Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody

Abstract

This article is available online at http://www.jlr.org atherosclerosis ( 7,8 ). Conversely, loss-of-function mutations are associated with reduced LDL-C and reduced risk of cardiovascular diseases ( 9-11 ).PCSK9 is expressed in multiple tissues, including liver, intestine, kidney, and cerebellum, of which the liver appears to be the major source of the circulating protein (12)(13)(14). It is synthesized as a 74 kDa proprotein, which is activated prior to secretion by the autocatalytic cleavage of its N-terminal prodomain ( 15 ). PCSK9 that is secreted from cells is comprised of the 14 kDa prodomain associated noncovalently with a 60 kDa mature domain, the latter consisting of an N-terminal catalytic domain and a cysteine-and histidine-rich C-terminal domain. In addition to this 74 kDa form (intact PCSK9), a smaller form (truncated PCSK9) is found in serum in which the N terminus of the catalytic domain is truncated by 7-8 kDa. This truncated PCSK9 represents up to 40% of the total circulating PCSK9 in humans (16)(17)(18). The site of cleavage in PCSK9 was identifi ed as Arg218 of the catalytic domain ( 16,17 ). Furin appears to be responsible for most of the cleavage in vivo, based on studies in mice with hepatocytespecifi c inactivation of the protease ( 18 ). The observation of reduced plasma levels of the truncated PCSK9 in humans heterozygous for the R218S gain-of-function mutation is also consistent with furin-mediated cleavage, as this mutation disrupts the RXXR sequence recognized by furin ( 18 ).Several reports have addressed the question of whether the truncated PCSK9 is active in the regulation of LDL-C. Studies in furin knockout mice found reduced circulating levels of truncated PCSK9 and less hepatic LDLR than in Abstract Lilly PCSK9 antibody LY3015014 (LY) is a monoclonal antibody (mAb) that neutralizes proprotein convertase subtilisin-kexin type 9 (PCSK9). LY decreases LDL cholesterol in monkeys and, unlike other PCSK9 mAbs, does not cause an accumulation of intact PCSK9 in serum. Comparing the epitope of LY with other clinically tested PCSK9 mAbs, it was noted that the LY epitope excludes the furin cleavage site in PCSK9, whereas other mAbs span this site. In vitro exposure of PCSK9 to furin resulted in degradation of PCSK9 bound to LY, whereas cleavage was blocked by other mAbs. These other mAbs caused a signifi cant accumulation of serum PCSK9 and displayed a shorter duration of LDL-cholesterol lowering than LY when administered to mice expressing the WT human PCSK9. In mice expressing a noncleavable variant of human PCSK9, LY behaved like a cleavage-blocking mAb, in that it caused signifi cant PCSK9 accumulation, its duration of LDL lowering was reduced, and its clearance (CL) from serum was accelerated. Thus, LY neutralizes PCSK9 and allows its proteolytic degradation to proceed, which limits PCSK9 accumulation, reduces the CL rate of LY, and extends its duration of action.PCSK9 mAbs with this property are likely to achieve longer durability and require lower doses than mAbs that cause antigen...

Safety and efficacy of LY3015014, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9): a randomized, placebo-controlled Phase 2 study

“…This reduced accumulation of LY may reflect a difference in LY binding to PCSK9 compared with other PCSK9 antibodies in development. 4 , 5 As the LY-binding epitope does not include the furin cleavage site, the antibody may bind while allowing normal proteolytic degradation of PCSK9, minimizing accumulation of PCSK9. 11 …”

“…LY3015014 (LY) is a humanized immunoglobulin G4 (IgG4) monoclonal antibody characterized by a comparatively long duration of action demonstrated in preclinical studies. 4 , 5 The durability in LDL-C reduction may result from LY binding to a site that permits normal proteolytic cleavage of PCSK9, which may result in a reduction of target-mediated drug disposition. Based on these data, we hypothesized that administration of LY every 8 weeks might be sufficient for LDL-C reduction and offer a significant dosing advantage over other anti-PCSK9 antibodies that require dosing every 2 or 4 weeks.…”

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

“…Finally, the fact that PCSK9 is inactivated by some proteases such as furin (Benjannet et al, 2006;Essalmani et al, 2011) might open new strategies to enhance this inactivation mechanism, and thus lower the levels of active PCSK9. Indeed, recently a new mAb was reported that inhibits PCSK9 activity but still allows furin to inactivate it, resulting in a longer halflife of the antibody (Schroeder et al, 2015). The future will tell which strategies targeting PCSK9, including longer lasting mAbs, RNA interference (Fitzgerald et al, 2014), and small-molecule inhibitor approaches (Seidah, 2013), will find their way in dyslipidemia, cardiology, and sepsis in clinics worldwide, which would result in affordable and safe treatments and/or prevention of lifethreatening conditions.…”

Quantitative characterization of the mechanism of action and impact of a ‘proteolysis-permitting’ anti-PCSK9 antibody

“…We recently described a PCSK9 antibody with a novel mechanism of action. 11 Unlike other reported catalytic domain PCSK9 inhibitory antibodies, LY3015014 (LY) binds to a PCSK9 epitope that allows cleavage of the intact active form of PCSK9 to the inactive, 52-kDa form by protease cleavage at Arg218 in the catalytic domain. LY binds N-terminally to the cleavage site, so LY will bind and inhibit full-length (FL) PCSK9 binding to LDL-receptor, but since it allows cleavage of the protein, FL PCSK9 levels do not accumulate in the serum of the animals.…”

“…Other PCSK9 antibodies caused significant accumulation of FL PCSK9 in humans and animals. 11,12 It was shown that LY allowance of PCSK9 cleavage led to increased potency and durability of effect on LDL-C levels compared with antibodies that did not allow this cleavage. It was also shown that PCSK9-mediated clearance of LY was diminished relative to other anti-PCSK9 antibodies.…”