with human genetics led to the discovery of PCSK9, the 9th and last member of the family of proprotein convertases related to subtilisin and kexin (3), and the demonstration that the PCSK9 gene represents the 3rd locus of autosomal domin...

The novel llama‐human chimeric antibody has potent effect in lowering LDL‐c levels in hPCSK9 transgenic rats

“…To further verify the effect of the B11-Fc on lowering serum lipids in vivo, the hPCSK9 transgenic (Tg+) rat model was produced and induced by a high-fat diet. Before the formal experiment, we did a pre-experiment to probe an optimal dosage (20 mg/kg) according to the previous study [33]. In the formal experiment, the dual injections were on day 0 and day 7.…”

The Proprotein Convertases in Hypercholesterolemia and Cardiovascular Diseases: Emphasis on Proprotein Convertase Subtilisin/Kexin 9

“…Our data revealed that the C-terminal Cys-His-rich domain (CHRD) of PCSK9, which contains three repeat structures called M1, M2, and M3 (Cunningham et al, 2007), is needed for the PCSK9-induced degradation of the PCSK9-LDLR complex (Nassoury et al, 2007), including the repeat domain M2 (Saavedra et al, 2012), which has 14 His (Cunningham et al, 2007) and a number of natural mutations associated with it . This critical importance of the CHRD in regulating the ability of PCSK9 to induce the degradation of the LDLR was confirmed in two independent studies (Zhang et al, 2008;Poirier et al, 2016) and was shown to be inhibited by mAbs (Ni et al, 2010;Schiele et al, 2014) or singledomain antibodies that target the CHRD, without inhibiting the PCSK9-LDLR complex formation (Weider et al, 2016).…”

Hepatitis C virus and proprotein convertase subtilisin/kexin type 9: a detrimental interaction to increase viral infectivity and disrupt lipid metabolism

“…The structure of CHRD is organized in three Cys/His‐rich modules, termed M1, M2 and M3 53, 54. PCSK9 is synthesized as a zymogen and, in the ER, it undergoes autocatalytic cleavage, the latter process being required for PCSK9 maturation and secretion 55.…”

“…PCSK9 is a 692-amino acid secreted glycoprotein that consists of a signal sequence followed by a prodomain, a catalytic domain and a cysteine-and histidine-rich C-terminal domain (CHRD). The structure of CHRD is organized in three Cys/His-rich modules, termed M1, M2 and M3 [53,54]. PCSK9 is synthesized as a zymogen and, in the ER, it undergoes autocatalytic cleavage, the latter process being required for PCSK9 maturation and secretion [55].…”