JPET Fast Forward. Published on October 21, 2011 as DOI:10.1124/jpet.111.187419
JPET #187419
PCSK9 Antagonism Reduces LDL-cholesterol in Statin-treated Hypercholesterolemic Non-human Primates
Hong Liang, Javier Chaparro-Riggers, Pavel Strop, Tao Geng, Janette E. Sutton, Daniel Tsai, Lanfang Bai, Yasmina Abdiche, Jeanette Dilley, Jessica Yu, Si Wu, S. Michael Chin, Nicole A. Lee, Andrea Rossi, John C. Lin, Arvind Rajpal, Jaume Pons, and David L. Shelton
Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA 94080, USA
Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics.
JPET #187419
Running title: PCSK9 Antagonism and Statin in Hypercholesterolemic Monkey
Correspondence should be addressed to: Hong Liang, Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA 94080. Phone: 650-615-7331; Fax: 650-615-7350; E-mail:
[email protected].
Number of text pages: 26 Number of tables: 1 Number of figures: 4 Number of references: 43 Number of words in the Abstract: 139 Number of words in the Introduction: 823 Number of words in the Discussion: 1,308
Abbreviations: PCSK9, Proprotein Convertase Substilisin Kexin type 9; LDLR, Low Density Lipoprotein receptor; LDL-C, LDL-cholesterol; mAb, monoclonal antibody; ECD, extracellular domain; HDL-C, HDL-cholesterol; CHD, cardiovascular heart disease; ER, endoplasmic reticulum; EGF-A, epidermal growth factor-like repeat A; SREBP2, steroid response element binding protein 2; HNF alpha, hepatocyte nuclear factor 1 alpha; ATP, Adult Treatment Plan; NHP, Non-Human Primates; PCSK9-/-, PCSK9 knockout; huPCSK9, human PCSK9;
Recommended section assignment: Drug Discovery and Translational Medicine
JPET #187419
Abstract Proprotein Convertase Substilisin Kexin type 9 (PCSK9) promotes the degradation of Low Density Lipoprotein receptor (LDLR) and thereby increases serum LDL-cholesterol (LDL-C). We have developed a humanized monoclonal antibody (mAb) that recognizes the LDLR binding domain of PCSK9. This antibody, J16 and its precursor mouse antibody, J10 potently inhibit PCSK9 binding to LDLR extracellular domain (ECD) and PCSK9-mediated down regulation of LDLR in vitro. In vivo, J10 effectively reduces serum cholesterol in c57bl/6 mice fed with normal chow. J16 reduces LDL-C in healthy and diet-induced hypercholesterolemic cynomologous monkeys, but does not significantly affect HDL-cholesterol (HDL-C). Furthermore, J16 greatly lowered LDL-C in hypercholesterolemic monkeys treated with HMG-CoA reductase inhibitor simvastatin. Our data demonstrates that anti-PCSK9 antibody is a promising LDL-C lowering agent that is both efficacious and potentially additive to current therapies.
JPET #187419
Introduction Proprotein Convertase Subtilisin Kexin type 9 (PCSK9) has emerged in recent years as a promising therapeutic target for LDL-cholesterol (LDL-C) lowering. In human, gainand loss-of-function PCSK9 variants associate with hyper- or hypo-cholesterolemia, respectively (Abifadel et al., 2009). These mutations have been found to reduce or increase cardiovascular heart disease (CHD) risk. Furthermore, humans who are null for PCSK9 appear to be healthy and normal (Horton et al., 2007; Abifadel et al., 2009). PCSK9 is a secreted serine protease that is made mostly by the liver and intestine. It consists of a signal peptide, a prodomain, a catalytic domain and the histidine-rich Cterminal domain. The prodomain is self-cleaved in the endoplasmic reticulum (ER) after synthesis, and covalently bound to the mature protein thereafter. PCSK9 binds to the epidermal growth factor-like repeat A (EGF-A) domain on LDLR in a calcium dependent manner (Zhang et al., 2007). X-ray crystallography studies of co-crystals show that a region of the PCSK9 catalytic domain, distinct from the actual catalytic site, makes direct contact with the EGF-A domain (Kwon et al., 2008). Data show that PCSK9 asserts its function mainly through binding to hepatocyte LDLR and preventing LDLR recycling to the cell surface following endocytosis. This results in reduced LDLR levels, decreased cellular uptake of LDL-C and higher LDL-C levels in blood (reviewed in (Horton et al., 2009)).
Some evidence suggests that this occurs primarily on the cell surface of
hepatocytes, after PCSK9 has been secreted.
However, it has been suggested that
intracellular PCSK9 may target LDLR for degradation before it is chaperoned to the cell surface (Poirier et al., 2009), providing an additional mechanism to reduce LDLR levels. Thus the site of action of PCSK9 in vivo remains an open question. Elimination or reduction of PCSK9 activity via genetic deletion or pharmaceutical intervention, such as anti-sense, RNAi or monoclonal antibodies, has been shown to cause an increase in hepatocyte LDLR, and a subsequent reduction in serum LDL-C levels (Rashid et al., 2005; Graham et al., 2007; Frank-Kamenetsky et al., 2008; Chan et al., 2009; Gupta et al., 2010; Ni et al., 2011). Some of these interventions have also been shown to effectively reduce LDL-C in non-human primates (NHP) with normal diet and serum lipid levels.
JPET #187419
Statins are currently the most successful and widely used class of therapy for hyperlipidemia. Through inhibiting HMG-CoA reductase and cholesterol synthesis, they activate the transcription factor steroid response element binding protein 2 (SREBP2) and subsequently induce LDLR expression. SREBP2 also controls the expression of other genes involved in cholesterol metabolism, including PCSK9. While statin treatment increases LDLR transcription, it also increases PCSK9 transcription. In addition, statins have been found to up-regulate hepatocyte nuclear factor 1 alpha (HNF1alpha), which also induces PCSK9 expression (Dong et al., 2010). These increased levels of PCSK9, in turn, increase the degradation of LDLR protein, thus likely dampening the full potential effect of statin treatment (Dubuc et al., 2004; Park et al., 2004). It is therefore speculated that statins and PCSK9 inhibitors may have an additive or even synergistic effect on LDL-C levels. Indeed, mice lacking PCSK9 have been shown to be hypersensitive to statins (Rashid et al., 2005). Epidemiological studies have shown increased PCSK9 levels upon statin use and correlated statin responsiveness to PCSK9 loss of function mutants (Berge et al., 2006; Thompson et al., 2009; Konrad et al., 2011). Reports also indicated other lipid lowering therapies, such as fenofibrate and the Niemann-Pick C1Like 1 (NPCL1) inhibitor Ezetimibe, can increase circulating PCSK9 protein levels (Konrad et al., 2011). siRNA mediated knockdown of Pcsk9 led to a greater reduction in serum non-HDL-C in mice in combination with ezetimibe (Ason et al., 2011), suggesting that a PCSK9 inhibitor may have an additive effect with these major classes of current lipid-lowering medications as well. Population-based studies and pivotal clinical trials have shown that for most people there is a clear heart health benefit of lowering blood LDL-C to levels possibly even lower than the current Adult Treatment Plan (ATP) III guidelines (reviewed in (O'Keefe et al., 2004)). Current therapies, while well tolerated and efficacious in most incidences, are insufficient to provide optimal lowering of LDLC in a subset of patients due to inadequate efficacy, tolerability, or safety (reviewed in (Costet, 2010)). CHD is still the number one cause of death in the developed world, even with the advent and wide adoption of current lipid lowering therapies. There is clearly a need for new additive or replacement therapies that are effective and safe.
JPET #187419
We have identified a monoclonal antibody J16, which bind to PCKS9 and neutralizes its effect on LDLR degradation. Structural studies on co-crystals of this antibody bound to PCSK9 show that it binds to the same site on PCSK9 as LDLR. This LDLR-binding site is evolutionally conserved amongst rodent, NHP, and human, making J16 a potent cross species PCSK9 inhibitor. By using this antibody in mice and comparing its efficacy to that of genetic deletions we could directly test the hypothesis that little, if any, effect on LDLR is mediated by intracellular PCSK9 under normal physiological conditions. We show that J16 effectively reduces serum cholesterol in mice, to a level similar to that observed in PCSK9 -/- mice, demonstrating that the majority, if not all, of PCSK9 function in vivo is mediated via circulating PCSK9.
We also show that J16 can
selectively lower LDL-C in Non-Human Primates (NHP) with normal or elevated cholesterol levels. Finally, we were able to test and confirm the hypothesis that PCSK9 antagonism can provide further LDL-C lowering when co-administered with a statin.
JPET #187419
Methods Generation of Proteins and antibodies Recombinant human PCSK9 protein was produced as described in (Cunningham et al., 2007). Mouse and cynomolgus PCSK9 protein was produced by cloning the cDNA sequences into mammalian expression vector PRK5 with the addition of a 6-His tag at the C-terminus,
transiently
LipofectamineTM
transfected
(Invitrogen
and
Corp)
expressed following
in
HEK293
manufacturer’s
cells
using
instructions.
Recombinant proteins were purified from conditioned media using a Ni column (Qiagen Inc) using standard techniques. LDLR extracellular domain (ECD) was purchased from R&D system. Mouse antibodies to human PCSK9 were generated by immunizing wild type Balb/c and 129/bl6 PCSK9-/- mice (Rashid et al., 2005) with recombinant human PCSK9. Immortalized lymphocytes were generated from spleen by fusion with an established cell line using standard hybridoma technology. Clones were screened for ability to bind huPCSK9 by ELISA, and purified from hybridoma cultures using mAb select beads (Pierce) and further screened for effects on huPCSK9-mediated LDLR down regulation in Huh7 cells using Western blot analysis (see below). Mouse antibody 5A10 was humanized and affinity matured to antibody 5A10-AFM using standard humanization and affinity maturation strategies (Bostrom et al., 2009). Animal Studies C57BL/6 were purchased from Charles River labs; LDLR -/- and LDLR -/+ mice were purchased from Jackson Labs. Mice were acclimated to the facility for two weeks prior to the start of experiments. PCSK9 -/- mice (Rashid et al., 2005) were purchased from Jackson Labs and bred at the facility.
Animals were housed conventionally under
ambient conditions, with free access to water and standard rodent chow unless otherwise specified. HF (60% kcal fat, 20% kcal protein, 20% kcal carbohydrate) and HF/HC (40% kcal fat, 20% kcal protein, 40% kcal carbohydrate, 2.8mg/kcal cholesterol) diet were purchased from Research Diets (cat # 12492, , and cat # D12108, respectively). Mice were fasted for 4 hours prior to collecting serum samples.
JPET #187419
A total of 16 male adult cynomolgus macaque (Macaca fascicularis) ranged from 5-9 years of age, 4 to 6 kgs of weight were used for the dose response of J16. Animals were randomized based on LDL-C levels at day-7 and weight, n=4/group. Animals were housed in stainless steel individual cages and fed ad libitum with Harlan Primate Diet 2050, 20% protein and water, and fasted overnight prior to collecting plasma samples. 12 female adult cynomolgus macaque (Macaca fascicularis) ranging from 3.5 to 4 kgs of weight, that were fed a primate high fat diet (62.1% kcal fat, 21.7% kcal carbohydrate, 16.2% kcal protein, and 0.1mg/kcal cholesterol) ( Harlan Primate Diet TD.06278) for at least 18 months were used in this study. Animals were housed in individual cage and maintained on the 35% fat diet with ad libitum access to water.
Animals were
randomized into 3 groups based on LDL-c levels at day -7 and weight (n=4/group). Groups 2 and 3 were administered via oral gavages with Crestor (Rosuvastatin) during study days 1 – 55 as a whole tablet at 10mg/animal; 10mg/kg on days 63 – 76 as a fine suspension in a 5% Gum Arabic vehicle; and Zocor (simvastatin) administered to the animals on study days 84 – 133 as a fine suspension in a 5% Gum Arabic vehicle at 50mg/kg. 3mg/kg J16 were administered via i.v. bolus injection in group 1 and 2 animals on day 14, and group 3 animals on day 105. Fasted plasma samples were taken at indicated timepoints starting from day -7 to day 133.
Crestor (Rosuvastatin Calcium,
AstraZeneca PLC) and Zocor (Simvastatin, Merck & Co., Inc) were purchased at local pharmacy. All animal maintenance and handling were conducted in accordance with the Institutional Animal Care and Use Committees in AALAAC accredited facilities. Serum and plasma samples were analyzed for lipid measurements (total cholesterol, HDL-C, LDL-C, triglyceride) using Ace Alera clinical chemistry System (Alfa Wassermann).
Results of lipid measurements were analyzed and graphed using
GraphPad Prism software. PCSK9-mediated Down Regulation of LDLR in Huh7 Cells Huh7 cells were allowed to grow to approximately 80% confluency in RPMI media containing 10% FBS and 4 mM glutamine, then changed to one containing 10% delipidated FBS for 8-16 hrs to induce LDLR expression. Cells were then incubated for 6-
JPET #187419
16 hours with 293 expression media supplemented with 6 μg/ml of human or mouse PCSK9, with or without test antibodies. Cells were lysed with lysis buffer (50 mM glycerol phosphate, 10 mM HEPES pH 7.4, 1% Triton X-100, 20 mM NaCl, and a cocktail of protease inhibitors (Roche)) by shaking at 4 C for an hour. Cell lysates were collected and analyzed for LDLR protein levels via Western blot analysis. PCSK9 and LDLR binding assay Recombinant mouse or human PCSK9 protein was biotinylated using a biotinylation kit (Pierce). Standard Elisa techniques were used to coat (1 μg/ml LDLR ECD), block, wash, and incubate with indicated concentrations of biotinylated PCSK9. LDLR-PCSK9 binding was stabilized by adding 50 μl of 4% formaldehyde + 4% sucrose + PBS solution for 5 min.
Plates were detected using HRP conjugated Strepavidin (Invitrogen),
developed using TMB substrate and signal was read at 450 nm. Western blot Analysis Western blot analysis of liver and Huh7 cell lysates was performed using either goat antimouse LDLR antibody (R&D systems), mouse anti-human LDLR antibody (R&D systems), or goat anti-GAPDH antibody (R&D systems). Signal was detected using Pierce ECL Western blot substrate (Thermo Scientific). Bands were quantified using ImageJ software (http://rsbweb.nih.gov/ij/). Affinity Determination of Antibodies The binding kinetics and affinities of PCSK9 antibodies, J10 and J16, to recombinantn human, cynolmolgus monkey, and mouse PCSK9 were measured on a surface plasmon resonance Biacore 2000 biosensor (GE Healthcare). The binding affinity of J16 to recombinant human PCSK9 was also determined using a KinExA 3000 system (Sapidyne Instruments Inc.). Detailed methods are provided in the supporting information. Crystallization, Data Collection, and Refinement hPCSK9:J16 Fab complex crystals were grown by the hanging drop method at 25 °C using a precipitant solution containing 100 mm Hepes, pH 7.2, and 7% PEG 10000. Plate shaped crystals appeared in a few days and were cryoprotected with 2-methyl-2,4pentanediol before flash freezing in liquid nitrogen. Diffraction data were collected at
JPET #187419
Advanced Light Source (ALS) beamline 5.0.2. The crystals belong to the space group P21 with unit cell dimensions a = 82.4 Å, b = 70.8 Å, c = 109.4 Å, and β = 96.14° and diffracted to 2.75 Å resolution (Table S3). All diffraction data were processed with DENZO and SCALEPACK (Otwinowski and Minor, 1997).
The structure of the
PCSK9:J16 Fab complex was solved by molecular replacement using PCKS9 structure (PDB code 2P4E) (Cunningham et al., 2007) and a model of J16 Fab using PHASER (McCoy et al., 2005). Alternate cycles of model building using the program COOT (Emsley and Cowtan, 2004), positional, TLS, and individual restrained thermal factor refinement in REFMAC (Murshudov et al., 1997), and addition of water molecules reduced the R and free R values to 22.5 and 28.2%, respectively, for all of the reflections. The coordinates for the structure have been deposited in the Protein Data Bank, PDB ID 3SQO. Epitope identification and buried area calculations: The epitope residues for J16 on the PCSK9 protein were identified by calculating the difference in accessible surface area between the hPCSK9:J16 crystal structure and PCSK9 structure alone. PCSK9 residues that show buried surface area upon complex formation with J16 were defined as being part of the epitope. The solvent accessible surface of a protein was defined as the locus of the centre of a probe sphere (representing a solvent molecule of 1.4 Å radius) as it rolls over the Van der Waals surface of the protein. The solvent accessible surface area was calculated by generating surface points on an extended sphere about each atom (at a distance from the atom centre equal to the sum of the atom and probe radii), and eliminating those that lie within
JPET #187419
equivalent
spheres associated with neighboring atoms as implemented in program
AREAIMOL (Briggs, 2000). Results Identification of Antagonistic Anti-PCSK9 Antibodies We screened for anti-PCSK9 monoclonal antibodies among hybridoma cell lines generated by fusing the spleen of various wild type and PCSK9 knockout (PCSK9 -/-) mice immunized with recombinant human PCSK9 (huPCSK9) protein.
Over 800
antibodies that were positive for PCSK9 binding by ELISA were tested for their ability to block recombinant huPCSK9 mediated LDLR-degradation in Huh7 cells. Approximately 60 clones (7%) showed some degree of recovery of LDLR protein levels in the presence of PCSK9. Only four antibodies produced by hybridoma cell lines showed complete block of PCSK9 activity. Interestingly, these were all obtained from PCSK9 -/- mice and all were also cross-reactive to mouse PCSK9. These four can mutually block each other’s binding to PCSK9 in ELISA and biosensor assays, indicating that they share overlapping epitopes. One of them, J10, binds to recombinant human, cynomolgus monkey and mouse PCSK9 with approximate affinities of 0.3nM, 0.5nM and 2.7nM, respectively, as determined by biosensor binding assays (Supplemental Figure 1). Figure 1A shows J10 was able to completely rescue LDLR levels in Huh7 cells treated with exogenous mouse and human PCSK9. J10 also completely and dose-dependently blocks binding of 1.4nM human PCSK9 to immobilized huLDLR ECD with IC50 of 1nM (Figure 1B). We engineered J10 into a human IgG2deltaA (Armour et al., 2002) and kappa chain antibody, and further improved its antigen binding affinity. The resulting antibody, J16, contains fully human sequence outside of the CDR regions.
It binds to recombinant
human PCSK9 molecules with a KD of approximately 5 pM as determined by KinExA (Supplemental Figure 2), to cynomolgus monkey with KD to be less than 100 pM, and to mouse PCSK9 with KD of 35pM, as determined by biosensor binding assays (Supplemental Figure 1). J16 completely blocks human and mouse PCSK9 binding to immobilized LDLR ECD, and can fully reverse the PCSK9-mediated LDLR suppression in Huh7 cells.
JPET #187419
Epitope Determination of J16 The co-crystal structure of the Fab region of J16 with recombinant human PCSK9 revealed that J16 binds mainly to the catalytic domain of PCSK9, and partly to the Cterminus of the prodomain (Figure 2A). J16 binds to a non-linear, three-dimensional epitope that almost perfectly overlaps with the LDLR EGF-A domain binding site on PCSK9 (Kwon et al., 2008) (Figure 2B). The interaction between PCSK9 and J16 is mediated by both light and heavy chains, with main contribution coming from heavy chain CDR3. The surface area covered by J16 is approximately 788 Å2, while the LDLR EGF-A domain buries 456 Å2. Crystallographic data table and methods can be found in the supporting information. Anti-PCSK9 Antibodies Reduce Cholesterol in Mice and Monkey The ability of the mouse antibody J10 to modulate blood cholesterol levels in vivo was tested in mice. Since rodents carry LDLR binding lipoprotein in their HDL particles, the increase of LDLR that would occur via PCSK9 inhibition is expected to cause a decrease in LDL-C, HDL-C and total cholesterol in the rodent, as was seen in the PCSK9 -/- mice (Rashid et al., 2005). Due to the very low levels of LDL-C in wild-type mice, that are difficult to reliably measure and calculate, we observed the most consistent and significant effect of PCSK9 neutralization in mice on total and HDL-cholesterol levels. When 10 mg/kg of J10 was administered as a single intraperitoneal (IP) dose to C57BL/6 mice fed a normal diet, serum cholesterol levels were maximally reduced to an average of 49.7mg/dl at day 7 post treatment, a 46% reduction, compared to 92.5mg/dl in saline treated controls (table 1). HDL-C level was on average 33.4mg/dl in the J10 treated group compared to 58.4mg/dl in saline treated controls, representing a 43% reduction. There was no significant change in triglyceride levels. Two sets of experiments were carried out to explore the mechanism of this effect on serum lipids. Liver LDLR levels were significantly increased (2.3 fold, p=0.001) in mice 7 days post treatment of 10mg/kg of J10 (Supplemental Figure 3). In addition, total cholesterol, HDL-C and LDL-C levels in LDLR -/- mice were not affected by 10mg/kg J10 treatment. J10 treatment was partially effective in LDLR-/+ mice, with a 22% (27mg/dl) reduction in total cholesterol, a modest but statistically significant 13%
JPET #187419
reduction in HDL-C, and a preferential 70% reduction in LDL-C (table 1). These data are consistent with the effect of J10 being mediated by inhibition of the effect of endogenous PCSK9 on LDLR. To understand the differential effect of PCSK9 inhibition on HDL- and LDL-cholesterol in primates, we administered humanized J16 into cynomolgus monkey fed a normal diet. J16 was administered as a single intravenous (IV) dose at 0.1, 1, 3 and 10 mg/kg (n=4/group). J16 significantly and rapidly reduced LDL-cholesterol at all doses (Figure 3A).
The magnitude, duration and accumulative percentage reduction over time
calculated by AUC of the LDL-C lowering was dose dependent (Figure 3A and supplemental Figure 4). Administration of 0.1mg/kg J16 caused a transient 50% drop in LDL-cholesterol levels at day 2 that quickly recovered by day 5. 1mg/kg dosing reached a maximum effect of 71% reduction in LDL-cholesterol on day 5, and began to recover immediately thereafter, reaching pre-dose levels by day 14. 3 mg/kg and 10mg/kg dosing maintained the 70% reduction in LDL-cholesterol levels until day 10 and day 21 postdosing, respectively, and fully recovered by day 21 and 31, respectively. Total cholesterol levels were also significantly reduced in all dosing groups, with a dose-response and time course similar to that of LDL-C, and a maximum reduction seen in the 10mg/kg group (up to 39%) lasting 21 days. There were no dose related changes observed in HDLcholesterol (Figure 3B) or triglyceride levels throughout the study. Effect of Anti-PCSK9 Antibodies in Hypercholesterolemic Models We tested the effect of anti-PCSK9 antibodies on mice fed with a High Fat (HF, 60% Kcal fat) or a High Fat/High Cholesterol (HF/HC, 40% Kcal fat with 2.8mg/kcal cholesterol) diet. 6 weeks old C57BL/6 mice were fed with HF or HF/HC diet for 6 weeks prior to the antibody treatments, which raised total cholesterol to 135mg/dl and 202mg/dl, respectively, compared to the ~90mg/dl we typically observe for animals fed on normal chow. A single 10mg/kg injection of J10 was given to mice fed with HF diet, and 4 consecutive weekly injections at 10mg/kg were given to mice fed with HF/HC diet. To our surprise, neither treatment paradigm had a significant effect on serum total cholesterol, HDL-C or LDL-C (table S1 and S2). Furthermore, PCSK9-/- mice fed on a high fat/high cholesterol diet also did not exhibit a reduced cholesterol levels compared
JPET #187419
to those on normal chow in our hands (Supplemental Figure 5). To further investigate the cause of the unresponsiveness of these diet-induced hypercholesterolemic mice to PCSK9 antagonism, we measured serum PCSK9 concentrations in these animals. Serum PCSK9 was reduced by 64% in mice fed with HF diet for 6 weeks (14.4 + 1.6 ng/ml) compared to those on normal chow (40.4 + 4.6ng/ml), n=5/group. We then investigated the efficacy of J16 in hypercholesterolemic non-human primates. Prior to the initiation of the study, the LDL-C levels of a cohort of cynomolgus monkeys were elevated to an average of 120 mg/dL, compared to the normal average levels of 50 mg/dL, by feeding with a diet containing 62% kcal fat and 0.1mg/kcal cholesterol for over 18 months. A single dose of 3 mg/kg J16 administered to these animals effectively lowered serum LDL-C levels by 64% by day 3 post-treatment; serum LDL-C levels gradually returned to pre-dosing levels by about 2.5 to 3 weeks post-treatment (Figure 4A). The percentage reduction and the duration of the effect are very similar to that observed in monkeys fed on a normal chow and treated with the same dose (3mg/kg) of J16 (Figure 3A). HDL-C levels were not affected by the treatment (Figure 4B). Total cholesterol followed a similar time course as LDL-C, with maximum lowering of 41% (Figure 4C). There were no significant changes related to J16 treatment in fasting triglyceride levels throughout the study. J16 Reduces LDL-C in Statin-treated Hypercholesterolemic Monkeys To test the pharmacodynamic interactions between J16 and HMG-CoA reductase inhibiting statins, we administered Crestor (Rosuvastatin calcium) or Zocor (simvastatin), daily to hypercholesterolecmic monkeys. Surprisingly, no effect was observed on serum levels of total cholesterol or LDL-C after the daily administration of a low-dose (10 mg/animal) of Crestor for 6 weeks, or a subsequent daily administration of high-dose (20 mg/kg) for 2 weeks. Liquid chromatography-mass spectrometry was used to measure plasma Rosuvastatin concentrations (Martin et al., 2002). 4+4 ng/ml and 23+12 ng/ml Rosuvastatin were detected in plasma samples taken on day 10 of low- or high- Crestor dosing, respectively, confirming proper drug exposure in these animals. Upon switching the animals to daily administration of high dose (50 mg/kg) Zocor, their LDL-C levels reached a maximal reduction of 43% at day 5, and stabilized thereafter (Figure 4D).
JPET #187419
After 3 weeks of 50mg/kg/day Zocor administration, these animals were treated with vehicle or a single dose of 3mg/kg J16 while still receiving 50mg/kg/day Zocor. J16 dosing caused an additional 65% reduction in LDL-C by day 5, and returned to predosing levels within 2 weeks (Figure 4D), demonstrating that the PCSK9 antagonist can greatly reduce LDL-C levels on top of a statin. Daily Zocor treatment caused a trend of gradual reduction in HDL-C. Treatment of J16 had no significant effect on HDL-C levels (Figure 4E). Total cholesterol levels gradually reduced after dialy Zocor treatment, reaching a maximal 42% reduction at day 14. Single injection of J16 produced an additional 36% maximal lowering 5 days after dosing (Figure 4F).
The animals were
closely monitored throughout the duration of the study for clinical signs, body weight, food intake, hematology, and serum chemistry.
No meaningful changes related to
Crestor, Zocor or J16 treatment were observed. More specifically, liver function, as judged by serum ALT and AST levels, kidney function, as judged by serum creatinine, urea, and electrolytes levels were not altered by statin or J16 treatment.
JPET #187419
Discussion Since the discovery of its role in cholesterol metabolism and LDLR regulation and the realization that PCSK9 is a promising therapeutic target for LDL-C lowering, the domains and structures important for PCSK9 function have been under intense investigation. The protease activity has been shown to be necessary for self-cleavage, folding and secretion of the mature PCSK9 protein, but is not involved in the LDLR degradation (Li et al., 2007; McNutt et al., 2007). The binding epitope of PCSK9 to EGF-A of the LDLR has been localized to a conserved patch on the catalytic domain (Cameron et al., 2008; Kwon et al., 2008). The pro-domain and the c-terminus have also been implicated in PCSK9 activity or even direct binding to LDLR (Kwon et al., 2008; Mayer et al., 2008; Zhang et al., 2008). Self-association through an unknown mechanism has been observed to enhance the LDLR degrading activity(Fan et al., 2008). We have searched for complete blockers for PCSK9 among more than 800 ELISA positive antibodies.
While approximately 7% of antibodies showed some blocking
activity, potent blockers that can fully inhibit PCSK9 function were only derived from immunizations in a PCSK9 -/- mouse. One of those antibodies was humanized and affinity matured and the x-ray structure of the Fab/PCSK9 complex shows the antibody binds to a 3-dimensional epitope that is nearly identical to that of the EGF-A domain of LDLR.
These results suggest that the LDLR binding epitope is critical in fully
antagonizing PCSK9 function.
This epitope is evolutionarily conserved from
invertebrate to vertebrate (Cameron et al., 2008), hence it may be difficult to raise antibodies against it in wild-type mice. In fact, the residues of the J16 epitope on PCSK9 are 100% identical between mouse and human PCSK9, compared to the 78% overall identity and the 69% identity of the remaining surface residues.
Our experience
demonstrates the power of using knockout mice in generating functional antibodies. Consistent with our observations, monoclonal antibodies that have been reported to have potent PCSK9 blocking activities sterically block the binding of LDLR (Chan et al., 2009; Duff et al., 2009; Ni et al., 2011). One of these antibodies was generated by screening of human libraries; another was generated in mouse engineered to express fully
JPET #187419
human antibodies, suggesting the immune tolerance and repertoire of these ‘humanized’ mice are distinct from that of a wild type mouse. The critical cellular location where PCSK9 asserts its function under physiological conditions is still not clearly established. Many studies have demonstrated an important role for secreted PCSK9 in LDLR down regulation. In vitro, recombinant PCSK9 binds to LDLR ECD (Cunningham et al., 2007), and exogenous PCSK9 protein effectively reduces both LDLR protein levels and LDL uptake in cultured cells (Lalanne et al., 2005; Maxwell et al., 2005). In vivo, injection or infusion of recombinant PCSK9 or blood from paired parabiotic mice overexpressing PCSK9 as a transgene, can reduce LDLR protein levels in the liver and consequently elevate cholesterol in mice (Lagace et al., 2006; Grefhorst et al., 2008; Schmidt et al., 2008). Evidence also points to the ability of intracellular PCSK9 to degrade LDLR. While inhibition of PCSK9 using a neutralizing antibody can reduce LDL cholesterol in non human primates by 80%, a level that might be expected for a complete or near complete inhibition of PCSK9 function, the same antibody, with 160pM binding affinity to mouse PCSK9, only showed partial reduction of total cholesterol in wild-type mice (36%) compared with the reported 52% for the PCSK9 -/- mice (Rashid et al., 2005; Chan et al., 2009). In vitro, an antibody-mediated reversal of the PCSK9 effect on LDL uptake was lower in cultured cells when PCSK9 was overexpressed in the cells rather than being added exogenously as a purified protein(Duff et al., 2009). Overexpression of PCSK9 in HepG2 cells has been shown to increase the degradation of the precursor LDLR in addition to mature LDLR(Maxwell et al., 2005). Moreover, transfection of a PCSK9 expressing construct can reduce LDLR levels in PCSK9 expressing cells, but not in surrounding non-transfected cells (Poirier et al., 2009). Finally, two PCSK9 mutants, S127R and D129G were reported to have reduced or apparently abolished secretion but are associated with familial hypercholesterolemia, strongly implying that intracellular PCSK9 can still participate in LDLR degradation (Benjannet et al., 2006; Cameron et al., 2006; Homer et al., 2008). In our experiments, the degree of reduction (46%) in J10 treated mice is very similar to that observed in PCSK9-/- mice in our hands (45%) and reported by others (52%) (Rashid et al., 2005), as well as that reported for anti-sense
JPET #187419
knockdown of PCSK9 (50%) (Graham et al., 2007; Frank-Kamenetsky et al., 2008; Gupta et al., 2010). We show that this reduction in serum cholesterol is through PCSK9 inhibition and consequently, LDLR up-regulation, since J10 does not have any detectable effect in mice lacking LDLR, and that LDLR protein level is increased in J10 treated animals compared to controls. Since we are not aware of evidence suggesting antibodies can interfere with the intracellular function of a protein, our results indicate that neutralizing circulating PCSK9 via a monoclonal antibody can remove most PCSK9 function under normal physiological conditions, and strongly suggests that the vast majority if not all of PCSK9 mediated cholesterol modulation effect is carried out via secreted PCSK9, at least in the mouse. We show that PCSK9 antagonism can effectively lower LDL-C levels without significantly
affecting
HDL-C
or
triglyceride
levels,
in
both
normal
and
hypercholesterolemic monkeys. While several means of PCSK9 antagonism have been reported to reduce LDL-C levels by 50-80% in normal monkeys (Frank-Kamenetsky et al., 2008; Chan et al., 2009; Ni et al., 2011), we show for the first time that PCSK9 antagonism is effective in a hypercholesterolemic monkey model caused by a long term high-fat diet. An anti-sense oligo specific to mouse PCSK9 has been reported to reduce serum cholesterol by 53% in mice fed with a Western diet (60% lard) (Graham et al., 2007). However, our anti-PCSK9 mAb has no significant effect on mice fed with HF or HF/HC diet, nor did we see a significant difference in PCSK9 -/- fed with HF/HC diet. Differences in diet, experimental and housing conditions may be responsible for this discrepancy. In our case, HF diet decreased serum PCSK9 levels by 64%, possibly partly explaining the lack of effect of further PCSK9 inhibition or knockout in diet-induced hypercholesterolemic mice.
Our data is also consistent with reports that dietary
cholesterol inhibits PCSK9 expression in rat liver (Persson et al., 2009). Unlike rodents where hypercholesterolemia caused by a diet high in cholesterol suppresses PCSK9 levels, studies have found that hypercholesterolemic humans have elevated PCSK9 levels (Lakoski et al., 2009; Welder et al., 2010). It is likely that the unresponsiveness to dietinduced hypercholesterolemia is a property of rodents but not primates, since we have shown that both the degree and duration of LDL-C reduction are similar in normal and
JPET #187419
hypercholesteromic monkeys treated with the same dose of antagonistic PCSK9 antibody J16. Finally, we show that PCSK9 antagonism result in effective LDL-C in statin-treated hyperlipidemic monkeys.
Interestingly one of the most potent statins commercially
available, Rosuvastatin, had no effect on plasma cholesterol levels in these monkeys with a daily dose of 20mg/kg. Treatment with a high dose (50mg/kg/day) of Simvastatin caused a 43% reduction in LDL-C and a 32% reduction in HDL-C. Addition of a single administration of J16 on top of Simvastatin treatment caused an additional 65% lowering of LDL-C with no significant effect on HDL-C. The percentage LDL-C lowering is similar to the 64% lowering observed with J16 mono-therapy in these monkeys, demonstrating a potential additive effect of treatment with a PCSK9 monoclonal antibody paired with that of statin. Importantly, we have not observed any significant changes related to statin or J16 treatment in body weight, clinical signs, liver or kidney functions throughout the study. Our results provide further confidence that PCSK9 antagonism, particularly anti-PCSK9 monoclonal antibodies can be promising new lipid modifying therapies that warrant further clinical testing.
JPET #187419
Acknowledgments We thank Shirish Shenolikar, Julie Hawkins, Yuli Wang, Kieran Geoghegan, and Xiayang Qiu for providing protocols, recombinant human PCSK9 protein, and discussions; Leila Boustany for assistance in construct generation, editing the manuscript, and discussions; Kathy Tsui and Zea Melton for their assistance with the mouse hybridoma work; Terri Martin for assistance in expressing J16; Daniel Malashock, Alanna Pinkerton and Kevin Lindquist for their assistance with biosensor work; Duc Tien, Joyce Chou, Ariel Pios, German Vergara, Ikeem Meriwether, Ronald Ong, Teresa Radcliffe and Gustavo Ruiz for their assistance in animal work.
JPET #187419
Authorship Contributions: Participated in research design: Liang, Strop, Abdiche, Lin, Rajpal, Pons, and Shelton. Conducted experiments: Liang, Chaparro-Riggers, Strop, Geng, Sutton, Tsai, Bai, Dilley, Yu, and Wu. Contributed new reagents or analytic tools: Abdiche, Chin, and Lee. Performed data analysis: Liang, Strop, Abdiche, and Rossi.
JPET #187419
References Abifadel M, Rabes JP, Devillers M, Munnich A, Erlich D, Junien C, Varret M and Boileau C (2009) Mutations and polymorphisms in the proprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol metabolism and disease. Hum Mutat 30:520-529. Armour KL, Atherton A, Williamson LM and Clark MR (2002) The contrasting IgGbinding interactions of human and herpes simplex virus Fc receptors. Biochem Soc Trans 30:495-500. Ason B, Tep S, Davis HR, Jr., Xu Y, Tetzloff G, Galinski B, Soriano F, Dubinina N, Zhu L, Stefanni A, Wong KK, Tadin-Strapps M, Bartz SR, Hubbard B, Ranalletta M, Sachs AB, Flanagan WM, Strack A and Kuklin NA (2011) Improved efficacy for ezetimibe and rosuvastatin by attenuating the induction of PCSK9. J Lipid Res 52:679-687. Benjannet S, Rhainds D, Hamelin J, Nassoury N and Seidah NG (2006) The proprotein convertase (PC) PCSK9 is inactivated by furin and/or PC5/6A: functional consequences of natural mutations and post-translational modifications. J Biol Chem 281:30561-30572. Berge KE, Ose L and Leren TP (2006) Missense mutations in the PCSK9 gene are associated with hypocholesterolemia and possibly increased response to statin therapy. Arterioscler Thromb Vasc Biol 26:1094-1100. Bostrom J, Lee CV, Haber L and Fuh G (2009) Improving antibody binding affinity and specificity for therapeutic development. Methods Mol Biol 525:353-376, xiii. Briggs P (2000) http://www.ccp4.ac.uk/newsletters/newsletter38/03_surfarea.html. CCP4 Newsletter 28. Cameron J, Holla OL, Berge KE, Kulseth MA, Ranheim T, Leren TP and Laerdahl JK (2008) Investigations on the evolutionary conservation of PCSK9 reveal a functionally important protrusion. Febs J 275:4121-4133. Cameron J, Holla OL, Ranheim T, Kulseth MA, Berge KE and Leren TP (2006) Effect of mutations in the PCSK9 gene on the cell surface LDL receptors. Hum Mol Genet 15:1551-1558. Chan JC, Piper DE, Cao Q, Liu D, King C, Wang W, Tang J, Liu Q, Higbee J, Xia Z, Di Y, Shetterly S, Arimura Z, Salomonis H, Romanow WG, Thibault ST, Zhang R, Cao P, Yang XP, Yu T, Lu M, Retter MW, Kwon G, Henne K, Pan O, Tsai MM, Fuchslocher B, Yang E, Zhou L, Lee KJ, Daris M, Sheng J, Wang Y, Shen WD, Yeh WC, Emery M, Walker NP, Shan B, Schwarz M and Jackson SM (2009) A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates. Proc Natl Acad Sci U S A 106:98209825. Costet P (2010) Molecular pathways and agents for lowering LDL-cholesterol in addition to statins. Pharmacol Ther 126:263-278. Cunningham D, Danley DE, Geoghegan KF, Griffor MC, Hawkins JL, Subashi TA, Varghese AH, Ammirati MJ, Culp JS, Hoth LR, Mansour MN, McGrath KM, Seddon AP, Shenolikar S, Stutzman-Engwall KJ, Warren LC, Xia D and Qiu X (2007) Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia. Nat Struct Mol Biol 14:413-419.
JPET #187419
Dong B, Wu M, Li H, Kraemer FB, Adeli K, Seidah NG, Park SW and Liu J (2010) Strong induction of PCSK9 gene expression through HNF1alpha and SREBP2: mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters. J Lipid Res 51:1486-1495. Dubuc G, Chamberland A, Wassef H, Davignon J, Seidah NG, Bernier L and Prat A (2004) Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 24:1454-1459. Duff CJ, Scott MJ, Kirby IT, Hutchinson SE, Martin SL and Hooper NM (2009) Antibody-mediated disruption of the interaction between PCSK9 and the lowdensity lipoprotein receptor. Biochem J 419:577-584. Emsley P and Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126-2132. Fan D, Yancey PG, Qiu S, Ding L, Weeber EJ, Linton MF and Fazio S (2008) Selfassociation of human PCSK9 correlates with its LDLR-degrading activity. Biochemistry 47:1631-1639. Frank-Kamenetsky M, Grefhorst A, Anderson NN, Racie TS, Bramlage B, Akinc A, Butler D, Charisse K, Dorkin R, Fan Y, Gamba-Vitalo C, Hadwiger P, Jayaraman M, John M, Jayaprakash KN, Maier M, Nechev L, Rajeev KG, Read T, Rohl I, Soutschek J, Tan P, Wong J, Wang G, Zimmermann T, de Fougerolles A, Vornlocher HP, Langer R, Anderson DG, Manoharan M, Koteliansky V, Horton JD and Fitzgerald K (2008) Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc Natl Acad Sci U S A 105:11915-11920. Graham MJ, Lemonidis KM, Whipple CP, Subramaniam A, Monia BP, Crooke ST and Crooke RM (2007) Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice. J Lipid Res 48:763-767. Grefhorst A, McNutt MC, Lagace TA and Horton JD (2008) Plasma PCSK9 preferentially reduces liver LDL receptors in mice. J Lipid Res 49:1303-1311. Gupta N, Fisker N, Asselin MC, Lindholm M, Rosenbohm C, Orum H, Elmen J, Seidah NG and Straarup EM (2010) A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR expression in vitro and in vivo. PLoS One 5:e10682. Homer VM, Marais AD, Charlton F, Laurie AD, Hurndell N, Scott R, Mangili F, Sullivan DR, Barter PJ, Rye KA, George PM and Lambert G (2008) Identification and characterization of two non-secreted PCSK9 mutants associated with familial hypercholesterolemia in cohorts from New Zealand and South Africa. Atherosclerosis 196:659-666. Horton JD, Cohen JC and Hobbs HH (2007) Molecular biology of PCSK9: its role in LDL metabolism. Trends Biochem Sci 32:71-77. Horton JD, Cohen JC and Hobbs HH (2009) PCSK9: a convertase that coordinates LDL catabolism. J Lipid Res 50 Suppl:S172-177. Konrad RJ, Troutt JS and Cao G (2011) Effects of currently prescribed LDL-C-lowering drugs on PCSK9 and implications for the next generation of LDL-C-lowering agents. Lipids Health Dis 10:38.
JPET #187419
Kwon HJ, Lagace TA, McNutt MC, Horton JD and Deisenhofer J (2008) Molecular basis for LDL receptor recognition by PCSK9. Proc Natl Acad Sci U S A 105:18201825. Lagace TA, Curtis DE, Garuti R, McNutt MC, Park SW, Prather HB, Anderson NN, Ho YK, Hammer RE and Horton JD (2006) Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice. J Clin Invest 116:2995-3005. Lakoski SG, Lagace TA, Cohen JC, Horton JD and Hobbs HH (2009) Genetic and metabolic determinants of plasma PCSK9 levels. J Clin Endocrinol Metab 94:2537-2543. Lalanne F, Lambert G, Amar MJ, Chetiveaux M, Zair Y, Jarnoux AL, Ouguerram K, Friburg J, Seidah NG, Brewer HB, Jr., Krempf M and Costet P (2005) Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells. J Lipid Res 46:1312-1319. Li J, Tumanut C, Gavigan JA, Huang WJ, Hampton EN, Tumanut R, Suen KF, Trauger JW, Spraggon G, Lesley SA, Liau G, Yowe D and Harris JL (2007) Secreted PCSK9 promotes LDL receptor degradation independently of proteolytic activity. Biochem J 406:203-207. Martin PD, Mitchell PD and Schneck DW (2002) Pharmacodynamic effects and pharmacokinetics of a new HMG-CoA reductase inhibitor, rosuvastatin, after morning or evening administration in healthy volunteers. Br J Clin Pharmacol 54:472-477. Maxwell KN, Fisher EA and Breslow JL (2005) Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment. Proc Natl Acad Sci U S A 102:2069-2074. Mayer G, Poirier S and Seidah NG (2008) Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels. J Biol Chem 283:31791-31801. McCoy AJ, Grosse-Kunstleve RW, Storoni LC and Read RJ (2005) Likelihood-enhanced fast translation functions. Acta Crystallogr D Biol Crystallogr 61:458-464. McNutt MC, Lagace TA and Horton JD (2007) Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells. J Biol Chem 282:20799-20803. Murshudov GN, Vagin AA and Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53:240-255. Ni YG, Di Marco S, Condra JH, Peterson LB, Wang W, Wang F, Pandit S, Hammond HA, Rosa R, Cummings RT, Wood DD, Liu X, Bottomley MJ, Shen X, Cubbon RM, Wang SP, Johns DG, Volpari C, Hamuro L, Chin J, Huang L, Zhao JZ, Vitelli S, Haytko P, Wisniewski D, Mitnaul LJ, Sparrow CP, Hubbard B, Carfi A and Sitlani A (2011) A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo. J Lipid Res 52:78-86.
JPET #187419
O'Keefe JH, Jr., Cordain L, Harris WH, Moe RM and Vogel R (2004) Optimal lowdensity lipoprotein is 50 to 70 mg/dl: lower is better and physiologically normal. J Am Coll Cardiol 43:2142-2146. Otwinowski Z and Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode,. Methods Enzymology 276 307-326. Park SW, Moon YA and Horton JD (2004) Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver. J Biol Chem 279:50630-50638. Persson L, Galman C, Angelin B and Rudling M (2009) Importance of proprotein convertase subtilisin/kexin type 9 in the hormonal and dietary regulation of rat liver low-density lipoprotein receptors. Endocrinology 150:1140-1146. Poirier S, Mayer G, Poupon V, McPherson PS, Desjardins R, Ly K, Asselin MC, Day R, Duclos FJ, Witmer M, Parker R, Prat A and Seidah NG (2009) Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation: evidence for an intracellular route. J Biol Chem 284:28856-28864. Rashid S, Curtis DE, Garuti R, Anderson NN, Bashmakov Y, Ho YK, Hammer RE, Moon YA and Horton JD (2005) Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc Natl Acad Sci U S A 102:5374-5379. Schmidt RJ, Beyer TP, Bensch WR, Qian YW, Lin A, Kowala M, Alborn WE, Konrad RJ and Cao G (2008) Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo. Biochem Biophys Res Commun 370:634-640. Thompson JF, Hyde CL, Wood LS, Paciga SA, Hinds DA, Cox DR, Hovingh GK and Kastelein JJ (2009) Comprehensive whole-genome and candidate gene analysis for response to statin therapy in the Treating to New Targets (TNT) cohort. Circ Cardiovasc Genet 2:173-181. Welder G, Zineh I, Pacanowski MA, Troutt JS, Cao G and Konrad RJ (2010) High-dose atorvastatin causes a rapid sustained increase in human serum PCSK9 and disrupts its correlation with LDL cholesterol. J Lipid Res 51:2714-2721. Zhang DW, Garuti R, Tang WJ, Cohen JC and Hobbs HH (2008) Structural requirements for PCSK9-mediated degradation of the low-density lipoprotein receptor. Proc Natl Acad Sci U S A 105:13045-13050. Zhang DW, Lagace TA, Garuti R, Zhao Z, McDonald M, Horton JD, Cohen JC and Hobbs HH (2007) Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation. J Biol Chem 282:1860218612.
JPET #187419
Footnotes
Disclosure of Potential Conflicts of Interest All authors: employment and ownership interest, Pfizer, Inc
This work was supported by Pfizer Inc.
Reprint request should be addressed to: Hong Liang, Rinat Laboratories, Pfizer Inc., 230 East Grand Avenue, South San Francisco, CA 94080. Phone: 650-615-7331; Fax: 650-615-7350; E-mail:
[email protected].
JPET #187419
Legends of Figures Figure 1. Inhibition of PCSK9 function by J10 in vitro. A) Western blot analysis showing the effect of J10 on the ability of mouse and human recombinant PCSK9 to down regulate LDLR protein levels in cultured Huh7 cells. B) The dose-response of J10 (circle) and an isotype control (diamond) to block the binding of recombinant biotinylated huPCSK9 to immobilized recombinant LDLR ECD in vitro. Grey dotted line: signal with no biotinylated PCSK9 added; Black dotted line: signal with no antibody added. The average signal from duplicate samples are plotted.
Figure 2. J16 binds to the same region of PCSK9 as the LDLR EGF-A domain. A) Crystal structure of the PCSK9 bound to the J16 Fab. PCSK9 is shown in gray surface representation, while J16 Fab is shown in magenta (light chain) and blue (heavy chain) cartoon representation. B) View of epitope residues for J16 Fab and for LDLR EGF-A on the PCSK9. PCSK9 is shown in gray surface representation. Residues buried upon J16 binding are shown in blue, while outline of the buried surface for the PCSK9:LDLR complex is shown in red. Buried surface areas were calculated as described in methods.
Figure 3. A dose-response of J16 in cynomolgus monkeys. Percentage of LDL-C (A), HDL-C (B) and total cholesterol (C) compared to baseline levels at day -3 in cynomolgus monkey were plotted. Animals were treated with 0.1 mg/kg (circle), 1 mg/kg (square), 3 mg/kg (triangle) or 10mg/kg (diamond) of J16 on day 0 via i.v. injection. Results are expressed as mean ± SEM, n=4/group.
Figure 4. Effect of J16 in hypercholesterolemic cynomolgus monkeys. Percentages of LDLC (A and D), HDL-C (B and E) and total cholesterol (C and F) compared to baseline levels at day -7 (A, B and C) and day 77 (D, E and F) were plotted. A, B and C: Hypercholesterolemic cynomulgus monkey treated with 3 mg/kg of J16 (green square) and vehicle (blue triangle) on day 14 via i.v. injection. D, E and F: Hypercholesterolemic cynomulgus monkeys treated with 3 mg/kg of J16 (green quare) or vehicle (blue triangle) on day 105 via i.v. injection, on top of daily
JPET #187419
50mg/kg Simvastatin treatment from days 84 through 126 . Results are expressed as mean ± SEM, n=4/group.
JPET #187419
Table 1. Total cholesterol (TC), LDL-C and HDL-C levels (mg/dl) + standard error in mice fed with normal chow treated with 10mg/kg J10 and vehicle control through i.v. injection (n=9-10/group). Serum samples were taken 7 days post treatment and measured for lipid concentrations. LDL-C in wild type mice were under detection limit. J10-treated
Vehicle-treated
Genotype TC
HDL-C
LDL-C
TC
HDL-C
LDL-C
Wild type
92.5+2.5
58.4+1.4
n/a
49.7+2.0*
33.4+0.9*
n/a
LDLR +/-
123.1+1.7
70.2+1.1
32.2+2.1
96.0+2.6*
61.1+1.4*
9.7+1.6*
LDLR -/-
253.6+11.2
77.8+2.5
139.6+8.7
266.5+9.9
82.3+2.2
145.7+9.4
*: p
