EA044927B1 - COMPOSITIONS CONTAINING PCSK9-BINDING MOLECULES AND METHODS OF APPLICATION - Google Patents

Description

Related applications

This application claims benefit and priority to U.S. Provisional Application No.

62/672187, filed May 16, 2018, which is incorporated herein by reference in its entirety.

Field of technology to which the invention relates

The present disclosure relates to fibronectin-based proteins with scaffolding domains that bind proprotein convertase subtilisin/kexin-9 (PCSK9), as well as pharmaceutical compositions and methods of use thereof.

Prerequisites for creating the invention

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme encoded by the human PCSK9 gene on chromosome 1. PCSK9 binds to the low-density lipoprotein (LDL) particle receptor. The LDL receptor (LDLR) on the membranes of liver and other cells binds to and initiates the endocytosis of LDL particles from the extracellular fluid into the cells, thereby reducing the concentrations of circulating LDL particles. If PCSK9 is blocked, more LDLR is recycled and present on the cell surface to remove LDL particles from the extracellular fluid. Therefore, blocking PCSK9 may reduce LDL particle concentrations in the blood.

The PCSK9 inhibitors, the monoclonal antibodies alirocumab and evolocumab, have been approved as biweekly or monthly subcutaneous injections or infusions to reduce LDL particle concentrations when statins and other drugs have been ineffective or poorly tolerated. Injections once a month require several milliliters of medication to achieve the desired dose. Although formulations having higher concentrations of active agent may provide more desirable dosage regimens and volumes, this is hampered by limitations associated with solubility, increased viscosity, and instability of biologics, including the tendency to aggregate and form particulates. Carpenter JF, et al., Overlooking subvisible particles in therapeutic protein products: Gaps that may compromise product quality. Journal of Pharmaceutical Sciences Vol. 98, Issue 4 (2008).

What is needed are pharmaceutical compositions that target PCSK9 and have the potential for more desirable dosing regimens, smaller volumes and/or increased efficacy while maintaining an overall safe and well-tolerated profile.

The essence of the invention

The present disclosure provides compositions containing fibronectin-based scaffolding proteins that bind proprotein convertase subtilisin/kexin type 9 (PCSK9) with high affinity, and which can be stably formulated at high concentrations for maximum biological effect and more convenient dosing regimens, dosage volumes and convenient for patient delivery devices. The PCSK9 binding fusion protein contains a PCSK9 binding motif and the amino acid sequence of human serum albumin (HSA). The PCSK9 binding motif and the HSA amino acid sequence can be expressed as a genetic fusion or chemically conjugated.

The PCSK9 binding motif described herein is based on adnectin, a family of proteins derived from human fibronectin type III domain 10 (10Fn3) designed to bind with high affinity to the target. According to this disclosure, the PCSK9 binding fusion protein is stably formulated at high concentrations to provide maximum biological activity as well as convenient dosage regimens and volumes. The concentration of the PCSK9 binding fusion protein in the composition is at least 100 mg/ml. In some embodiments, the concentration of the PCSK9 binding fusion protein in the composition is at least about 200 mg/ml. In some embodiments, the concentration of the PCSK9 binding fusion protein in the composition is at least about 250 mg/ml, or at least about 275 mg/ml, or at least about 300 mg/ml, or at least about 350 mg/ml . In some embodiments, the PCSK9 binding fusion protein is administered at a unit dose of about 275 mg to about 325 mg (eg, about 300 mg).

The PCSK9 binding motif contains or consists of the amino acid sequence (SEQ ID NO: 1) or a variant thereof, as described herein. The PCSK9 binding motif binds to human PCSK9 with sub-nanomolar affinity in a concentration-dependent manner. The PCSK9 binding motif has a chemical conjugation or fusion at the C-terminus to the amino acid sequence of human serum albumin (HSA). In various embodiments, the PCSK9 binding motif has a fusion of an HSA amino acid sequence at the C-terminus and may comprise a linking amino acid sequence between the PCSK9 binding motif and the HSA amino acid sequence.

The PCSK9-binding fusion protein can be stably formulated in solution form at high concentration. The formulation does not exhibit significant degradation or particle formation under normal long-term storage conditions and short-term storage under accelerated and stressful conditions. Exemplary formulations contain or consist primarily of (in addition to the active agent as described):

- 1 044927

L-histidine, L-histidine monohydrochloride, sodium chloride and optionally polysorbate-80.

The pharmaceutical compositions of the present disclosure may conveniently be presented in unit dosage forms containing a predetermined amount of the active agent of the disclosure per dose. In some embodiments, unit doses are no more than about 1.5 ml by volume or no more than about 1 ml by volume. In some embodiments, unit doses are no more than 0.8 ml by volume or no more than 0.7 ml by volume. In still other embodiments, the composition is administered as a microdose, for example, in a volume of less than 0.5 ml, or less than about 0.25 ml, or less than about 0.15 ml. In various embodiments, the composition is delivered in a unit dose containing from about 20 to about 450 mg of PCSK9 binding fusion protein. For example, in some embodiments, a dose of 20 to about 75 mg is administered as a weekly microdose (eg, in a volume of less than about 0.25 ml or less than about 0.15 ml). In other embodiments, a dose of about 200 to about 450 mg is administered approximately every two weeks, once a month, or every other month in a volume ranging from about 0.7-1.5 mL.

The composition or formulation is suitable for administration by the subcutaneous, intramuscular, intradermal or intravenous route. The high concentration of the formulation allows for the use of less frequent dosing regimens and smaller dosing volumes suitable for subcutaneous administration. As shown herein, maximum inhibition of PCSK9 is achieved at a relatively low concentration of PCSK9-binding fusion protein, and higher concentrations provide the opportunity to increase the duration of inhibition. In some embodiments, the subject receives a unit dose of the composition about once a week, once every 2 weeks, or about once every 3 weeks, or about once every 4 weeks (for example, about once a month), or about once every 6 weeks, or about once every 8 weeks (about once every 2 months).

In some embodiments, the subject receives a microdose of the composition, such as a volume of about 50 to about 250 μl, about every week or about every two weeks.

The composition can be administered to treat a disorder associated with PCSK9 in a human subject. In some embodiments, the patient requires a reduction in LDL (eg, LDL-C). In some embodiments, the subject may exhibit a cholesterol-related disease, such as hypercholesterolemia and/or atherosclerosis. In some embodiments, the subject has familial hypercholesterolemia. In some embodiments, the subject has or is at high risk for cardiovascular disease (eg, atherosclerotic coronary artery disease).

In some embodiments, the composition is administered in conjunction with statin therapy or other oral lipid-lowering therapy, or in some embodiments, the composition is administered as monotherapy for hypercholesterolemia, that is, without oral lipid-lowering therapy (eg, statin therapy).

Brief description of drawings

Fig. 1 illustrates the structure of a PCSK9 binding fusion protein.

Fig. 2A illustrates the pharmacokinetic and pharmacodynamic (PK-PD) model of PCSK9 in non-human primates (NHP) and humans. LIB-003 is a PCSK9-binding fusion protein.

Fig. 2B illustrates Gadkar's model for predicting the effect of targeting PCSK9 on LDLC. Gadkar K et al., A Mechanistic Systems Pharmacology Model for Prediction of LDL Cholesterol Lowering by PCSK9 Antagonism in Human Dyslipidemic Populations, CPT Pharmacometrics Syst. Pharmacol. 2014; 3(11).

Detailed Description of the Invention

The present disclosure provides compositions containing fibronectin-based scaffolding fusion proteins that bind proprotein convertase subtilisin/kexin-9 (PCSK9) with high affinity, and which can be stably formulated at high concentrations for maximum biological effect and more convenient dosage regimens and volumes, and also delivered using patient-friendly delivery devices such as a syringe or auto-injector. The PCSK9 binding fusion protein contains a PCSK9 binding motif and an amino acid sequence encoding human serum albumin (HSA) at the C-terminus.

Proprotein convertase subtilisin/kexin type 9 is a circulating protein secreted primarily by the liver that plays an important role in hepatic LDLR recycling and has been identified as a validated drug target for lowering LDLc. LDLR is the main pathway for clearing LDL-cholesterol (LDL-C) from the bloodstream. PCSK9 in plasma binds to hepatic LDLR as well as LDL-C, which targets the receptor for endocytosis and degradation, thereby reducing the ability of LDLR to clear LDL-C from the circulation. By inhibiting PCSK9 binding to LDLR, LDLR degradation is prevented, LDLR recycling is increased, LDL-C clearance is enhanced, and circulating LDL-C levels are decreased.

The PCSK9-binding motif described herein is based on adnectin, a family of proteins derived from the 10th domain of human fibronectin type III, designed for high-affinity binding to the target. Adnectins are small (<12 kDa) compact

- 2 044927 proteins without sequence homology with immunoglobulins, but having a β-sheet folding structure with various loops similar to the variable regions of the antibody. Adnectins are disulfide-free and non-glycosylated, have high thermal stability and monomeric behavior in solution, and are efficiently produced using bacterial, yeast or mammalian expression systems. By modifying the sequence and length of the variable loop while maintaining essentially constant backbone residues, sub-nanomolar target binding affinities can be achieved while maintaining structural stability. Given their size, adnectins are rapidly filtered by the kidney and therefore require modification to enhance pharmacokinetics (PK) for in vivo applications. Exemplary PCSK9 binding motifs (adnectins) are disclosed in US Pat. No. 8,420,098; 9234027; and 9856309, each of which is incorporated herein by reference in its entirety.

According to this disclosure, the PCSK9 binding fusion protein is stably formulated at high concentrations to provide maximum biological activity as well as convenient dosage schedules and volumes. The concentration of the PCSK9 binding fusion protein in the composition is at least 100 mg/ml. In some embodiments, the concentration of the PCSK9 binding fusion protein in the composition is at least about 150 mg/ml, or in some embodiments, at least about 175 mg/ml, or at least about 200 mg/ml, or at least about 225 mg /ml. In some embodiments, the concentration of the PCSK9 binding fusion protein in the composition is at least about 250 mg/ml, or at least about 275 mg/ml, or at least about 300 mg/ml, or at least about 350 mg/ml. In some embodiments, the concentration of the PCSK9 binding fusion protein in the composition is from about 250 mg/ml to about 350 mg/ml. In some embodiments, the concentration of the PCSK9 binding fusion protein in the composition is about 250 mg/ml or about 300 mg/ml.

The PCSK9 binding motif contains or consists of the amino acid sequence (SEQ ID NO: 1):

VSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQEFTVPVSKGTA

TISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRTE.

PCSK9-binding loops are shown underlined. In some embodiments, the PCSK9 binding motif is a variant of SEQ ID NO: 1 having one to five amino acid substitutions, deletions, or insertions with respect to SEQ ID NO: 1. In some embodiments, the amino acid changes are made outside the binding loops. In some embodiments, 1, 2, or 3 amino acid changes are made within the binding loops.

The PCSK9-binding motif is designed to specifically target PCSK9 while essentially retaining the wild-type (WT) Fn3 ¹⁰ sequence to minimize its inherent immunogenicity. See US Pat. No. 8,420,098, which is incorporated herein by reference in its entirety. The PCSK9-binding motif binds to human PCSK9 with subnanomolar affinity in a concentration-dependent manner.

In various embodiments, the PCSK9 binding motif has a C-terminal fusion to the amino acid sequence of human serum albumin (HSA). In some embodiments, the HSA comprises a sequence having at least 80% identity, or at least 85% identity, or at least 90% identity, or at least 95% identity, or at least 98% identity, or at least 99% identity with the amino acid sequence set forth in SEQ ID NO: 2. For example, the amino acid sequence of HSA may contain from one to ten or from one to five modifications independently selected from amino acid substitutions, deletions and insertions relative to SEQ ID NO: 2 . In some embodiments, the HSA amino acid sequence contains an alanine residue at a position corresponding to position 34 of SEQ ID NO: 2, as illustrated in SEQ ID NO: 2. In various embodiments, the HSA amino acid sequence is at least 500 amino acids in length.

In some embodiments, the PCSK9 binding motif and the HSA amino acid sequence are chemically conjugated using any known chemical conjugation approach.

HSA is a multidomain protein composed of helical clusters and containing 17 pairs of disulfide bridges; only one cysteine residue, Cys34, exists as a free sulfhydryl group in natural HSA. In some embodiments, this Cys is replaced by an alanine residue in the PCSK9-binding fusion protein. The HSA fragment serves to increase the circulating half-life of the PCSK9-binding fusion protein. In various embodiments, a PCSK9 binding fusion protein comprising a PCSK9 binding motif and an HSA amino acid sequence has a molecular weight of about 77 kDa.

In some embodiments, the HSA amino acid sequence is a variant described in US Pat. No. 9,493,545, US Pat. 9,821,039, US Pat. 9,944,691, or

- 3 044927 US publication 2014/0315817, each of which is incorporated herein by reference in its entirety.

In some embodiments, the PCSK9 binding motif and the HSA amino acid sequence are linked by genetic fusion, for example, to the HSA amino acid sequence at the C terminus of the molecule. A short amino acid linker can connect the PCSK9 binding domain and the HSA amino acid sequence. For example, the linker may contain from 2 to 20 amino acids, or, in some embodiments, from 4 to 10 amino acids. In some embodiments, the PCSK9 binding motif and the HSA amino acid sequence are joined via a 6 amino acid linker. The linker may consist predominantly of the amino acids serine, glycine, threonine and alanine. For example, the linker may be a serine/glycine linker. In some embodiments, the linker comprises or consists of the amino acid sequence GSGSGS.

The PCSK9-binding fusion protein can be stably formulated in solution form at high concentration. For example, the formulation does not exhibit significant degradation or particle formation under long-term storage conditions, such as around 5°C (eg, 2 to 8°C), or under short-term storage conditions at ambient conditions (eg, 1 to 6 months at a temperature of 25±3°C). The PCSK9 binding fusion protein is suspended in a suitable physiological solution, eg, saline or other pharmacologically acceptable solvent or buffer solution, and may optionally contain a surfactant (eg, a nonionic surfactant). In some embodiments, the composition contains a buffering agent, an isotonic agent, optionally a surfactant, and a solvent.

Pharmaceutically acceptable carriers include water, saline, glycerin. In some embodiments, the composition may contain fatty oils, polyethylene glycol, propylene glycol, or other solvents. In some embodiments, the solvent is water.

The composition is usually a buffer solution. As used herein, the term buffer refers to a chemical agent that is capable of absorbing a certain amount of acid or base without undergoing a large change in pH. Examples of buffers include citrate buffer, phosphate buffer, acetate buffer, succinate buffer and bicarbonate buffer. In some embodiments, the buffering agent may comprise L-histidine/L-histidine monohydrochloride. For example, when using the L-histidine/L-histidine monohydrochloride buffer system, L-histidine/L-histidine monohydrochloride may be present in an amount of from about 1 mg/ml to about 10 mg/ml, for example, from about 2 mg/ml to about 5 mg/ml. In some embodiments, the buffer maintains the pH of the formulation in the range of about pH 5.5 to about pH 7.2, such as about pH 6.8 (eg, pH 6.6 to 7.0). In some embodiments, the pH of the composition is adjusted, for example, using hydrochloric acid and/or sodium hydroxide. In some embodiments, the L-histidine/L-histidine monohydrochloride ratio is such that no pH adjustment is required.

In various embodiments, isotonic agents that may be used alone or in combination include dextrose, sucrose, glycerol, trehalose, mannitol, sorbitol, arginine, sodium chloride, or potassium chloride. In some embodiments, the isotonic agent contains or consists of sodium chloride. For example, the formulation may contain from about 2 to about 20 mg/ml sodium chloride (for example, from 6 to 12 mg/ml sodium chloride), or one or more isotonic agents in an amount equivalent to an osmolality of from 2 to 20 mg/ml chloride sodium (or 6 to 12 mg/ml sodium chloride).

In some embodiments, the composition contains a surfactant that can act as a solubilizing agent. In some embodiments, the surfactant is a nonionic surfactant. Examples of nonionic surfactants include polysorbate surfactants such as polysorbate 20, 40, 60 or 80. For example, the formulation may contain polysorbate 80. Other pharmaceutically acceptable nonionic surfactants may also be used, alone or in combination. In some embodiments, the composition does not contain a surfactant.

Exemplary formulations include citrate buffer (eg, 10 to 50 mM, pH 5.6 to 6.0), histidine buffer (eg, 10 mM to 50 mM, pH 6.0 to 7.0), or succinate buffer ( e.g. 10 to 50 mM, pH 5.5 to 6.0). In some embodiments, the formulation contains an excipient selected from arginine (eg, 100 to 200 mM), NaCl (eg, 100 to 200 mM), sorbitol (eg, 100 to 300 mM), or sucrose (eg, 100 to 300 mM). up to 300 mM). In some embodiments, the formulation contains a surfactant such as polysorbate 80 (eg, 0.01 to 0.5 mg/ml). In some embodiments, the composition does not contain a surfactant.

In some embodiments, the composition further contains a preservative such as phenol, meta-cresol or sodium benzoate.

In some embodiments, the composition of the PCSK9 binding fusion protein consists primarily of (in addition to the active agent as described): L-histidine, L-histidine monohydrochloride, sodium chloride, and polysorbate-80. Exemplary embodiments are shown in the following Table 1.

Component Range Exemplary Embodiments (±10%) Function PCSK9 binding fusion protein 100 - 300 mg/ml 250 mg/ml 300 mg/ml Active Ingredient L-Histidine 1-5 mg/ml 2.6 mg/ml Buffer agent L-Histidine monohydrochloride 0.5-2 mg/ml 0.7 mg/ml Buffer agent Sodium chloride 2-20 mg/ml 8.8 mg/ml Isotonic agent Polysorbate-80 0.0-0.5 mg/ml 0.2 mg/ml Surface-active substance

The pharmaceutical compositions of the present disclosure may conveniently be presented in unit dosage forms containing a predetermined amount of the active agent of the disclosure per dose.

In some embodiments, the composition is contained in a pen syringe. Auto-injectors, such as pens, are spring-loaded syringes designed to deliver a dose of a specific drug. By design, pen syringes are easy to use and are designed for self-administration by patients or administration by untrained personnel. Pen syringes are designed to overcome the hesitancy associated with self-administration using a needle-based drug delivery device. The pen keeps the needle tip capped until injection and also has a passive safety mechanism to prevent accidental injection. The injection depth can be adjusted or fixed, and the needle cap removal function can be enabled. When you press a button, the syringe needle is automatically inserted into the subcutaneous tissue and the drug is delivered. Once the injection is complete, some pen syringes have a visual or audible indication to confirm that the full dose has been administered.

In some embodiments, the pen contains 1 to 10 unit doses or 1 to 5 unit doses. In some embodiments, unit doses are no more than about 1.5 ml or about 1 ml by volume (whether contained or delivered by a pen). In some embodiments, unit doses are no more than 0.8 ml by volume or no more than 0.7 ml by volume. In some embodiments, the pen delivers a microdose, for example, having a volume ranging from about 50 μL to about 500 μL, or a volume ranging from about 75 μL to about 250 μL. In some embodiments, the microdose has a volume of 100 to 200 μL. In various embodiments, the pen or other subcutaneous delivery device provides a dose containing from 30 to about 450 mg of PCSK9 binding fusion protein. In various embodiments, the unit dose is from about 50 to about 400 mg, or from about 50 to about 300 mg. In some embodiments, the unit dose is at least 200 mg, or at least 250 mg, or at least 300 mg. In some embodiments, the unit dose is from about 250 mg to about 350 mg (eg, about 300 mg). The amount of active agent delivered per unit dose can be adjusted depending on the desired frequency of administration. For example, in some embodiments, a dose of 20 to about 75 mg is administered as a weekly microdose (eg, in a volume of less than about 0.25 ml or less than about 0.15 ml). In other embodiments, a dose of about 200 to about 450 mg is administered approximately every two weeks, once a month, or every other month, with a volume ranging from about 0.7 to 1.5 mL. In some embodiments, a dose of about 275 mg to about 350 mg (eg, about 300 mg) is administered once every four weeks by subcutaneous injection, the volume being about 1.5 ml or less, or about 1 ml or less.

The composition or formulation is suitable for administration by the subcutaneous, intramuscular, intradermal or intravenous route. The high concentration, relatively low viscosity and proper osmolality of the formulation ensure patient tolerance, less frequent dosing regimens and smaller

- 5 044927 volumes. As demonstrated herein, maximum inhibition of PCSK9 is achieved at a relatively low concentration of PCSK9-binding fusion protein, and higher concentrations allow longer duration of inhibition. In some embodiments, the subject receives a unit dose of the composition about once every 1 week (e.g., by administering a once-weekly microdose), or about once every 2 weeks, or about once every 3 weeks, or about once every 4 weeks ( for example, about once a month), or about once every 6 weeks, or about once every 8 weeks (about once every 2 months).

The composition can be administered to treat a PCSK9-related disorder in a human subject. Infringements related to PCSK9 are described in US patents 8420098, 9238027 and 9856306, which are incorporated herein by reference in their entirety. In some embodiments, the patient requires a reduction in LDL (eg, LDL cholesterol). In some embodiments, the subject may exhibit a cholesterol-related disease, such as hypercholesterolemia and/or atherosclerosis. In various embodiments, the subject exhibits a condition selected from a lipid disorder, hypercholesterolemia, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, coronary artery disease, atherosclerosis, and diabetes mellitus. In some embodiments, the subject has familial hypercholesterolemia. In some embodiments, the subject has or is at high risk for cardiovascular disease (eg, atherosclerotic coronary artery disease).

Hypercholesterolemia is a condition characterized by elevated serum cholesterol levels. Elevated serum cholesterol levels occur in a significant portion of the population and are an important risk factor for the development of atherosclerosis and myocardial infarction. Patients with hypercholesterolemia are usually given cholesterol-lowering drugs such as HMG-CoA reductase inhibitors (statins). Familial hypercholesterolemia (FH) is a genetic disorder characterized by high cholesterol levels, particularly very high levels of low-density lipoproteins (eg, LDL cholesterol) in the blood, and early cardiovascular disease. For individuals with FH, high cholesterol levels are less responsive to traditional cholesterol control methods (such as statin therapy).

Familial hypercholesterolemia (FH) is an autosomal dominant metabolic disorder characterized by mutation or mutations in the LDL receptor (LDL-R) gene or other genes involved in lipid regulation, significantly elevated LDL-C levels, and premature onset of atherosclerosis. In some embodiments, the hypercholesterolemia is homozygous familial hypercholesterolemia, or HoFH, which is a condition characterized by a mutation in both the maternal and paternal LDL-R genes.

In some embodiments, the subject has heterozygous FH. Heterozygous FH is usually treated with statins, bile acid sequestrants, or other lipid-lowering agents that lower cholesterol levels.

In some embodiments, hypercholesterolemia is polygenic hypercholesterolemia, which is a condition characterized by elevated cholesterol levels that result from multiple genetic factors. In some embodiments, polygenic hypercholesterolemia may be exacerbated by dietary lipid intake.

In some embodiments, the composition is administered in conjunction with statin therapy or other oral lipid-lowering therapy. The composition in such embodiments will provide additional reduction in LDL-C. In some embodiments, the composition is provided as monotherapy for hypercholesterolemia, that is, without statin therapy or other oral lipid-lowering therapy. For example, in such embodiments, the subject may be intolerant to statins. Statin intolerance occurs when a patient is unable to continue taking statins due to the development of a side effect or abnormality indicating liver function or muscle function (creatine kinase) after a blood test. In some embodiments, statin intolerance may be partial (ie, only some statins at some doses) or complete (ie, all statins at any dose). In some embodiments, statin intolerance results in muscle aches, pain, weakness, or cramps (ie, myalgia); occurs in 15% of treated patients.

Unless otherwise specified, the term about as used herein means ±10% of the corresponding numerical value.

Embodiments of the invention will now be described using the following examples.

Examples

Example 1. PCSK9-binding fusion protein and PK-PD behavior.

The PCSK9-binding fusion protein contains a modified fibronectin domain (adnectin) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) and human serum albumin (Fig. 1). The fusion protein has a total molecular weight of approximately 77,000 Daltons. The PCSK9-binding fusion protein has high binding affinity to human PCSK9 and in

- 6 044927 >100 times weaker affinity for cynomolgus PCSK9. Despite this difference in binding affinity, the non-human primate (NHP) is considered a suitable species for testing the safety and pharmacology of the PCSK9-binding fusion protein because NHPs achieve the greatest suppression of free PCSK9 and the greatest reduction of LDL-C.

A PCSK9 binding fusion protein has been developed for subcutaneous (SC) administration to treat, for example, hypercholesterolemia, including in patients with familial hypercholesterolemia or hypercholesterolemia and atherosclerotic coronary artery disease (CHD). Two major determinants of the pharmacokinetics (PK) of PCSK9-binding fusion protein are the interaction with its target, PCSK9, and the ability to recycle through the neonatal Fc receptor (FcRn) and minimize renal filtration, thereby reducing PCSK9-binding fusion protein clearance and increasing half-life. Studies in wild-type mice cannot evaluate the effect of each of these determinants on the PK of PCSK9-binding fusion protein because PCSK9-binding fusion protein does not bind efficiently to mouse PCSK9 and HSA does not interact with mouse FcRn. Thus, the cynomolgus macaque provides the most suitable in vivo model for evaluating the PK-PD PCSK9-binding fusion protein, since binding to PCSK9 in this model results in a decrease in both free PCSK9 and LDL-C; in addition, HSA can be efficiently recycled through the FcRn receptor NHP.

An hPCSK9 transgenic mouse was used to examine the in vivo binding affinity of the PCSK9 binding fusion protein to hPCSK9, and the ability of the PCSK9 binding fusion protein to recycle through hFcRn was also assessed in the hFcRn mouse. This mouse model of human FcRn carries a null mutation of the mouse FcRn gene and a transgene expressing the human FcRn a-chain under the control of its natural human promoter. Therefore, these mice serve as a model to evaluate the pharmacokinetics of human monoclonal antibodies and serum albumin.

There is extensive evidence supporting a relationship between suppression of systemic levels of free PCSK9 and reduction in serum LDL-C for anti-PCSK9 antibodies. Gadkar K et al., A Mechanistic Systems Pharmacology Model for Prediction of LDL Cholesterol Lowering by PCSK9 Antagonism in Human Dyslipidemic Populations, CPT Pharmacometrics Syst. Pharmacol. 2014; 3(11): 1-9; Squizzato A, et al., PCSK9 inhibitors for treating dyslipidemia in patients at different cardiovascular risk: a systematic review and meta-analysis. Intern. Emerg. Med. 2017; Jul:el-11. See fig. 2B. To estimate the human dose of PCSK9 binding fusion protein (LIB003, SEQ ID NO:3) required to achieve the desired target level of LDL-C reduction, allometric scaling of the data obtained in the above studies was incorporated into a translational semi-mechanistic model that incorporated the known relationship between PCSK9 and LDL-C. Fig. 2A. The expected dose-response relationship for LIB003 and serum LDL-C was constructed using this model. TK-PD in non-human primates.

The behavior of TK-PD LIB003 was characterized following intravenous (IV) and subcutaneous (SC) administration in a non-GLP dose range finding (DRF) study with single dose administration (IV doses of 10, 30, 100, 200 mg/kg; SC dose 200 mg/kg) and after repeated administration in GLP toxicity studies for 4 weeks (IV dose 100 mg/kg; SC doses 30 and 100 mg/kg), 12 weeks (SC doses 30 and 100 mg/kg) or 26 weeks (SC doses 30 and 100 mg/kg).

The TK assay for LIB003 was performed as an electrochemical luminescent target capture assay, with hPCSK9 as the capture reagent and ruthenium-labeled rabbit polyclonal anti-HSA antibody as the detection reagent. In NHPs, the assay measures total LIB003 (due to stronger affinity for the capture reagent, hPCSK9, compared to NHP PCSK9). Alternatively, the TK assay used a LIB003-specific mAb as the capture reagent and a ruthenium-labeled rabbit polyclonal anti-HSA antibody as the detection reagent. This analysis format also measures total LIB003. The results of these studies indicate that, over this dose range, after intravenous and subcutaneous administration, the kinetics of total PCSK9-binding fusion protein are approximately linear and dose proportional.

In the DRF study, durable anti-drug antibody (ADA) responses were present at all dose levels above 10 mg/kg. Following a single dose of LIB003, the presence of anti-drug antibodies (ADA) in this study was associated in most cases with rapid loss of LIB003 exposure and preceded loss of target acquisition (decreased free PCSK9). Although anti-drug antibodies (ADA) were detected in most animals following weekly administration of PCSK9-binding fusion protein (4-week, 12-week, and 26-week GLP toxicity studies), exposure to PCSK9-binding fusion protein was maintained throughout the interval dosing and the effect of anti-drug antibodies (ADA) on TK/PD was significant in only two animals (4-week study), one animal (12-week study) and one animal (26-week study).

In the absence of anti-drug antibody (ADA) exposure, LIB003 was cleared slowly and the terminal serum half-life ranged from 8.3 to 10.4 days (median 9 days) in the DRF study and from 7.6 to 9.2 days (on average 8.3 days) in recovered animals in

- 7 044927

4-week GLP toxicity study; consistent with the behavior of an albumin-like molecule in non-human primates. Similar results were observed in the 12-week and 26-week GLP toxicity studies. In the DRF study, after a single IV dose, clearance (CL) ranged from 5.63 to 7.65 mL/day/kg (mean 6.64 mL/day/kg), and volume of distribution (Vz) ranged from 67. 6-103.8 ml/kg (mean 86.4 ml/kg) in animals in which this could be determined.

Total concentrations of PCSK9 (bound and unbound to LIB003) increased slowly, with a peak occurring approximately 7 days after dosing, at which point total PCSK9 concentrations were <10% of circulating LIB003 concentrations. Target acquisition, as measured by free PCSK9 and LDL reduction, is greatest at all doses in the DRF study, indicating maximum target inhibition at the lowest dose tested (10 mg/kg). The results indicate that in the absence of anti-drug antibodies (ADA), increasing the dose of LIB003 increased the duration of maximum target acquisition.

Regardless of dose or route of administration, serum LDL was suppressed by approximately 60%, corresponding to the maximum pharmacodynamic effect at all dose levels. As expected, loss of LDL suppression and return to baseline levels corresponded with loss of maximal PCSK9 uptake. Overall, these studies demonstrate that maximum pharmacodynamic effect was achieved and higher doses would not result in greater suppression of free PCSK9 or LDL.

Following a subcutaneous dose, _Cmax concentrations were lower and _Tmax occurred later compared with slow intravenous (IV) bolus at the same dose level, indicating absorption from the injection site into the systemic circulation. Absolute bioavailability by subcutaneous administration is estimated to be about 76% in the DRF study and 66.5-89.7% in the 4-week GLP study, although both estimates are compromised by the presence of anti-drug antibodies (ADA) and the inability to characterize the total area under the curve (AUC) after subcutaneous administration. A combined analysis of both studies using a population PK model estimates bioavailability after subcutaneous administration to be 92% and is likely to be a more reliable estimate since the model estimates the overall AUC for both routes of administration.

Predicted behavior of PK-PD in humans

Data from multiple studies were combined to predict the expected behavior of PK-PD LIB003 in humans. Specifically, a population two-compartment model of PK-PD binding describing LIB003 exposure and PCSK9 inhibition was constructed based on NHP data (DRF study and 4-week GLP toxicity study). When translating these parameters to humans, in vivo binding affinity for hPCSK9 was obtained from PK-PD studies in hPCSK9 transgenic mice. These data were adequately described using a single-compartmental PK model because only data after subcutaneous administration were available; the model was otherwise equivalent to that used for the NHP data. The observed difference in binding affinity of LIB003 to PCSK9 NHP and hPCSK9 was consistent with both in vitro and in vivo data, and the KD value obtained in vivo in the hPCSK9 transgenic mouse was used for prediction in humans.

Table 2

Kinds Binding affinity for PCSK9 in vitro (nM) Binding affinity for PCSK9 in vivo (nM) NHP 8.1 56.4 Human 0.06 0.15

LIB003 clearance in both NHP and hFcRn mice was consistent with expected HSA clearance in these animals, and LIB003 clearance was therefore predicted to mimic human HSA clearance (half-life 19 days; allometric exponent 0.74). Other parameters were scaled allometrically using the expected coefficients for a therapeutic albumin fusion protein of approximately 77 kDa.

The general structure of the NHP and human PK-PD binding model describing the uptake and inhibition of free PCSK9 is shown in FIG. 2A and 2B. A quantitative systems pharmacology model was previously established to describe the mechanism of action of statins and anti-PCSK9 antibodies in humans. Gadkar K et al., A Mechanistic Systems Pharmacology Model for Prediction of LDL Cholesterol Lowering by PCSK9 Antagonism in Human Dyslipidemic Populations, CPT Pharmacometrics Syst. Pharmacol. 2014; 3(11). This model was used to provide an association between predicted human PCSK9 suppression and LDL-C reduction over time following single subcutaneous and intravenous doses of LIB003 in the First in Human (FIH) study.

Example 2: Composition and stability assessment.

The effectiveness of various buffers and excipients in PCSK9 was assessed

- 8 044927 binding fusion protein.

To determine the appropriate buffer and pH to use in the formulation, 18 different buffers and pH conditions using 6 different buffers at 2 mg/mL LIB003 were analyzed using DSC to assess thermal stability and DLS to assess aggregate formation. pH values below 5 and above 7 were excluded from these experiments due to the fact that for these pH values protein unfolding began at <50°C (Tonset).

The buffer/pH combinations analyzed are shown in the table below. 3. Combinations selected for further screening are indicated by the pH value in bold and underlined.

Table 3

After identifying an acceptable pH range and a suitable buffer, the excipients were tested in combination with specific buffers and pH values. A total of 15 different buffer/excipient combinations were prepared at a LIB003 concentration of 2 mg/mL and analyzed by DSC to assess thermal stability and DLS to assess aggregate formation. Three buffer/excipient combinations were excluded because the initial Tonset temperature was below 55°C. Additionally, DLS data suggested that the use of sucrose, sorbitol, and low concentrations of NaCl resulted in aggregation and therefore their use was limited in subsequent formulations.

The buffer/excipient combinations analyzed are shown in the table below. 4. Combinations selected for further screening are indicated by excipient in bold and underlined.

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Table 4

Screening buffer/excipient

Buffer/pH Excipient 20 mM Succinate, pH 5.5 50 MMNaCl 150 mM NaCl 150 mM Arginine 250 mM Sorbitol 250 mM Sucrose 20 mM Citrate, pH 6.0 50 mM NaCl 150 mM NaCl 150 mM Arginine 250 mM Sorbitol 250 mM Sucrose 20 mM Histidine, pH 7.0 50 mM NaCl 150 mM NaCl 150 mM Arginine 250 mM Sorbitol 250 mM Sucrose

To determine suitable buffer and excipient combinations to maintain stability of LIB003 formulated at high concentrations, solubility screening was performed on the 12 different formulations listed in Table 1 above. 4. These formulations were concentrated to target concentrations of 200 mg/ml, 250 mg/ml, 300 mg/ml and 340 mg/ml. Turbidity was similar across all formulations and no aggregation was observed when assessed by SEC-HPLC. Based on these parameters, these formulations were considered equivalent.

To evaluate the potential benefit of adding a surfactant to the LIB003 candidate formulations, the eight buffer/excipient combinations from Table. 4 were evaluated with or without polysorbate 80 (PS80) for a total of 16 formulations at 250 mg/mL LIB003 (Table 5). Because PS80 may be effective in preventing aggregation and particle formation, candidate formulations were evaluated for their ability to withstand repeated freeze-thaw and agitation stresses. No differences were observed for release tendency, turbidity or aggregation (as assessed by DLS and SEC). All formulations containing PS80 were improved due to the perceived benefits of PS80 in long-term storage and product handling.

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Table 5

Screening for Buffer/Excipient and Surfactant Combinations

Buffer/pH Excipient Surfactant (PS80) 20 mM Succinate, pH 5.5 150 mM NaCl - + 250 mM Sorbitol - + 250 mM Sucrose - + 20 mM Citrate, pH 6.0 150 mM Arginine 20 mM Citrate, pH 6.0 - + 250 mM Sorbitol - + 250 mM Sucrose - + 150 mM NaCl - + 20 mM Histidine, pH 7.0 150 mM NaCl - +

The stability of LIB003 and the lack of differences between formulations under normal storage conditions and shear stresses, even at high concentrations of LIB003, suggest that an alternative approach to identifying a formulation for LIB003 that will withstand long-term storage conditions is required. Formulations identified from screening buffer/excipient combinations (Table 4) at target concentrations of 340 mg/ml were added with 0.02% PS80, stored at 2-8°C or 50°C for three weeks and analyzed using SEC-HPLC. Overall, low levels of high molecular weight (HMW) and low molecular weight (LMW) compounds were observed after 3 weeks of storage at 50°C. Formulations containing citrate were narrowed down to the excipients arginine and sorbitol due to the low percentage of HMW compounds in these formulations. From the formulations containing histidine, 150 mM NaCl was selected for additional experiments based on the highest percentage of compounds corresponding to the main peak. Finally, succinate was excluded from further consideration due to the elevated levels of HMW and LMW compounds observed in most succinate-containing formulations stored at 50°C compared to the comparator control stored at 2-8°C.

The results of these analyzes are shown in the table below. 6; preferred formulations are shown in bold and underlined.

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Table 6

Evaluation of LIB003 at high concentration stored at 50°C for 3 weeks by SEC-HPLC

Buffer/pH Excipient %HMW % Main compound %LMW 20 mM Succinate, pH 5.5 150 mM NaCl 3.1 96.2 0.8 150 mM Arginine 1.7 97.7 0.6 250 mM Sorbitol 3.9 95.4 0.7 250 mM Sucrose 3.3 96.1 0.6 20 mM Citrate, pH 6.0 50 mM NaCl 1.8 97.9 Below LOQ 150 mM NaCl 2.0 97.6 Below LOQ 150 mM Arginine 1.1 98.3 Below LOQ 250 mM Sorbitol 1.1 98.6 Below LOQ 250 mM Sucrose 1.2 98.5 Below LOQ 20 mM Histidine, pH 7.0 50 mM NaCl 2.6 97.0 Below LOQ 150 mM NaCl 1.0 98.6 Below LOQ 150 mM Arginine 1.0 98.3 0.8

HMW = high molecular weight; LMW = low molecular weight; LOQ = limit of quantitation; mM = millimolar; NaCl = sodium chloride.

To further explore candidate formulations, LIB003 was formulated at 250 mg/mL in three buffer/excipient combinations with PS80 at 3 different pH values (Table 7). Samples were stored at 2-8°C or 50°C for three weeks to induce degradation and analyzed for LIB003 (A280) concentration/release, particulate (DLS), aggregation (SEC-HPLC), charge profile (icIEF), clipping ( CE-SDS), activity, turbidity and thermal stability (DSC). Comparison of charge profiles, aggregation/particulate propensity and relative activity after storage at 50°C for 3 weeks demonstrated some differences between the formulations (Tables 8, 9, 10 and 11). CE-SDS profiles, release, turbidity, and thermal stability were comparable between formulations.

Table 7 _________________________Candidate compositions before storage_________________________

Compound Target pH Actual pH Concentration (mg/ml) SEC-HPLC %HMW/%Och./%LMW icIEF %Acid/%Basic/%Alkali 20 mM Citrate, 150 mM Arginine, 0.02% PS80 5.5 5.6 274.4 0.8 99.2 ND 30.1 68.3 1.6 5.7 259.0 0.6 99.3 ND 30.2 68.1 1.7 6.0 6.1 271.6 0.9 99.2 ND 30.8 67.4 1.8 6.1 254.1 0.8 99.2 ND 31.6 66.3 2.1 6.5 6.5 260.2 0.9 99.1 ND 30.8 67.4 1.8 6.5 267.9 ι,ο 99 ND 31.1 67.1 1.8 20 mM Citrate, 250 mM Sorbitol, 0.02% PS80 5.5 5.6 259.6 0.8 99.2 ND 30.7 67.7 1.6 5.6 258.9 0.6 99.3 ND 32.7 65.5 1.8 6.0 6.0 268.3 1.1 98.9 ND 31.1 67.3 1.6 6.0 271.0 0.8 99.2 ND 30.6 67.8 1.7 6.5 6.5 266.8 ι,ο 99 ND 33.3 65.6 1.1 6.5 261.4 0.7 99.3 ND 30.7 67.7 1.6 20 mM Histidine, 150 mM NaCl, 0.02% PS80 6.0 6.0 248.7 0.7 99.3 ND 30.1 68.1 1.8 6.0 263.9 0.9 99.1 ND 31.3 66.3 2.4 6.5 6.5 260.4 0.7 99.3 ND 31.6 66.1 2.4 6.5 260.5 0.9 99.2 ND 29.7 68.5 1.8 7.0 6.9 267.3 0.9 99.1 ND 29.8 68.0 2.1 7.0 267.2 0.8 99.3 ND 30.1 68.1 1.9 ND = not defined

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Table 8

Change in charge distribution after storage at 50°C for 3 weeks according to icIEF

Compound pH Changing charge types (storage at 50 °C versus 28 °C) Acid Basic Alkaline 20 mM Citrate, 150 mM Arginine, 0.02% PS80 5.6 0.8 -22.0 21.2 5.7 3.2 -23.3 20.1 6.1 4.6 -8.3 3.7 6.1 5.1 -7.2 2.1 6.5 6.9 -11.1 4.3 6.5 4.4 -9.7 5.3 20 mM Citrate, 250 mM Sorbitol, 0.02% PS80 5.6 -1.5 -31.2 32.7 5.6 2.4 -30.3 27.9 6.0 5.5 -12.8 7.3 6.0 6.0 -12.8 6.8 6.5 4.3 -8.5 4.4 6.5 6.8 -9.1 2.3 20 mM Histidine, 150 mM NaCl, 0.02% PS80 6.0 2.6 -19.5 16.7 6.0 4.9 -15.5 10.5 6.5 9.2 -10.7 1.4 6.5 10.4 -13.3 2.8 6.9 10.3 -11.6 1.4 7.0 12.4 -15.0 2.5

Table 9

Change in HMW and LMW compounds after storage at 50°C for 3 weeks according to SEC-HPLC

Compound pH Change at 50°C compared to 2-8°C H.M.W. Basic LMW 20 mM Citrate, 150 mM Arginine, 0.02% PS80 5.6 25.5 -26.4 1.0 5.7 20.7 -21.7 1.0 6.1 5.2 -6.2 1.0 6.1 4.8 -5.8 1.0 6.5 2.6 -3.8 1.1 6.5 2.5 -3.2 0.7 20 mM Citrate, 250 mM 5.6 42.0 -42.5 0.5 5.6 38.2 -38.9 0.7 Sorbitol, 0.02% PS80 6.0 12.3 -13.1 0.8 6.0 11.6 -12.5 0.9 6.5 4.2 -5.0 0.8 6.5 3.6 -4.7 1.0 20 mM Histidine, 150 mM NaCl, 0.02% PS80 6.0 20.9 -22.1 1.2 6.0 16.5 -17.4 0.9 6.5 5.7 -6.9 1.2 6.5 6.4 -7.5 1.1 6.9 2.4 -3.5 1.0 7.0 3.9 -5.2 1.3

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Table 10

Activity depending on storage temperature

Compound % Relative activity Change in % relative activity 2-8 °C 50°C 20 mM Citrate, 150 mM Arginine, 0.02% PS80, pH 6.0 113.7 97.8 -15.9 93.5 80.2 -13.3 20 mM Citrate, 250 mM Sorbitol, 0.02% PS80 pH 6.0 106 70.5 -35.6 103.4 75.1 -28.3 20 mM Histidine, 150 mM NaCl, 0.02% PS80 pH 6.5 98.9 78.5 -20.5 100 82.1 -17.9

Given the goal of delivering LIB003 as a 0.25-1.5 mL subcutaneous (SC) injection using an autoinjector with a 27G or smaller gauge needle, additional experiments were conducted to determine whether LIB003 formulated at a concentration of 250 mg/mL would have properties suitable for SC injection via auto-injector. The top three formulations at 250 mg/mL with corresponding center pH were assessed for viscosity, osmolality and particulate matter. Targets were <15 cP for viscosity, 250–350 mOsm for osmolality, and particle levels well below regulatory limits. The results are shown in the table below. 11. Particle levels were relatively low in all formulations.

Table 11 _____________Viscosity and osmolality analysis results_____________

Compound Rev. Osmolality (mOsm) Viscosity (cP) Concentration (mg/ml) 20 mM Citrate 150 mM Arginine pH 6.0 1 355 I.5 250.2 2 365 14.4 266.0 20 mM Citrate 250 mM Sorbitol pH 6.0 1 415 17.5 254.5 2 420 16.9 267.8 20 mM Histidine 150 mM NaCl pH 6.5 1 335 10.2 242.8 2 329 from 269.0

For additional stability studies, the PCSK9-binding fusion protein was formulated as a sterile solution for injection (subcutaneous) in 20 mM histidine, 150 mM NaCl, 0.02% (w/v) polysorbate-80, pH 6.8. These stability studies included evaluations under long-term storage conditions, accelerated conditions, and under stress conditions. The expected long-term storage temperature of the PCSK9 binding fusion protein product was 5±3°C, and the short-term storage temperature (<1-6 months) was 25°C±3°C.

Stability data for 1 month were obtained at the assumed storage temperature (5°C±3°C), as well as accelerated (25±2°C/60±5% relative humidity (RH)) and stress (40±2°C /75±5% RH) storage conditions.

For a batch of an exemplary PCSK9 binding fusion protein product, data for all parameters evaluated, including parameters indicating stability (icIEF, activity, CE-SDS, and SEC-HPLC) in the stability study protocol, were within the acceptance criteria at the time point studied ( 1 month) and under each storage condition. In addition, under long-term storage conditions there was no significant increase in the amount of aggregating or degrading compounds, or a decrease in activity. A small increase in aggregates (~1%) was observed by SEC-HPLC for accelerated and stress conditions, however the results were within the acceptance criteria for the main peak by SEC-HPLC and no changes in activity were observed. Also, a small increase in fragments (~2%) was observed by CE-SDS analysis under reducing conditions under stress conditions, but again, no concomitant change in activity was observed. A small increase (~1-4%) of variants differing in acidic charges, with a concomitant decrease in compounds corresponding to the main peak, was observed by icIEF at each storage condition (notably, the opposite trend was observed for the drug substance, indicating that perhaps the observed changes are within the variability of the method). No changes in appearance, physicochemical parameters or activity were observed. Taken together, the stability data demonstrate that the PCSK9-binding fusion protein product is stable for 1 month when evaluated under long-term storage, accelerated and stress storage conditions.

In addition, long-term stability data were obtained for 18 months at the intended storage temperature (5°C±3°C), as well as for 9 months at accelerated storage conditions (25±2°C/60±5% RH) and for three months under stressful storage conditions (40±2°C/75±5% RH).

For a batch of an exemplary PCSK9 binding fusion protein product, data for all parameters evaluated, including parameters indicating stability (icIEF, activity, CE-SDS, and SEC-HPLC), in the stability study protocol were within the acceptance criteria at the time points studied and under each storage condition. In addition, under long-term storage conditions, there was no significant increase in the amount of aggregating or degrading compounds, nor a decrease in activity. A small increase in the number of aggregates (~1%) was observed by SEC-HPLC under all storage conditions, however, the results were within the acceptable criteria for the main peak by SEC-HPLC, and no changes in activity were observed (within the assay variability). Also, a slight increase in fragment abundance (up to ~2%) was observed when CE-SDS was analyzed under reducing conditions under long-term storage conditions, but again, a corresponding change in activity was not observed and can be considered to be within method variability. The increase in fragment number by CE-SDS analysis under reducing and non-reducing conditions correlated with increasing time and temperature (up to ~5% change under stress conditions). Fluctuations (up to ~8%) of the compounds corresponding to the acidic and main peaks according to icIEF were observed under long-term storage conditions, but these fluctuations were within the variability of the method. icIEF results under accelerated and stress conditions initially indicated a potential positive trend for acidic compounds (up to ~7%) and a corresponding negative trend for compounds corresponding to the major peaks, although no concomitant change in activity was observed. However, additional time points indicate that the small changes observed could also be fluctuations due to method variability. Invisible particles appear to fluctuate slightly over time, but are within acceptable limits (it should be noted that an atypical result was observed for particles >2 µm and >5 µm after 9 months in both long-term and accelerated conditions; however, the number of particles returned to the expected level at the 12-month and 18-month time points). No changes in appearance, physicochemical parameters or activity were observed.

In addition, long-term stability data were obtained for 12 months at the intended storage temperature (5°C ± 3°C), as well as for 12 months at accelerated storage conditions (25 ± 2°C/60 ± 5% RH).

For an exemplary batch of PCSK9 binding fusion protein product, data for all parameters evaluated, including stability parameters (icIEF, activity, CE-SDS, and SEC-HPLC) in the stability study protocol, were within the acceptance criteria at the time points studied and under each storage condition. In addition, for long-term storage conditions there was no significant increase in the amount of aggregating or degrading compounds, nor a decrease in activity. A slight increase in the number of aggregates (up to ~1%) was observed by SEC-HPLC under both storage conditions, however the results were within the acceptable criteria for the main peak by SEC-HPLC, and no changes in activity were observed (within the assay variability) . Also, a slight increase in the number of fragments during long-term storage (up to ~2%) and under stressful storage conditions (~3%) was observed using CE-SDS under reducing conditions and using CE-SDS under non-reducing conditions under stressful storage conditions (~3 %), but, again, no corresponding change in activity (within the variability of the assay results) was observed. An increase in acidic-charged variants (up to ~5%) with a concomitant decrease in compounds corresponding to the main peak was observed by icIEF under both storage conditions (which may be due to assay variability as later time points had a lower percentage of acidic compounds). No changes in appearance, physicochemical parameters or activity were observed.

Example 3: First human study.

LIB003 was studied in a phase 1 Single Ascending Dose study (SAD) involving 63 subjects aged >18 and <70 years, 24 women and 39 men, 45 receiving LIB003 and 18 placebo, followed post-dose for at least 43 days. Researched

- 15 044927 The study was placebo-controlled and double-blind. Each of the 9 cohorts had 7 subjects: 5 patients receiving LIB-003 and 2 patients receiving placebo, resulting in 43 patients receiving LIB003 and 18 receiving placebo. Subcutaneous doses of LIB003 25 mg, 75 mg, 150 mg, 300 mg, and 600 mg were administered to healthy subjects on a stable diet without lipid-lowering therapy and with baseline LDL-C levels >100 and >190 mg/dL. Doses of 150 mg and 300 mg were also administered subcutaneously (SC) to patients on stable statin therapy who had a baseline LDL-C level >100 mg/dL. All subjects had TG >250 mg/dL. Two additional cohorts of healthy subjects not receiving lipid-lowering therapy, with the same lipid inclusion criteria as the SC cohorts, received LIB003 at a dose of 300 mg and 600 mg intravenously (IV).

Security Results Review

All 63 subjects completed the study, with no patients dropping out or discontinuing the study before day 43. Overall, LIB003 was safe and well tolerated after single subcutaneous (SC) and intravenous (IV) administration in healthy subjects and hypercholesterolemic patients receiving statin therapy.

Review of pharmacodynamic analysis (efficacy) results

Mean reductions in free PCSK9 were rapid at all doses, reaching greater than 99% at 12 hours, and were sustained in virtually all subjects for at least 3 weeks (day 22) in lipid-lowering treatment cohorts receiving >150 mg LIB003. While the 300 mg dose of LIB003 maintained 99% suppression of free PCSK9 for 29 days, in subjects receiving the 150 mg dose not undergoing lipid-lowering therapy, free PCSK9 decreased by 12% and in those receiving statins by 54 % of the initial level. Lower reductions in free PCSK9 were reflected in reductions in LDL-C and apo B, with greater reductions persisting in subjects not receiving lipid-lowering therapy. However, they were not sustained after 4 weeks (day 29) in patients taking statins. A single 300 mg dose, in both statin-naïve and statin-treated subjects, provided more consistent and maximal reductions in free PCSK9, LDL-C, and apo B. Additionally, based on previous data from studies using monoclonal antibodies (mAbs), it is possible hypothesize that repeated dosing would result in longer-lasting suppression of free PCSK9 and reduction in LDL-C levels. In addition, extensive prior data indicate that patients receiving high-intensity statin therapy and patients with FH have higher baseline PCSK9 levels and likely increased PCSK9 synthesis and would require doses of 300 mg or higher to completely suppress both free PCSK9 and LDL-C for 4 weeks. Summary of Pharmacokinetic Analysis Results _Cmax , _{AUC0_t} and _AUCinf for total LIB003 following SC doses increased dose proportionally in the range of 75 mg to 300 mg LIB003 and demonstrated non-significant supradose proportionality in the range of 25-75 mg (4-5 fold increase) and in the range of 300-600 mg (3-fold increase). Similarly, statin-treated subjects exhibited dose-proportional increases in total LIB003 over the 150-300 mg dose range, although exposure to total LIB003 (AUCo-t and AUC _inf ) was generally lower than exposure in subjects not receiving statins. _Cmax , AUC0 _-t, and AUCinf values for total LIB003 administered intravenously (IV) increased in a dose-proportional manner.

The median _Tmax of total LIB003 ranged from 72 to 168 hours (range varied: 36220 hours) for all SC dose levels. T-HALF, CL/F and Vz/F were also similar at all SC doses tested, as well as when LIB003 was administered with statins. The median Tmax of total LIB003 varied from 0.33 to 1.08 hours for both IV dose levels. T-HALF, CL and Vz were similar for both IV doses tested.

The absolute bioavailability of total LIB003 varied from 67 to 111% following single SC doses of 300 mg and 600 mg LIB003, respectively.

Rationale for Phase 2 Study Dose Levels

Based on free PCSK9 and LDL-C data obtained in phase 1 and aiming to achieve at least Q4W dosing at a volume equivalent to a single subcutaneous (SC) injection using an auto-injector (<1 .5 mL), a phase 2 dose-finding study was planned in approximately 80 patients with ASCVD or high-risk ASCVD, or HeFH without CVD, stable on statins and/or ezetimibe. The doses selected for Q4W dosing in this phase 2 dose-finding study included 150 mg, 300 mg, and 350 mg. All 3 doses are expected to be safe due to a lack of data regarding human exposure to LIB003 in Phase 1 600 mg dose, both subcutaneously and intravenously, and levels achieved in the 12-week GLP toxicology study in primates , not related to humans.

Example 4: A double-blind, randomized, placebo-controlled, phase 2 dose-finding study to evaluate the efficacy and safety of LIB003 in patients receiving stable lipid-lowering therapy requiring additional LDL-C lowering.

LIB003 was studied in a 12-week, double-blind, randomized, placebo-controlled study

- 16 044927 a phase 2 dose-finding study followed by a four-week follow-up period to assess the percentage change from baseline in LDL-C levels as the mean of weeks 10 and 12 and week 12 calculated using the Friedwald formula, with dosing times per month (Q4W) of various doses of LIB003. The study included a total of 81 men and women aged >18 years who had atherosclerotic cardiovascular disease (ASCVD) or were at high risk of ASCVD (>10% 5-year risk or >7.5% 10-year risk), and for subjects at risk for ASCVD or CVD had an estimated LDL-C level of >80 mg/dL, or for subjects with heterozygous familial hypercholerstolemia and no CVD, an estimated LDL-C level of >100 mg/dL. All subjects had TG <400 mg/dL during stable oral lipid-lowering therapy such as statins, with or without ezetimibe. These subjects were divided into 3 active treatment groups and 1 matched placebo treatment group. In each group, three subjects receiving LIB003 were randomized to every one subject receiving placebo, i.e. 20 LIB003-treated subjects per treatment group and 20 placebo-treated subjects). Subcutaneous doses of 150 mg, 300 mg, or 350 mg of LIB003 or placebo were administered subcutaneously once monthly (Q4W) to hypercholesterolemic subjects receiving a stable diet and oral LDL-C-lowering drug therapy.

Summary demographic data of the subjects included in this study are shown in Table. 11 below.

Table 11

Patient Cohort Demographics

Treatment group Placebo LIB003 150 mg Q4W LIB003 300 mg Q4W LIB003 350 mg Q4W Number of patients N=20 N=21 N=19 N=21 Age: mean number of years (SD) 66.6 (7.4) 65(13) 61.9(10) 65.5 (7.1) Gender: male/female 10/10 12/9 10/9 6/15 Ethnicity: Caucasian/African American 14/6 15/6 12/7 19/2 Average BMI (kg/ ^m2 ) (SD) 29.6 (4.6) 31.1 (4.6) 29.6(3.3) 29.4 (5.4) Cardiovascular disease, N (%) 9 (45%) 10 (48%) 7 (37%) 10 (48%) Diabetes mellitus, N (%) 8 (40%) 11 (52%) 10 (53%) 10 (48%) Current statin use, N (%) 16 (80%) 18(86%) 12 (63%) 19 (91%) Initial LDL-C level* (mean±8P) mg/dL 119.2(34) 119.5 (37.5) 128.9 (39.3) 115.7(30.5) *Calculated using the Friedwald formula

Review of Security Analysis Results

Overall, LIB003 was safe and generally well tolerated in this study when given as a subcutaneous dose of up to 350 mg Q4W. All doses of LIB003, 150 mg, 300 mg, and 350 mg, were well tolerated and did not reveal any safety concerns. Of the 81 subjects, 79 subjects completed the study and two subjects withdrew from the study. Study discontinuations were not associated with adverse events. Additionally, there were no adverse events resulting in death in this study.

A summary of treatment-emergent adverse events (TEAEs) experienced by subjects during the study is shown in Table 12 below.

Table 12

TEAEs occurring in subjects during the study

Treatment group Placebo LIB003 150 mg Q4W LIB003 300 mg Q4W LIB003 350 mg Q4W Treatment-emergent events (TEAE) 10 (50%) 9 (42.9%) I (58%) 13 (62%) Drug-associated TEAEs 2 (10%) 1 (4.3%) 1 (5.2%) 2 (9.5%)

A total of 43 of 81 subjects (53%) had TEAE, with 10 of 20 (50%) receiving placebo and 33 of 61 (54%) in the combined LIB003 groups. Most TEAEs were mild or moderate in severity. The most commonly reported TEAEs included fatigue, injection site bruising, upper respiratory tract infection, and shortness of breath. All other TEAEs were reported by <3 subjects.

Only six of 81 subjects (7%) had study drug-related TEAEs, with two of 20 (10%) subjects receiving placebo and four of 61 (7%) in the LIB003-treated groups combined. Most study drug-associated TEAEs were mild in severity; none of them were considered serious. The most common manifestation of study drug-associated TEAE was injection site erythema. All other TEAEs associated with the study drug were reported by <1 subject.

Six of the 81 subjects (7%) had a serious adverse event (SAE), one (5%) received placebo and five (8%) in the combined LIB003 groups, none of which were considered related to the study drug. Five serious adverse events (SAEs), one (5%) in the placebo group and four (7%) in the combined LIB003 groups were severe, and one (2%) in the LIB003 group was moderate in intensity.

There were no TEAEs associated with laboratory abnormalities. There were no clinically significant increases or trends in liver function tests (ALT, AST, or bilirubin) in any treatment group or differences between the placebo or LIB003 groups. Specifically, no subjects had an increase in ALT or AST >3x ULN, and none had bilirubin >2x ULN. A number of subjects across all treatment groups experienced transient increases in CK that were associated with exercise or activity, none of which exceeded 5 x ULN. There were no clinically significant increases or differences between treatment groups in renal function, glucose, other chemical or hematological parameters.

No clinically significant data were found regarding vital signs, ECG, and physical examination findings. A total of five cases of injection site erythema were reported 15 minutes after study drug administration. One subject in the placebo group also reported itching at the injection site 15 minutes after dosing on the first day. All injection site reactions that occurred 15 minutes after dosing were mild in severity: one in the placebo group and four in the combined LIB003-treated groups.

Review of Effectiveness Research Results

All doses of LIB003 tested, 150 mg, 300 mg, and 350 mg, caused rapid, sustained, and significant mean reductions in LDL-C and free PCSK9 levels. The greatest mean reduction in LDLC was observed in the cohort receiving the 300 mg dose of LIB003. Consistent with previous studies, the higher dose of 350 mg LIB003 did not further reduce LDL-C. The 150 mg dose was found to be insufficient to achieve maximum LDL-C reduction for a full four weeks between doses.

Summary data on the effectiveness obtained during the study are presented in table. 13 below.

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Table 13

Efficiency data

Treatment group Placebo LIB003 150 mg Q4W LIB003 300 mg Q4W LIB003 350 mg Q4W Mean (SD) % change in LDL-C at week 12* 7.3 (21.3) -26.4 (24.1) -71 (17.7) -62.8 (19.5) Mean (SD) % change in LDL-C at week 12* 5.7(20.3) -24.7 (25) -66.2 (16.8) -62.2 (16.7) Alipopretein % change week 12 5.2(13.2) -22.4 (22.7) -54.0 (16.0) -53.0 (11.7) Week 10/Week 12 LOCF* LIB003 versus placebo, mean LS (SE) -48.0 (5.37) ^Δ -76.1 (5.51) ^Δ -67.0 (5.37) _δ 95%CI (-58.7, -37.3) (-87.1, -65.1) (-77.7, -56.3) Week 12 LOCF* LIB003 versus placebo, mean LS (SE) -33.5 (6.80) ^Δ -77.3 (6.97) ^Δ -67.1 (6.80) ^Δ 95% CI (-47.0, -19.9) (-91.2, -63.5) (-80.6, -53.6) * Calculated using the Friedwald formula 7 By ultracentrifugation ¥ Primary endpoint (ANOVA) Δ p-value <0.0001

There was a large sustained mean decrease in LDL-C levels from baseline to mean change at weeks 10 and 12 of LOCF and week 12 of LOCF, as calculated by the Friedwald formula, following LIB003 dosing. Maximum difference in mean percentage changes determined by least squares (LS) (95% CI) between the LIB003 cohorts and the placebo group in LDL-C from baseline to mean at weeks 10 and 12 of LOCF and at week 12 week LOCF observed in the LIB003 300 mg cohort was -76.1% ([-86.0%, -66.2%], p<0.0001) and 77.3% ([-90 .5%, -64.1%], p<0.0001), respectively. Estimation of the mean percentage change in LDL-C levels using preparative ultracentrifugation and the Hopkins formula yielded similar results.

Consistent with previous studies showing that no additional reduction in LDL-C occurs after maximal PCSK9 suppression is achieved, a higher dose of 350 mg LIB003 did not cause an additional reduction in LDL-C levels. Although the 150 mg dose provided the same reduction as the 300 mg and 350 mg doses every two weeks after dosing, it was not sufficient to maintain the maximum reduction in LDL-C for the full four weeks between doses.

There was a large, sustained mean decrease in free PCSK9 from baseline to the mean at weeks 10 and 12 LOCF and at week 12 LOCF following LIB003 doses. The difference in mean percent LS change (95% CI) between the LIB003 and placebo cohorts in free PCSK9 from baseline to the mean at weeks 10 and 12 LOCF and at week 12 LOCF was observed in the 300 mg cohorts and 350 mg, which was -89.7% ([-100.0%, -79.4%], p<0.0001) and -92.8% ([-102.9%, -82.6 %], p<0.0001), respectively. The difference in mean LS percent change (95% CI) between the LIB003 cohorts and the placebo group in free PCSK9 from baseline to week 12 LOCF was -84.1% ([-99.5%, -68.7%], p<0.0001) and -90.2% ([-105.4%, -75.0%], p<0.0001) for the 300 mg and 350 mg cohorts, respectively. Although the 350 mg dose of LIB0003 suppressed slightly more PCSK9 than the 300 mg dose at week 12 (90.2% vs. 84.1%, respectively), it did not result in greater efficacy against LDL-C.

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In addition, there was a large and sustained mean increase in total PCSK9 levels from baseline to the mean at weeks 10 and 12 LOCF and at week 12 LOCF following LIB003 doses. The mean percentage change from baseline to mean for weeks 10 and 12 of LOCF was 95.555%, 90.273%, and 90.080% for the cohorts receiving LIB003 at doses of 150 mg, 300 mg, and 350 mg, respectively. The mean percentage change from baseline to week 12 LOCF was 75.165%, 78.188%, and 86.811% for cohorts receiving LIB003 150 mg, 300 mg, and 350 mg, respectively. In contrast, placebo showed the smallest mean percentage change (<3.044%) at the same time points.

In addition, there was a large and sustained mean decrease in non-HDL-C levels and a moderate and sustained mean decrease in TC levels from baseline to mean at weeks 10 and 12 of LOCF and at week 12 of LOCF. However, in both the LIB003 and placebo cohorts, only minimal mean changes in HDL-C levels were observed from baseline to the mean at weeks 10 and 12 of LOCF and at week 12. Additionally, comparison of the percentage change in VLDL-C and TG levels between each LIB003-treated cohort and the placebo group using an ANOVA model showed a small mean decrease in VLDL-C and TG levels from baseline to the mean at 10 and 12 weeks of LOCF and week 12 of LOCF.

Additionally, there was a significant and sustained mean reduction in apo B levels from baseline to week 12 LOCF following LIB003 doses. The maximum difference in mean percent change in LS (95% CI) between the LIB003 and placebo cohorts for apo B occurred for the 300 mg cohort and was -58.4% ([-68.9%, -48.0 %], p<0.0001).

Moreover, there was a moderate and sustained decrease in Lp(a) levels from baseline to week 12 of LOCF after all doses of LIB003. The maximum difference in mean percent change in LS (95% CI) between the LIB003 and placebo cohorts in Lp(a) occurred for the 300 mg cohort and was -28.7% ([-42.6%, -14 .8%], p<0.0001). This reduction is consistent with the reduction achieved with anti-PCSK9 monoclonal antibodies at equivalent doses and dosing frequencies, which inhibited free PCSK9 and LDL-C similarly to LIB003 at 300 mg Q4W.

Apo A1 levels showed minimal mean change from baseline to week 12 LOCF for the LIB003 and placebo cohorts.

Based on data obtained from the phase 2 study discussed above, a dose of 300 mg LIB003 administered subcutaneously by Q4W was selected for the open-label extension phase 3 study.

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Claims (39)

Sequences. PC5K9-BINING MOTIF VSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRTE (SEQ Ш NO: 1) AMINO ACID SEQUENCE OF HSA DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVAD ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNL PRLVRPEVDVMCTAFHDNEETFLI<I<YLYEIARRHPYFYAPELLFFAI<RYI<AAFTECC QAADKAACLLPKLDELRDEGKASSAK QRLKCASLQKFGERAFKAWAVARLSQRFPK AEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKP LLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRH PDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCEL F EQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAED YLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTF HADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKE TCFAEEGKKLVA ASQAALGL (SEQ IDNO: 2) PC5K9-BINDING FUSION PROTEIN VSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRTEGSGSGSDAHKSEVAHRFKD LGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLF GDKLCTVATLRETYGEMADCCAKQEPERNEC FLQHKDDNPNLPRLVRPEVDVMCT AFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKL DELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL TKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEND EMPADLP SLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKT YETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHE KTPVSDRVTKCCTESLVNRRPCFSALE VDETYVPKEFNAETFTFHADICTLSEKERQIK KQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAAS QAALGL (SEQ Ш NO: 3). CLAIM 1. A pharmaceutical composition containing one or more unit doses of a PCSK9 binding fusion protein comprising the amino acid sequence of SEQ ID NO: 3, where the unit dose contains from about 250 mg to about 350 mg of PCSK9 binding fusion protein formulated at a concentration of at least 200 mg /ml; and a pharmaceutically acceptable carrier. 2. The composition of claim 1, comprising at least about 250 mg/ml PCSK9 binding fusion protein. 3. The composition of claim 1, comprising at least about 300 mg/ml PCSK9 binding fusion protein. 4. The composition of claim 1, comprising a unit dose of from about 275 mg to about 325 mg of PCSK9 binding fusion protein. 5. The composition according to claim 4, containing a unit dose of about 300 mg of PCSK9-binding fusion protein. 6. The composition according to any one of claims 1 to 5, wherein the pharmaceutically acceptable carrier contains a buffering agent, an isotonic agent, optionally a surfactant and a solvent. 7. The composition according to claim 6, where the buffering agent is an amino acid buffer, citrate - 21 044927 buffer, phosphate buffer, acetate buffer, succinate buffer and/or bicarbonate buffer. 8. The composition according to claim 6 or 7, where the buffering agent contains L-histidine/L-histidine monohydrochloride. 9. The composition according to any one of claims 6 to 8, wherein the isotonic agent contains one or more of sodium chloride, dextrose, sucrose, glycerol, mannitol, sorbitol, arginine or potassium chloride. 10. The composition according to claim 9, wherein the isotonic agent is sodium chloride. 11. The composition according to any one of claims 6 to 10, in which the surfactant is a polysorbate surfactant. 12. The composition according to claim 11, wherein the surfactant is polysorbate-80. 13. Composition according to any one of claims 6 to 10, characterized in that the composition does not contain a surfactant. 14. The composition according to any one of claims 6 to 13, wherein the solvent is water. 15. The composition according to any one of claims 1 to 14, further containing a preservative, optionally selected from phenol, metacresol and sodium benzoate. 16. The composition according to any one of claims 1 to 15, wherein the PCSK-9 binding fusion protein contains from 1 to 10 amino acid substitutions, insertions and/or deletions. 17. The composition according to any one of claims 1 to 16, characterized in that the composition is contained in a pen, syringe or auto-injector. 18. The composition of claim 17, wherein the pen, syringe, or auto-injector contains or delivers a unit dose of about 250 mg to about 350 mg of the PCSK9-binding fusion protein. 19. The composition of claim 18, wherein the pen, syringe, or auto-injector contains and delivers a unit dose of about 275 mg to about 300 mg of the PCSK9-binding fusion protein. 20. The composition of claim 19, wherein the pen, syringe or auto-injector contains and delivers a unit dose of about 300 mg of the PCSK9 binding fusion protein. 21. A composition according to any one of claims 18 to 20, characterized in that the unit doses have a volume of no more than about 1.5 ml or no more than about 1 ml. 22. The composition according to claim 21, characterized in that the standard doses have a volume of no more than 0.8 ml. 23. The composition according to claim 21, characterized in that the unit doses have a volume of no more than about 0.25 ml. 24. The composition according to claims 17-23, where the syringe pen, syringe or auto-injector contains from 1 to 10 standard doses. 25. A pharmaceutical composition containing at least one unit dose of a PCSK9 binding fusion protein comprising the amino acid sequence of SEQ ID NO: 3, where the unit dose contains from about 250 mg to about 350 mg of PCSK9 binding fusion protein formulated at a concentration of at least 200 mg/ml; and a pharmaceutically acceptable carrier containing from about 1 mg/ml to about 10 mg/ml L-histidine/L-histidine monohydrochloride maintaining a pH of about 6.8, and from about 6 mg/ml to about 12 mg/ml sodium chloride. 26. A method of treating a disorder associated with PCSK9, comprising administering a composition according to any one of claims 1 to 25 to a subject in need thereof. 27. The method of claim 26, wherein the subject is a human having a condition selected from a lipid disorder, hypercholesterolemia, dyslipidemia, coronary heart disease, atherosclerosis, high blood pressure, type 2 diabetes mellitus and cholestatic liver disease. 28. The method according to claim 27, characterized in that the subject has hypercholesterolemia. 29. The method of claim 27, wherein the subject has familial hypercholesterolemia. 30. The method of claim 27, wherein the subject has atherosclerotic cardiovascular disease (ASCVD) or high risk (ASCVD). 31. The method according to any one of claims 26-30, characterized in that the subject is not receiving statin therapy or receiving oral lipid-lowering therapy. 32. The method according to any one of claims 26-30, characterized in that the subject receives statin therapy or oral lipid-lowering therapy. 33. The method according to claim 31, characterized in that the subject is intolerant to statins. 34. The method according to any one of claims 26 to 33, wherein the composition is administered by subcutaneous, intramuscular, intradermal or intravenous administration. 35. The method according to any one of claims 26 to 34, wherein the subject receives a unit dose of the composition from once a week to once every two months. 36. The method of claim 35, wherein the subject receives a unit dose of the composition about once a month or about once every 4 weeks. 37. The method according to claim 36, in which the unit dose has a volume of from 0.7 ml to 1.5 ml. 38. The method of claim 35, wherein the subject receives a unit dose of the composition approximately once per week. 39. The method of claim 38, wherein the unit dose has a volume of from about 0.05 ml to about 0.25 ml. -