Compositions comprising molecules that bind PCSK9 and methods of use

Abstract

The present disclosure provides pharmaceutical compositions comprising a fibronectin-based scaffold domain protein that binds to, for example, proprotein convertase subtilisin kexin-9 (PCSK9).

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of May 16, 2019, application number 2019800475529, and invention name “Compositions containing molecules that bind PCSK9 and methods of use”.

[0002] Related applications

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 62 / 672,187, filed May 16, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0004] The present disclosure relates to fibronectin-based scaffold domain proteins that bind proprotein convertase subtilisin kexin-9 (PCSK9), as well as pharmaceutical compositions and methods of use thereof. Background Art

[0005] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is an enzyme encoded by the PCSK9 gene on human chromosome 1. PCSK9 binds to receptors for low-density lipoprotein (LDL) particles. LDL receptors (LDLRs) on the membranes of liver and other cells bind and initiate endocytosis of LDL particles from the extracellular fluid into cells, thereby reducing the concentration of circulating LDL particles. If PCSK9 is blocked, more LDLRs are recycled and present on the cell surface, thereby removing LDL particles from the extracellular fluid. Therefore, blocking PCSK9 can reduce the concentration of LDL particles in the blood.

[0006] When statins and other drugs are ineffective or poorly tolerated, the monoclonal antibody PCSK9 inhibitors alirocumab and evolocumab are approved for biweekly or monthly subcutaneous injection or infusion to reduce LDL particle concentrations. For monthly injections, several milliliters of drug product are required to achieve the desired dose. Although formulations with higher concentrations of active agent can provide more ideal dosing schedules and volumes, these formulations are hampered by solubility limitations, 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, No. 4 (2008).

[0007] There is a need for pharmaceutical compositions that target PCSK9 and have the potential to achieve more optimal dosing schedules, smaller volumes, and / or improvements in efficacy while maintaining a generally safe and well-tolerated profile. Summary of the Invention

[0008] The present disclosure provides compositions comprising a fibronectin scaffold protein that binds to proprotein convertase subtilisin kexin type 9 (PCSK9) with high affinity and can be stably formulated at high concentrations to achieve maximum biological effect and more convenient dosing schedules, dosing volumes, and patient-friendly delivery devices. The PCSK9-binding fusion protein comprises a motif that binds to PCSK9 and an amino acid sequence of human serum albumin (HSA). The motif that binds to PCSK9 and the HSA amino acid sequence can be expressed as a gene fusion or chemically conjugated.

[0009] The PCSK9-binding motifs described herein are based on adnectins, a family of proteins derived from the human 10th fibronectin type III domain (10Fn3) that have been engineered to achieve high-affinity target binding. According to the present disclosure, fusion proteins that bind to PCSK9 are stably formulated at high concentrations to achieve maximum biological activity and convenient dosing 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 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 / L. In some embodiments, the PCSK9-binding fusion protein is administered at a unit dose of about 275 mg to about 325 mg (e.g., about 300 mg).

[0010] As described herein, the PCSK9-binding motif comprises or consists of the amino acid sequence of (SEQ ID NO: 1) or a variant thereof. The PCSK9-binding motif binds to human PCSK9 with subnanomolar affinity in a concentration-dependent manner. The PCSK9-binding motif is chemically conjugated or C-terminally fused to a human serum albumin (HSA) amino acid sequence. In various embodiments, the PCSK9-binding motif is fused to the HSA amino acid sequence at the C-terminus and may include a linker sequence of amino acids between the PCSK9-binding motif and the HSA amino acid sequence.

[0011] Fusion proteins that bind PCSK9 can be stably formulated in solution at high concentrations. The formulations exhibit no significant degradation or particulate formation under conventional long-term storage conditions and short-term storage under accelerated and pressurized conditions. Exemplary formulations comprise or consist essentially of (in addition to the active agent): L-histidine, L-histidine monohydrochloride, sodium chloride, and optionally polysorbate 80.

[0012] Pharmaceutical compositions of the present disclosure can be conveniently present in unit dosage form, and the unit dosage form per dosage comprises a predetermined amount of activating agent of the present disclosure. In some embodiments, the volume of the unit dose is no more than about 1.5 mL, or the volume is no more than about 1 mL. In some embodiments, the volume of the unit dose is no more than 0.8 mL, or the volume is no more than 0.7 mL. In other embodiments, with a microdose, for example, with a volume less than 0.5 mL, or less than about 0.25 mL, or less than about 0.15 mL, the composition is administered. In various embodiments, the composition is delivered with a unit dose comprising about 20 to about 450 mg of the fusion protein in conjunction with PCSK9. For example, in some embodiments, a dosage of 20 to about 75 mg is administered with a weekly microdose (for example, with a volume less than about 0.25 mL or less than about 0.15 mL). In other embodiments, a dosage of about 200 to about 450 mg is administered with a volume within the range of about 0.7 to 1.5 mL, approximately every two weeks, monthly, or every other month.

[0013] The compositions or preparations are suitable for use by subcutaneous, intramuscular, intradermal or intravenous administration. High concentration preparations allow for less frequent dosing schedules and lower dosing volumes to be suitable for subcutaneous administration. As demonstrated herein, maximum PCSK9 inhibition is achieved under relatively low concentrations of the fusion protein in conjunction with PCSK9, and higher concentrations achieve longer inhibition durations. In some embodiments, the subject receives a unit dose of the composition approximately once a week, once every 2 weeks, or approximately once every 3 weeks, or approximately once every 4 weeks (e.g., approximately once a month), or approximately once every 6 weeks, or approximately once every 8 weeks (approximately once every 2 months).

[0014] In some embodiments, the subject receives a microdose of the composition about weekly or about biweekly, such as in a volume of about 50 to about 250 μL.

[0015] The compositions can be administered to treat PCSK9 related disorders in human subjects. In some embodiments, the patient needs to reduce LDL (e.g., LDL-C). In some embodiments, the subject may exhibit cholesterol related diseases, such as hypercholesterolemia and / or atherosclerosis. In some embodiments, the subject suffers from familial hypercholesterolemia. In some embodiments, the subject suffers from cardiovascular disease (e.g., atherosclerotic coronary heart disease) or is at high risk for cardiovascular disease.

[0016] In some embodiments, the composition is administered with statin therapy or another oral lipid-lowering therapy, or in some embodiments, the composition is provided as the sole therapy for hypercholesterolemia, i.e., without oral lipid-lowering therapy (e.g., statin therapy). BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The structure of the PCSK9-binding fusion protein is shown.

[0018] Figure 2 PCSK9 pharmacokinetic and pharmacodynamic (PK-PD) models in non-human primates (NHPs) and humans are shown. LIB-003 refers to a fusion protein that binds to PCSK9.

[0019] Figure 3 Gadkar model for predicting the effect of PCSK9 targeting on LDLC is shown. Gadkar K et al., A Mechanistic Systems PharmacologyModel for Prediction ofLDLCholesterolLowering by PCSK9 Antagonism Human Dyslipidemic Populations ,CPT PharmacometricsSyst.Pharmacol.2014;3(11). DETAILED DESCRIPTION

[0020] The present disclosure provides a composition comprising a fibronectin scaffold fusion protein that binds to proprotein convertase subtilisin kexin-9 (PCSK9) with high affinity and can be stably formulated at high concentrations to achieve maximum biological effect and more convenient dosing schedules and volumes, and delivered using a patient-friendly delivery device (such as a syringe or autoinjector). The PCSK9-binding fusion protein comprises a motif that binds to PCSK9 and an amino acid sequence encoding human serum albumin (HSA) at the C-terminus.

[0021] Proprotein convertase subtilisin / kexin type 9 is a circulating protein primarily secreted by the liver. It plays an important role in the recycling of the hepatic LDLR and has been identified as an effective drug target for lowering LDL-C. The LDLR is the primary pathway for clearing LDL-cholesterol (LDL-C) from the circulation. Plasma PCSK9 binds to the hepatic LDLR along with LDL-C, targeting the receptor for endocytosis and degradation, thereby reducing the effectiveness of the LDLR in clearing LDL-C from the circulation. Inhibiting the binding of PCSK9 to the LDLR can prevent LDLR degradation, increase LDLR recycling, improve LDL-C clearance, and reduce circulating LDL-C levels.

[0022] The PCSK9-binding motifs described herein are based on "adnectins," a family of proteins derived from the human fibronectin type III domain 10 that have been engineered to achieve high-affinity target binding. "Adnectins" are small (≤12 kDa), compact proteins that share no sequence homology with immunoglobulins but possess a β-pleated structure with diverse loops similar to antibody variable regions. Adnectins lack disulfide bonds and are not glycosylated, exhibit high thermal stability and monomeric solution behavior, and can be efficiently produced using bacterial, yeast, or mammalian expression systems. By modifying the variable loop sequences and lengths while keeping the scaffold residues essentially constant, subnanomolar target binding affinities can be achieved while maintaining structural stability. Due to their size, adnectins are rapidly filtered by the kidneys, necessitating pharmacokinetic (PK)-enhancing modifications for in vivo use. Exemplary PCSK9-binding motifs (adnectins) are disclosed in U.S. Patents 8,420,098; 9,234,027; and 9,856,309, each of which is incorporated herein by reference in its entirety.

[0023] According to the present disclosure, the fusion protein that binds PCSK9 is stably formulated at high concentrations to achieve maximum biological activity and convenient dosing schedules and volumes. The concentration of the fusion protein that binds PCSK9 in the composition is at least 100 mg / mL. In some embodiments, the concentration of the fusion protein that binds PCSK9 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 fusion protein that binds PCSK9 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 fusion protein that binds PCSK9 in the composition is about 250 mg / mL to about 350 mg / mL. In some embodiments, the concentration of the fusion protein that binds PCSK9 in the composition is about 250 mg / mL or about 300 mg / mL.

[0024] The PCSK9 binding motif comprises or consists of the amino acid sequence of (SEQ ID NO: 1):

[0025] VSDVPRDLEVVAATPTSLLISW DAPAEGYG YYRITYGETGGN SPVQEFTV PVSKG TATISGLKPGVDYTITVYAV EFDFPGAGYYHR P ISINYRTE.

[0026] The PCSK9-binding loop is 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 relative to SEQ ID NO: 1. In some embodiments, the amino acid changes are made outside the binding loop. In some embodiments, one, two, or three amino acid changes are made within the binding loop.

[0027] The PCSK9-binding motif was designed to specifically target PCSK9 while essentially maintaining wild-type (WT) 10 Fn3 sequences are used to minimize inherent immunogenicity. See U.S. Patent 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.

[0028] In various embodiments, the PCSK9 binding motif is fused to a human serum albumin (HSA) amino acid sequence at the C-terminus. In some embodiments, HSA comprises a sequence that is at least 80% identical, or at least 85% identical, or at least 90% identical, or at least 95% identical, or at least 98% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2. For example, the HSA amino acid sequence may comprise one to ten or 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 comprises an alanine residue at a position corresponding to position 34 of SEQ ID NO: 2, as set forth in SEQ ID NO: 2. In various embodiments, the HSA amino acid sequence is at least 500 amino acids in length.

[0029] In some embodiments, the PCSK9 binding motif and the HSA amino acid sequence are chemically conjugated using any known chemical conjugation method.

[0030] HSA is a multidomain protein composed of helical clusters and contains 17 pairs of disulfide bridges. In native HSA, only one cysteine ​​residue, Cys34, exists as a free sulfhydryl group. In some embodiments, this Cys is replaced with an alanine residue in the PCSK9-binding fusion protein. The HSA moiety is used to extend the circulating half-life of the PCSK9-binding fusion protein. In various embodiments, the PCSK9-binding fusion protein comprising a PCSK9-binding motif and the HSA amino acid sequence has a molecular weight of approximately 77 kDa.

[0031] In some embodiments, the HSA amino acid sequence is a variant described in U.S. Patent 9,493,545, U.S. Patent 9,821,039, U.S. Patent 9,944,691, or U.S. 2014 / 0315817, each of which is herein incorporated by reference in its entirety.

[0032] In some embodiments, the PCSK9-binding motif and the HSA amino acid sequence are linked by gene 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 can comprise 2 to 20 amino acids, or in some embodiments, 4 to 10 amino acids. In some embodiments, the PCSK9-binding motif and the HSA amino acid sequence are connected via a 6-amino acid linker. The linker can be primarily composed of serine, glycine, threonine, and alanine amino acids. For example, the linker can be a serine / glycine linker. In some embodiments, the linker comprises or consists of the amino acid sequence GSGSGS.

[0033] Fusion proteins that bind to PCSK9 can be stably formulated in solution at high concentrations. For example, under long-term storage conditions, such as a temperature of about 5°C (e.g., 2°C to 8°C) or under short-term storage conditions (e.g., at 25±3°C for 1 to 6 months) under ambient conditions, the formulation does not show significant degradation or particulate formation. The fusion protein that binds to PCSK9 is suspended in an appropriate physiological solution (e.g., saline or other pharmacologically acceptable solvent or buffer solution) and may optionally contain a surfactant (e.g., a nonionic surfactant). In some embodiments, the formulation comprises a buffer, an isotonic agent, an optional surfactant, and a solvent.

[0034] Pharmaceutically acceptable carriers include water, saline, glycerol. In some embodiments, the formulation may contain fixed oils, polyethylene glycol, propylene glycol or other solvents. In some embodiments, the solvent is water.

[0035] The preparation is typically a buffer solution. As used herein, "buffer" refers to a chemical agent that can absorb a certain amount of acid or alkali without experiencing a strong change in pH. Exemplary buffers include citrate buffer, phosphate buffer, acetate buffer, succinate buffer, and bicarbonate buffer. In some embodiments, the buffer may include L-histidine / L-histidine monohydrochloride. For example, when using an L-histidine / L-histidine monohydrochloride buffer system, L-histidine / L-histidine monohydrochloride can be about 1mg / mL to about 10mg / mL, such as about 2mg / mL to about 5mg / mL. In some embodiments, the pH of the preparation is maintained in the range of about pH 5.5 to about pH 7.2 by the buffer, such as about pH 6.8 (for example, pH 6.6 to 7.0). In some embodiments, the pH of the preparation is adjusted, for example, with hydrochloric acid and / or sodium hydroxide. In some embodiments, the ratio of L-histidine / L-histidine monohydrochloride does not require pH adjustment.

[0036] In various embodiments, the isotonic agent that can be used alone or in combination includes dextrose, sucrose, glycerol, trehalose, mannitol, sorbitol, arginine, sodium chloride or potassium chloride. In some embodiments, the isotonic agent comprises sodium chloride or consists of sodium chloride. For example, the formulation can include about 2 to about 20 mg / mL of sodium chloride (e.g., 6 to 12 mg / mL sodium chloride), or one or more isotonic agents equal to the amount of an osmotic concentration of 2 to 20 mg / mL sodium chloride (or 6 to 12 mg / mL sodium chloride).

[0037] In some embodiments, the formulation comprises a surfactant, which can act as a solubilizing agent. In some embodiments, the surfactant is a nonionic surfactant. Exemplary nonionic surfactants include polysorbate surfactants, such as polysorbate 20, 40, 60, or 80. For example, the formulation may comprise polysorbate 80. Other pharmaceutically acceptable nonionic surfactants may also be used alone or in combination. In some embodiments, the formulation does not comprise a surfactant.

[0038] Exemplary formulations include citrate buffer (e.g., 10-50 mM, pH 5.6 to 6.0), histidine buffer (e.g., 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 comprises an excipient selected from arginine (e.g., 100 to 200 mM), NaCl (e.g., 100 to 200 mM), sorbitol (e.g., 100 to 300 mM), or sucrose (e.g., 100 to 300 mM). In some embodiments, the formulation comprises a surfactant, such as polysorbate 80 (e.g., 0.01 to 0.5 mg / mL). In some embodiments, the formulation does not contain a surfactant.

[0039] In some embodiments, the formulation further comprises a preservative, such as phenol, m-cresol, or sodium benzoate.

[0040] In some embodiments, the fusion protein formulation that binds PCSK9 consists essentially of (in addition to the active agent) L-histidine, L-histidine monohydrochloride, sodium chloride, and polysorbate 80. Exemplary embodiments are described below. Table 1 Display:

[0041]

[0042] The pharmaceutical compositions of the present disclosure may conveniently be presented in unit dosage form containing a predetermined amount of the active agent of the present disclosure per dosage.

[0043] In some embodiments, the composition is contained in an injection pen. An automatic injector such as an "injection pen" is a spring-loaded syringe that is designed to deliver a certain dose of a specific drug. By design, the injection pen is easy to use and is intended to be administered by the patient themselves or by untrained personnel. The injection pen is designed to overcome the hesitation associated with the self-administration of needle-based drug delivery devices. The injection pen keeps the needle tip hidden before injection and has a passive safety mechanism to prevent accidental firing (injection). The injection depth is adjustable or fixed and can be combined with the function of removing the needle shield. By pressing a button, the syringe needle will automatically insert into the subcutaneous tissue and deliver the drug. After the injection is completed, some injection pens will have visual or auditory indications to confirm that the full dose has been delivered.

[0044] In some embodiments, the injection pen contains 1 to 10 unit doses or 1 to 5 unit doses. In some embodiments, the volume of the unit dose does not exceed about 1.5 mL or about 1 mL (whether or not contained or delivered by the injection pen). In some embodiments, the volume of the unit dose does not exceed 0.8 mL, or the volume does not exceed 0.7 mL. In some embodiments, the injection pen delivers a microdose, for example, having a volume in the range of about 50 μL to about 500 μL, or a volume in the range of about 75 μL to about 250 μL. In some embodiments, the microdose has a volume of 100 to 200 μL. In various embodiments, the injection pen or other devices for subcutaneous delivery provide a dose of a fusion protein that binds to PCSK9 of 30 to about 450 mg. In various embodiments, the unit dose is about 50 to about 400 mg, or 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 (e.g., about 300 mg). The amount of the active agent delivered per unit dose can be adjusted based on the desired frequency of administration. For example, in some embodiments, a dosage of 20 to about 75 mg is administered in a weekly microdose (e.g., with a volume less than about 0.25 mL or less than about 0.15 mL). In other embodiments, a dosage of about 200 to about 450 mg is administered approximately every two weeks, monthly, or every other month with a volume within the range of about 0.7 to 1.5 mL. In some embodiments, a dosage of about 275 mg to about 350 mg (e.g., about 300 mg) is administered by subcutaneous injection every four weeks with a volume of about 1.5 mL or less or about 1 mL or less.

[0045] The compositions or preparations are suitable for use by subcutaneous, intramuscular, intradermal or intravenous administration. The high concentration, relatively low viscosity and appropriate weight-gram molecular osmotic concentration of the preparation realize patient tolerance, less frequent dosing schedule and smaller volume. As demonstrated herein, maximum PCSK9 inhibition is achieved under relatively low concentrations of the fusion protein in conjunction with PCSK9, and higher concentrations realize longer inhibition duration. In some embodiments, the subject receives a unit dose of the composition approximately once per week (e.g., by administering a microdose weekly), or approximately once per 2 weeks, or approximately once per 3 weeks, or approximately once per 4 weeks (e.g., approximately once per month), or approximately once per 6 weeks, or approximately once per 8 weeks (approximately once every 2 months).

[0046] The composition can be used to treat PCSK9 related conditions in human subjects. PCSK9 related conditions are described in U.S. Patents 8,420,098, 9,238,027 and 9,856,306, which are incorporated herein by reference in their entirety. In some embodiments, the patient needs to reduce LDL (e.g., LDL-cholesterol). In some embodiments, the subject may show cholesterol related diseases, such as hypercholesterolemia and / or atherosclerosis. In various embodiments, the subject shows a disease selected from lipid disorders, hypercholesterolemia, hyperlipoproteinemia, hyperlipidemia, dyslipidemia, coronary heart disease, atherosclerosis and diabetes. In some embodiments, the subject suffers from familial hypercholesterolemia. In some embodiments, the subject suffers from cardiovascular disease (e.g., atherosclerotic coronary heart disease) or is at high risk for cardiovascular disease.

[0047] Hypercholesterolemia is a disease characterized by elevated serum cholesterol. Elevated serum cholesterol levels affect a significant portion of the population and are an important risk factor for atherosclerosis and myocardial infarction. Cholesterol-lowering drugs, such as HMG-CoA reductase inhibitors ("statins"), are conventionally administered to patients with hypercholesterolemia. "Familial hypercholesterolemia" (FH) is a hereditary disorder characterized by high cholesterol levels in the blood and early cardiovascular disease, particularly very high low-density lipoprotein (e.g., LDL-cholesterol) levels. For individuals suffering from FH, the response of high cholesterol levels to conventional cholesterol control methods (e.g., statin therapy) is relatively weak.

[0048] FH is an autosomal dominant inherited metabolic disorder characterized by one or more mutations in the LDL receptor (LDL-R) gene or other genes involved in lipid regulation, significantly elevated LDL-C, and premature onset of atherosclerosis. In some embodiments, the hypercholesterolemia is homozygous familial hypercholesterolemia, or HoFH, a condition characterized by mutations in both the maternal and paternal LDL-R genes.

[0049] In some embodiments, the subject has heterozygous FH. Heterozygous FH is typically treated with statins, bile acid sequestrants, or other lipid-lowering agents that lower cholesterol levels.

[0050] In some embodiments, hypercholesterolemia is polygenic hypercholesterolemia, which is a condition characterized by elevated cholesterol and is caused by the influence of multiple genetic factors. In certain embodiments, polygenic hypercholesterolemia can be exacerbated by dietary lipid intake.

[0051] In some embodiments, the composition is administered together with statin therapy or other oral lipid-lowering therapies. In such embodiments, the composition will provide a cumulative reduction in LDL-C. In some embodiments, the composition is provided as the only therapy for hypercholesterolemia, i.e., without statin therapy or other oral lipid-lowering therapies. For example, in such embodiments, the subject may have statin intolerance. "Statin intolerance" occurs when a patient is unable to continue using statins due to side effects or abnormalities in liver function or muscle function (creatine kinase) after a blood test. In some embodiments, statin intolerance can be partial (i.e., only certain statins at certain doses) or complete (i.e., all statins at any dose). In some embodiments, statin intolerance causes muscle aches, pains, weakness, or cramps (i.e., myalgia); occurs in up to 15% of patients receiving treatment.

[0052] Unless otherwise indicated, the term "about" as used herein means ±10% of the relevant numerical value.

[0053] Embodiments of the present invention will now be described by way of the following examples.

[0054] Example

[0055] Example 1: PCSK9-binding fusion protein and PK-PD behavior

[0056] The PCSK9-binding fusion protein comprises a modified fibronectin domain targeting proprotein convertase subtilisin / kexin type 9 (PCSK9) and human serum albumin ( Figure 1 ). The total molecular weight of the fusion protein is approximately 77,000 Daltons. The PCSK9-binding fusion protein has a high binding affinity for human PCSK9 and a >100-fold weaker affinity for cynomolgus monkey PCSK9. Despite this difference in binding affinity, non-human primates (NHPs) are considered an appropriate species for testing the safety and pharmacology of PCSK9-binding fusion proteins because the greatest degree of free PCSK9 inhibition and the greatest degree of LDL-C reduction are achieved in NHPs.

[0057] Fusion proteins that bind to PCSK9 are being developed for subcutaneous (SC) administration to treat, for example, hypercholesterolemia, including patients with familial hypercholesterolemia or hypercholesterolemia and atherosclerotic coronary heart disease (CHD). Two major determinants of the PK of fusion proteins that bind to PCSK9 are interaction with their target, PCSK9, and the ability to recycle via the neonatal Fc receptor (FcRn) and minimize renal filtration, thereby reducing the clearance of the PCSK9-bound fusion protein and increasing its half-life. Studies in wild-type mice cannot address the impact of these determinants on the PK of fusion proteins that bind to PCSK9 because fusion proteins that bind to PCSK9 do not effectively bind to mouse PCSK9, and HSA does not interact with mouse FcRn. Therefore, cynomolgus monkeys provide the most appropriate in vivo model for evaluating the PK-PD of fusion proteins that bind to PCSK9 because binding to PCSK9 in this model results in a decrease in free PCSK9 and LDL-C; in addition, HSA is effectively recycled via the NHP FcRn receptor.

[0058] The in vivo binding affinity of PCSK9-binding fusion proteins for hPCSK9 was investigated using hPCSK9 transgenic mice, and the ability of PCSK9-binding fusion proteins to be recycled by hFcRn was also assessed in hFcRn mice. This human FcRn mouse model carries a null mutation in the mouse FcRn gene and a transgene that expresses the human FcRn α chain under the control of its native human promoter. Therefore, these mice serve as a model for evaluating the pharmacokinetics of monoclonal antibodies and human serum albumin.

[0059] For anti-PCSK9 antibodies, there are extensive data sets supporting a relationship between inhibition of systemic free PCSK9 levels and reduction of serum LDL-C. 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;July:e1-11. Figure 3 To estimate the human dose of a PCSK9-binding fusion protein (LIB003, SEQ ID NO: 3) required to achieve the desired target LDL-C lowering levels, allometric scaling of the data from the above studies was incorporated into a translational semi-mechanistic model that included the known relationship between PCSK9 and LDL-C. Figure 2The model was used to construct the expected dose-effect relationship between LIB003 and serum LDL-C.

[0060] TK-PD in nonhuman primates

[0061] The TK-PD behavior of LIB003 was characterized in a single-dose non-GLP dose-ranging (DRF) study following intravenous (IV) and subcutaneous (SC) administration (IV doses of 10, 30, 100, 200 mg / kg; SC dose of 200 mg / kg) and in a GLP toxicity study following repeated administration for 4 weeks (IV dose of 100 mg / kg; SC doses of 30 and 100 mg / kg), 12 weeks (SC doses of 30 and 100 mg / kg), or 26 weeks (SC doses of 30 and 100 mg / kg).

[0062] The TK assay for LIB003 was constructed as a target capture electrochemiluminescent assay with hPCSK9 as the capture reagent and a ruthenium-labeled rabbit anti-HSA polyclonal antibody as the detection reagent. In NHPs, the assay measures total LIB003 (due to a stronger affinity for the capture reagent hPCSK9 compared to NHP PCSK9). Alternatively, the TK assay uses a LIB003-specific mAb as the capture reagent and a ruthenium-labeled rabbit anti-HSA polyclonal antibody as the detection reagent. This assay format also measures total LIB003. The results of these studies indicate that the kinetics of total PCSK9 binding to the fusion protein are approximately linear and dose-proportional following intravenous and subcutaneous administration within this dose range.

[0063] In the DRF study, there was a persistent anti-drug antibody (ADA) response at all dose levels above 10 mg / kg. After a single dose of LIB003, in most cases, the presence of ADA in this study was associated with a rapid loss of LIB003 exposure, and first of all, a loss of target capture (reduction of free PCSK9). Although ADA was detected in most animals after weekly administration of the fusion protein binding to PCSK9 (4-week, 12-week, and 26-week GLP toxicity studies), exposure to the fusion protein binding to PCSK9 was maintained throughout the dosing interval, and the effect of ADA on TK / PD was only evident in two animals (4-week study), one animal (12-week study), and one animal (26-week study).

[0064] In the absence of ADA effects, LIB003 is slowly cleared, with terminal serum half-lives ranging from 8.3 to 10.4 days (average 9 days) in the DRF study and 7.6 to 9.2 days (average 8.3 days) in recovered animals in the 4-week GLP toxicity study; this is consistent with the behavior of albumin-like molecules in non-human primates. Similar results were observed in 12-week and 26-week GLP toxicity studies. In the DRF study, after a single IV administration, clearance (CL) ranged from 5.63 to 7.65 mL / day / kg (average 6.64 mL / day / kg) and volume of distribution (Vz) ranged from 67.6 to 103.8 mL / kg (average 86.4 mL / kg) in animals that could be assayed.

[0065] Total PCSK9 concentrations (both bound and unbound to LIB003) increased slowly and peaked approximately 7 days after dosing, at which point total PCSK9 concentrations were less than 10% of circulating LIB003 concentrations. Target capture was maximal at all doses in the DRF study, as measured by free PCSK9 and LDL reduction, indicating that target inhibition was maximal at the lowest dose tested (10 mg / kg). The results indicate that, in the absence of ADAs, increasing the dose of LIB003 increases the duration of maximal target capture.

[0066] Regardless of dose or route of administration, serum LDL was suppressed by approximately 60%, consistent with the maximal pharmacodynamic effect at all dose levels. Predictably, loss of LDL suppression and return to baseline levels coincided with the loss of maximal PCSK9 capture. Overall, these studies suggest that the maximal pharmacodynamic effect has been achieved and that higher doses do not result in greater inhibition of free PCSK9 or LDL.

[0067] Compared with slow IV bolus at the same dose level, C max Lower and T max The late onset indicates absorption from the injection site into the systemic circulation. Following subcutaneous administration, the absolute bioavailability of the subcutaneous route was estimated to be approximately 76% in the DRF study and 66.5%-89.7% in the 4-week GLP toxicity study, but both estimates were affected by the presence of ADA and the inability to characterize the total area under the curve (AUC). A combined analysis of these two studies using a population PK model estimated the bioavailability after subcutaneous administration to be 92%, and this may be a more reliable estimate because the model generates a total AUC estimate for both routes of administration.

[0068] Predicted PK-PD behavior in humans

[0069] Data from multiple studies were combined to predict the expected PK-PD behavior of LIB003 in humans. That is, a population 2-compartment PK-PD binding model describing LIB003 exposure and PCSK9 inhibition was constructed from NHP data (DRF study and 4-week GLP toxicity study). When these parameters were extended to humans, the in vivo binding affinity for hPCSK9 was derived from PK-PD studies in hPCSK9 transgenic mice. Since only data after subcutaneous administration were available, a one-compartment PK model was sufficient to describe these data; the model was otherwise identical to the model used for NHP data. The observed differences in LIB003 binding affinity to NHP PCSK9 and hPCSK9 were consistent in both in vitro and in vivo derived data, and the in vivo derived K values ​​from hPCSK9 transgenic mice were used to describe the binding affinity of LIB003. D The value is used for prediction of people.

[0070] Table 2

[0071]

[0072] The clearance of LIB003 in NHP and hFcRn mice was consistent with the expected clearance of HSA in these animals, and therefore, the clearance of LIB003 was predicted to mimic the clearance of HSA in humans (half-life of 19 days; allometric exponent of 0.74). Other parameters were allometrically converted using the expected coefficients for a therapeutic albumin fusion protein of approximately 77 kDa.

[0073] The overall structure of the NHP and human PK-PD binding models describing the capture and inhibition of free PCSK9 is shown in Figure 2 and Figure 3 Quantitative systems pharmacology models have been previously developed to describe the mechanisms 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 a predicted link between PCSK9 inhibition and LDL-C reduction over time in humans following single subcutaneous and intravenous administration of LIB003 in a first-in-human (FIH) study.

[0074] Example 2: Formulation and stability evaluation

[0075] The performance of various buffers and excipients in the formulation of PCSK9-binding fusion proteins was evaluated.

[0076] To determine the appropriate buffer and pH for use in the formulation, 18 different buffer and pH conditions using 6 different buffers at 2 mg / mL LIB003 were analyzed by DSC to assess thermal stability and by DLS to assess aggregate formation. From these experiments, pH values ​​below 5 and above 7 were excluded because protein unfolding began at temperatures <50°C (Tonset) for these pH values.

[0077] The buffer / pH combinations analyzed are shown below in Table 3. Combinations selected for further screening are shown with bold and underlined pH values.

[0078] Table 3: Buffer / pH Screening

[0079]

[0080]

[0081] After confirming the acceptable pH range and appropriate buffer, excipient combinations were studied with defined buffers and pH. A total of 15 different buffer / excipient combinations were prepared at 2 mg / mL LIB003 and analyzed by DSC to evaluate thermal stability and by DLS to evaluate aggregate formation. Three of the buffer / excipient combinations were excluded due to onset temperatures below 55°C. In addition, DLS data indicated that the use of sucrose, sorbitol, and low concentrations of NaCl resulted in aggregation, thus limiting their use in subsequent formulations.

[0082] The buffer / excipient combinations analyzed are shown below in Table 4. Combinations selected for further screening are shown with bold and underlined excipients.

[0083] Table 4: Buffer / Excipient Screening

[0084]

[0085]

[0086] To determine the appropriate buffer and excipient combination for maintaining the stability of LIB003 formulated at high concentrations, solubility screening was performed on 12 different formulations identified in Table 4 above. These formulations were concentrated to target concentrations of 200 mg / mL, 250 mg / mL, 300 mg / mL, and 340 mg / mL. Turbidity was equivalent in all formulations, and no aggregation was observed when evaluated by SEC-HPLC. Based on these parameters, these formulations were considered equivalent.

[0087] To evaluate the potential benefits of adding surfactants to LIB003 candidate formulations, eight buffer / excipient combinations in 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 effectively prevents aggregation and particle formation, the ability of candidate formulations to withstand repeated freeze-thaw and agitation stress was evaluated. No differences were observed based on recovery, turbidity, or aggregation tendency (evaluated by DLS and SEC). Due to the expected benefits of PS80 after long-term storage and product handling, all formulations containing PS80 have made progress.

[0088] Table 5: Buffer / Excipient and Surfactant Screening

[0089]

[0090]

[0091] LIB003 is stable and there are no differences between formulations under normal storage conditions and shear stress, even at high LIB003 concentrations, suggesting the need for an alternative approach to identify LIB003 formulations that can withstand long-term storage conditions. Formulations identified from the buffer / excipient screen (Table 4) at a target concentration of 340 mg / mL were spiked with 0.02% PS80, stored at 2°C-8°C or 50°C for three weeks, and analyzed by SEC-HPLC. Generally, low levels of high molecular weight (HMW) and low molecular weight (LMW) species were observed after 3 weeks of storage at 50°C. Based on the low percentage of HMW species in these formulations, the formulations containing citrate were narrowed to the excipients arginine and sorbitol. In the histidine formulation, 150 mM NaCl was selected for further experiments based on the highest percentage of the main peak species. Finally, succinate was no longer considered due to the increased levels of HMW and LMW species observed in most succinate formulations stored at 50°C relative to the control samples at 2°C-8°C.

[0092] The results of these analyses are shown below in Table 6; preferred formulations are indicated in bold and underlined font.

[0093] Table 6: SEC-HPLC evaluation results of high concentration LIB003 stored at 50°C for 3 weeks.

[0094]

[0095]

[0096] To further explore candidate formulations, LIB003 was formulated at 250 mg / mL with three buffer / excipient combinations plus PS80 at 3 different pH values ​​(Table 7). Samples were stored at 2°C-8°C or 50°C for three weeks to induce degradation and analyzed for concentration / recovery (A280), microparticles (DLS), aggregation (SEC-HPLC), charge distribution (icIEF), clipping (CE-SDS), efficacy, turbidity, and thermal stability (DSC) of LIB003. Comparison of charge distribution, aggregation / microparticle formation tendency, and relative efficacy after storage at 50°C for 3 weeks demonstrated certain differences between the formulations (Tables 8, 9, 10, and 11). CE-SDS curves, recovery, turbidity, and thermal stability were comparable in the formulations.

[0097] Table 7. Candidate formulations before storage

[0098]

[0099]

[0100] Table 8. Changes in icIEF of charge distribution after storage at 50°C for 3 weeks

[0101]

[0102] Table 9. SEC-HPLC changes in HMW and LMW species after storage at 50°C for 3 weeks

[0103]

[0104] Table 10. Potency as a function of storage temperature

[0105]

[0106] Given that the goal was to deliver LIB003 as a 0.25-1.5 mL SC injection using an autoinjector with a 27G or smaller needle, additional experiments were conducted to determine whether LIB003 formulated at 250 mg / mL had properties suitable for SC injection via an autoinjector. The viscosity, osmolality, and microparticles of the first three formulations at 250 mg / mL with their respective center point pH were evaluated. The goals were viscosity <15 cP, osmolality of 250-350 mOsm, and microparticle levels well below regulatory limits. The results are shown in Table 11 below. Microparticle levels were quite low in all formulations.

[0107] Table 11: Results of viscosity and osmolality analysis

[0108]

[0109] The PCSK9-binding fusion protein was formulated as a sterile injectable solution (subcutaneous) in 20 mM histidine, 150 mM NaCl, 0.02% (w / v) polysorbate 80, pH 6.8 for further stability studies. These stability studies included evaluations under long-term storage conditions, accelerated conditions, and pressurized conditions. The expected long-term storage temperature for the PCSK9-binding fusion protein product is 5°C ± 3°C, and the temperature for short-term storage (≤ 1-6 months) is 25°C ± 3°C.

[0110] Stability data were obtained for 1 month at the expected storage temperature (5°C ± 3°C) as well as accelerated storage conditions (25°C ± 2°C / 60 ± 5% RH) and pressurized storage conditions (40°C ± 2°C / 75 ± 5% RH).

[0111] For the exemplary PCSK9-binding fusion protein product batch, data for all parameters evaluated in the stability protocol, including stability-indicating parameters (icIEF, potency, CE-SDS, and SEC-HPLC), were within acceptance criteria at the time point examined (1 month) and under each storage condition. In addition, no significant increase in aggregates or degradant species or decrease in potency was observed under long-term storage conditions. A slight increase in aggregates (approximately 1%) was observed by SEC-HPLC under accelerated and stressed conditions, but these results were well within the acceptance criteria for the main peak of SEC-HPLC, and no change in potency was observed. Furthermore, a slight increase in fragmentation (approximately 2%) was observed by reducing CE-SDS under stressed conditions, but again, no change in potency was observed. A slight increase in acidic charge variant species (approximately 1%-4%) was observed by icIEF under each storage condition, accompanied by a concomitant decrease in the main peak species (note that the opposite trend was observed for the drug substance, indicating that the observed changes were within the variability of the method). No changes in appearance, physicochemical parameters, or potency were observed. Taken together, the stability data demonstrate that the PCSK9-binding fusion protein product is stable for 1 month under the evaluated long-term, accelerated, and stressed storage conditions.

[0112] In addition, long-term stability data of 18 months were obtained at the expected storage temperature (5°C ± 3°C), long-term stability data of 9 months were obtained at accelerated storage conditions (25°C ± 2°C / 60±5% RH), and long-term stability data of three months were obtained at pressurized storage conditions (40°C ± 2°C / 75±5% RH).

[0113] For the exemplary PCSK9-binding fusion protein product batch, data for all parameters evaluated in the stability protocol, including stability-indicating parameters (icIEF, potency, CE-SDS, and SEC-HPLC), were within acceptance criteria at all time points and storage conditions examined. Furthermore, no significant increase in aggregates or degradant species or decrease in potency was observed under long-term storage conditions. A slight increase in aggregates (approximately 1%) was observed by SEC-HPLC under all storage conditions, but these results were well within the acceptance criteria for the main peak by SEC-HPLC, and no changes in potency were observed (within assay variability). Furthermore, a slight increase in fragmentation (up to approximately 2%) was observed by reducing CE-SDS under long-term storage conditions, but similarly, no corresponding changes in potency were observed, and this change is considered within the variability of the method. Increases in fragmentation observed by reducing and non-reducing CE-SDS correlated with increased time and temperature (up to approximately 5% variation under pressurized conditions). Fluctuations in acidic and main peak species (up to approximately 8%) were observed by icIEF under long-term storage conditions, but these fluctuations were within the variability of the method. icIEF results under accelerated and stressed conditions initially appeared to indicate a potential positive trend for acidic material (up to approximately 7%) and a corresponding negative trend for the main peak material, but no concomitant changes in potency were observed. However, additional time points indicated that the minor changes observed may also be fluctuations related to method variability. Subvisible particles appeared to fluctuate slightly over time but remained within acceptable limits (note that atypical results were observed for particles ≥2μm and ≥5μm at 9 months under both long-term and accelerated conditions; however, particle counts returned to expected levels at the 12-month and 18-month time points). No changes in appearance, physicochemical parameters, or potency were observed.

[0114] Additionally, long-term stability data were obtained for 12 months at the intended storage temperature (5°C ± 3°C) and for 12 months at accelerated storage conditions (25°C ± 2°C / 60 ± 5% RH).

[0115] For the exemplary PCSK9-binding fusion protein product batch, data for all evaluation parameters in the stability protocol, including stability-indicating parameters (icIEF, potency, CE-SDS, and SEC-HPLC), were within the acceptance criteria at the time points and under each storage condition examined. In addition, no significant increase in aggregates or degradation products or decrease in potency was observed under long-term storage conditions. A slight increase in aggregates (up to approximately 1%) was observed by SEC-HPLC under both storage conditions, but these results were well within the acceptance criteria for the main peak of SEC-HPLC, and no change in potency was observed (within the assay variability). In addition, a slight increase in fragmentation (up to approximately 2%) was observed under long-term storage conditions by reducing CE-SDS conditions, a slight increase in fragmentation (approximately 3%) was observed under pressurized storage conditions, and a slight increase in fragmentation (approximately 3%) was observed under pressurized storage conditions by non-reducing CE-SDS, but again no corresponding change in potency was observed (within the assay variability). An increase in acidic charge variant species (up to approximately 5%) was observed by icIEF under both storage conditions, with a concomitant decrease in the main peak species (this may be due to assay variability, as later time points had lower percentages of acidic species). No changes in appearance, physicochemical parameters, or potency were observed.

[0116] Example 3: First-in-human study

[0117] LIB003 was studied in a Phase 1 SAD study that included 63 subjects aged ≥18 years and ≤70 years, 24 women and 39 men, 45 of whom received LIB003 and 18 received placebo, and were monitored for at least 43 days after dosing. The study was placebo-controlled and double-blind. There were 7 subjects in each of 9 cohorts: 5 patients receiving LIB-003 treatment and 2 patients receiving placebo treatment, resulting in 43 subjects receiving LIB003 treatment and 18 receiving placebo treatment. Subcutaneous doses of 25 mg, 75 mg, 150 mg, 300 mg, and 600 mg of LIB003 were administered to healthy subjects who were on a stable diet, not receiving lipid-lowering therapy, and had a baseline LDL-C ≥100 and ≤190 mg / dL. In addition, 150 mg and 300 mg doses were administered SC to patients receiving stable statin therapy and with a baseline LDL-C ≥100 mg / dL. All subjects had TG ≤ 250 mg / dL. Two additional cohorts of healthy subjects not receiving lipid-lowering therapy and with the same lipid inclusion criteria as their SC cohort received LIB003 300 mg and 600 mg IV.

[0118] Summary of Safety Results

[0119] All 63 subjects completed the study, and no patient withdrew or terminated the study before Day 43. Overall, LIB003 was safe and well tolerated following a single SC and IV dose in healthy subjects and patients with hypercholesterolemia receiving statin therapy.

[0120] Summary of Pharmacodynamic (Efficacy) Results

[0121] The mean reduction in free PCSK9 was rapid at all doses and reached over 99% within 12 hours, and this reduction persisted for at least 3 weeks (Day 22) in almost all subjects in the group receiving ≥150 mg of LIB003 who were not on lipid-lowering therapy. The LIB003 300 mg dose maintained 99% free PCSK9 inhibition over 29 days, but free PCSK9 fell to 12% of baseline in subjects on the 150 mg dose who were not on lipid-lowering therapy and to 54% of baseline in subjects on statins. The lower degree of reduction in free PCSK9 was reflected in the decrease in LDL-C and apolipoprotein B, with subjects not on lipid-lowering therapy maintaining a greater degree of reduction. However, these results were not sustained over 4 weeks (Day 29) in patients receiving statins. A single 300 mg dose resulted in more consistent and maximal reductions in free PCSK9, LDL-C, and apolipoprotein B in both non-statin and statin-treated subjects. Furthermore, based on prior data from mAb studies, it is anticipated that multiple doses will result in longer-lasting inhibition of free PCSK9 and LDL-C lowering. Furthermore, extensive prior data suggest that patients receiving high-intensity statins and those with FH have higher baseline PCSK9 levels and potentially increased PCSK9 synthesis, necessitating doses of 300 mg or higher to achieve complete suppression of free PCSK9 and LDL-C for four weeks.

[0122] Overview of Pharmacokinetic Results

[0123] C of total LIB003 from SC dose max , AUC 0-t and AUC inf There was a dose-proportional increase in LIB003 between 75 mg and 300 mg, and minimal overdose proportionality was observed between 25 mg and 75 mg (a 4-fold to 5-fold increase) and between 300 mg and 600 mg (a 3-fold increase). Similarly, within the 150 mg to 300 mg dose range, statin-treated subjects showed a dose-proportional increase in total LIB003, but total LIB003 exposure (AUC 0-t and AUC inf) were generally lower than the exposure in subjects treated with non-statin drugs. max , AUC 0-t and AUC inf Increase in proportion to the dose.

[0124] The median T of total LIB003 at all SC dose levels was max The range was 72 to 168 hours (variable range: 36 to 220 hours). T-HALF, CL / F, and Vz / F were also similar at the SC doses tested and when LIB003 was administered with a statin. The median T of total LIB003 was 2.37 at both IV dose levels. max The range was 0.33 to 1.08 hours. T-HALF, CL, and Vz were similar at both IV doses tested.

[0125] The absolute bioavailability of total LIB003 ranged from 67% to 111% following a single SC dose of 300 mg and 600 mg LIB003, respectively.

[0126] Rationale for Phase 2 Dose Levels

[0127] Based on the free PCSK9 and LDL-C data from Phase 1 and the goal of achieving at least Q4W dosing of a volume (≤1.5 mL) consistent with a single SC injection using an autoinjector, a Phase 2 dose-finding study is planned in approximately 80 patients with ASCVD or high-risk ASCVD or HeFH without CVD who are receiving stable statins and / or ezetimibe. The doses selected for Q4W dosing in this Phase 2 dose-finding study include 150 mg, 300 mg, and 350 mg. All three doses are expected to be safe based on human LIB003 exposures up to 600 mg (SC and IV) in Phase 1 and the lack of exposure at levels achieved in a 12-week non-human primate GLP toxicology study.

[0128] Example 4: To evaluate LIB003 in patients receiving stable lipid-lowering therapy who require further LDL-C lowering Randomized, double-blind, placebo-controlled, phase 2 dose-finding study of efficacy and safety

[0129] LIB003 was studied in a 12-week, randomized, double-blind, placebo-controlled, dose-finding Phase 2 study, followed by a 4-week follow-up assessment period to assess the percent change from baseline in LDL-C levels at weeks 10 and 12, as calculated by the Friedewald formula, and averaged at week 12, at various doses of LIB003 administered monthly (Q4W). The study enrolled 81 men and women aged ≥18 years with atherosclerotic cardiovascular disease (ASCVD) or at high ASCVD risk (≥10% 5-year or ≥7.5% 10-year risk), and with a calculated LDL-C ≥80 mg / dL for subjects with ASCVD or CVD risk, or ≥100 mg / dL for subjects with heterozygous familial hypercholesterolemia and no CVD. All subjects had a TG ≤ 400 mg / dL while receiving stable oral lipid-lowering medication (such as statins with or without ezetimibe). These subjects were divided into three active substance groups and one matched placebo treatment group. In each group, three LIB003 subjects were assigned to each placebo subject, i.e., 20 LIB003 subjects and 20 placebo subjects per treatment group. Monthly (Q4W) subcutaneous doses of 150 mg, 300 mg, or 350 mg LIB003 or placebo were administered subcutaneously to subjects with hypercholesterolemia who were receiving a stable diet and oral LDL-C lowering medication.

[0130] A demographic summary of the subjects enrolled in the study is shown in Table 11 below.

[0131] Table 11: Demographics of the subject group

[0132]

[0133] Summary of Safety Results

[0134] Overall, in this study, LIB003 was safe and generally well tolerated 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 show any safety issues. Of the 81 subjects, 79 completed the study and 2 withdrew from the study. Withdrawal from the study was not due to adverse events. In addition, no adverse events led to death in this study.

[0135] A summary of treatment-emergent adverse events (TEAEs) experienced by subjects during the study is shown in Table 12 below.

[0136] Table 12: TEAEs of subjects during the study

[0137]

[0138]

[0139] A total of 43 of 81 subjects (53%) experienced TEAEs, including 10 of 20 subjects (50%) who received placebo and 33 of 61 subjects (54%) who received LIB003 in combination. Most TEAEs were mild to moderate in severity. The most frequently reported TEAEs were fatigue, injection site bruising, upper respiratory tract infection, and dyspnea. All other TEAEs were reported by ≤3 subjects.

[0140] Only 6 of 81 subjects (7%) had a TEAE related to study drug, including 2 of 20 subjects (10%) who received placebo and 4 of 61 subjects (7%) who received LIB003 in combination. Most TEAEs related to study drug were mild in severity; none were considered severe. The most frequently reported TEAE related to study drug was injection site erythema. All other TEAEs related to study drug were reported by ≤1 subject.

[0141] Six of 81 subjects (7%) had SAEs, one (5%) in the placebo group, and five (8%) in the LIB003 plus treatment group; none were considered related to study drug. Five SAEs (one (5%) in the placebo group and four (7%) in the LIB003 plus treatment group) were severe in severity, and one (2%) in the LIB003 plus treatment group was moderate in severity.

[0142] There were no TEAEs associated with abnormal laboratory values. There were no clinically meaningful increases or trends in liver function tests (ALT, AST, or bilirubin) in any treatment group, and there were no differences between the placebo group and the LIB003 treatment group. Specifically, no subject experienced an increase in ALT or AST>3×ULN, and no subject had a bilirubin>2×ULN. CK did not increase consistently in most subjects across all treatment groups (which is associated with exercise or activity), and none exceeded 5×ULN. There were no clinically significant increases or differences between treatment groups in renal function, glucose, other chemistry or hematology parameters.

[0143] There were no clinically significant findings in vital signs, ECG, or physical examination results. There were a total of five documented injection site erythema events at the 15-minute post-dose time point following study drug administration. One subject in the placebo group also reported injection site pruritus 15 minutes after the first day's dose. All injection site reactions occurring 15 minutes after the dose were mild in severity, with one in the placebo group and four in the LIB003 combination group.

[0144] Overview of Efficacy Results

[0145] All LIB003 doses tested (150 mg, 300 mg, and 350 mg) resulted in rapid, sustained, and significant mean decreases in LDL-C and free PCSK9 levels. The greatest mean decrease in LDL-C was observed in the 300 mg LIB003 dose group. Consistent with previous studies, the higher 350 mg LIB003 dose did not further reduce LDL-C. The 150 mg dose was found to be insufficient to achieve the maximum LDL-C reduction over the entire four weeks between doses.

[0146] A summary of the efficacy data from this study is shown below in Table 13.

[0147] Table 13: Efficacy data

[0148]

[0149]

[0150] Following dosing with LIB003, LDL-C levels continued to decrease significantly from baseline to the mean of Week 10 and Week 12 LOCF, as calculated by the Friedewald formula. The largest LS mean percentage change difference (95% CI) in LDL-C from baseline to the mean of Week 10 and Week 12 LOCF, as well as to Week 12 LOCF, between the LIB003 group and the placebo group occurred in the 300 mg LIB003 dose group and was -76.1% (-86.0%, -66.2%], p<0.0001) and -77.3% (-90.5%, -64.1%], p<0.0001), respectively. Similar findings were observed when the mean percentage change in LDL-C levels was assessed by preparative ultracentrifugation and the Hopkins formula.

[0151] Consistent with previous studies showing that once maximal PCSK9 inhibition is achieved, no further LDL-C reductions occur, the higher dose of 350mg LIB003 did not result in further reductions in LDL-C levels. Although the 150mg dose achieved similar reductions as the 300mg and 350mg doses after biweekly dosing, it was insufficient to maintain maximal LDL-C reductions across the four weeks between doses.

[0152] Following LIB003 dosing, there was a sustained and substantial mean decrease in free PCSK9 levels from baseline to the mean of Week 10 and Week 12 LOCF, and to Week 12 LOCF. The LS mean percentage difference (95% CI) in free PCSK9 levels from baseline to the mean of Week 10 and Week 12 LOCF, and to Week 12 LOCF, between the LIB003 and placebo groups was -89.7% ([-100.0%, -79.4%], p<0.0001) and -92.8% ([-102.9%, -82.6%], p<0.0001) in the 300 and 350 mg groups, respectively. The LS mean percentage difference (95% CI) in free PCSK9 levels from baseline to Week 12 in LOCF between the LIB003 group and the placebo group was -84.1% ([-99.5%, -68.7%], p<0.0001) and -90.2% ([-105.4%, -75.0%], p<0.0001) in the 300 and 350 mg groups, respectively. Although the 350 mg LIB0003 dose inhibited slightly more PCSK9 than the 300 mg dose at Week 12 (90.2% vs. 84.1%, respectively), this did not result in greater LDL-C efficacy.

[0153] In addition, after LIB003 dosing, total PCSK9 levels continued to decrease significantly from baseline to the mean of Week 10 and Week 12 LOCF, as well as to Week 12 LOCF. For the LIB003 150 mg, 300 mg, and 350 mg groups, the mean percentage changes from baseline to the mean of Week 10 and Week 12 LOCF were 95.555%, 90.273%, and 90.080%, respectively. For the LIB003 150 mg, 300 mg, and 350 mg groups, the mean percentage changes from baseline to Week 12 LOCF were 75.165%, 78.188%, and 86.811%, respectively. In contrast, placebo showed the smallest mean percentage change (≤3.044%) at the same time points.

[0154] In addition, large mean decreases in non-HDL-C levels continued from baseline to the mean of Week 10 and Week 12 LOCF, and to Week 12 LOCF, and moderate mean decreases in TC levels continued. However, there were minimal mean changes in HDL-C levels from baseline to the mean of Week 10 and Week 12 LOCF, and to Week 12, in both the LIB003 and placebo groups. Furthermore, comparisons of the percentage changes in VLDL-C and TG levels between each LIB003 group and the placebo group using ANOVA models showed small mean decreases in VLDL-C and TG levels from baseline to the mean of Week 10 and Week 12 LOCF, and to Week 12 LOCF.

[0155] Furthermore, following the LIB003 dose, apolipoprotein B levels continued to decrease significantly from baseline to LOCF Week 12. The largest LS mean percentage change difference (95% CI) in apolipoprotein B between the LIB003 and placebo groups occurred in the 300 mg group and was -58.4% ([-68.9%, -48.0%], p<0.0001).

[0156] In addition, Lp(a) levels continued to decline moderately from baseline to Week 12 LOCF after all LIB003 doses. The largest LS mean percentage change difference (95% CI) in Lp(a) between the LIB003 group and the placebo group occurred in the 300 mg group and was -28.7% ([-42.6%, -14.8%], p<0.0001). This reduction is consistent with the reductions achieved by PCSK9 monoclonal antibodies at equivalent doses and dosing frequencies, which similarly inhibited free PCSK9 and LDL-C as LIB003 300 mg Q4W.

[0157] Apolipoprotein A1 levels showed minimal mean changes from baseline to Week 12 LOCF for both the LIB003 population and the placebo group.

[0158] Based on the data from the above Phase 2 study, the LIB003 300 mg subcutaneous Q4W dose was selected for open-label extension and Phase 3 studies.

[0159] sequence

[0160] PCSK9 binding motif

[0161] VSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRTE(SEQ ID NO:1)

[0162] HSA amino acid sequence

[0163] (SEQ ID NO:2)

[0164] PCSK9-binding fusion protein

[0165] VSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRTE GSGSGS(SEQ ID NO:3).

Claims

1. A composition comprising at least 100 mg / mL of a fusion protein that binds to PCSK9, wherein the fusion protein comprises a PCSK9-binding motif of SEQ ID NO: 1 or a variant thereof, and a human serum albumin (HSA) amino acid sequence; and a pharmaceutically acceptable carrier.

2. The composition of claim 1, wherein the PCSK9 binding motif is genetically fused to the HSA amino acid sequence at the C-terminus of the fusion protein.

3. The composition of claim 1, wherein the PCSK9 binding motif is chemically conjugated to the HSA amino acid sequence.

4. The composition of any one of claims 1 to 3, comprising at least about 150 mg / mL of the PCSK9-binding fusion protein.

5. The composition of claim 4, comprising at least about 200 mg / mL of the PCSK9-binding fusion protein.

6. The composition of claim 4, comprising at least about 250 mg / mL of the PCSK9-binding fusion protein.

7. The composition of claim 4, comprising at least about 300 mg / mL of the PCSK9-binding fusion protein.

8. The composition of claim 4, comprising a unit dose of about 275 mg to about 325 mg of the PCSK9-binding fusion protein.

9. The composition of claim 8, comprising a unit dose of about 300 mg of the PCSK9-binding fusion protein.

10. The composition of any one of claims 1 to 9, wherein the pharmaceutically acceptable carrier comprises a buffer, an isotonicity agent, optionally a surfactant, and a solvent.

11. The composition of claim 10, wherein the buffer comprises an amino acid buffer, a citrate buffer, a phosphate buffer, an acetate buffer, a succinate buffer and / or a bicarbonate buffer.

12. The composition of claim 10 or 11, wherein the buffer comprises L-histidine / L-histidine monohydrochloride.

13. The composition of any one of claims 10 to 12, wherein the isotonic agent comprises one or more of sodium chloride, dextrose, sucrose, glycerol, mannitol, sorbitol, arginine, or potassium chloride.

14. The composition of claim 13, wherein the isotonic agent comprises sodium chloride.

15. The composition of any one of claims 10 to 14, wherein the surfactant comprises a polysorbate surfactant.

16. The composition of claim 15, wherein the surfactant is polysorbate-80.

17. The composition of any one of claims 10 to 14, wherein the composition does not comprise a surfactant.

18. The composition of any one of claims 10 to 17, wherein the solvent is water.

19. A composition according to any one of claims 1 to 18, further comprising a preservative, optionally selected from phenol, m-cresol and sodium benzoate.

20. The composition of any one of claims 1 to 19, wherein the PCSK9-binding fusion protein further comprises a linker between the PCSK-9-binding motif and the HSA amino acid sequence.

21. The composition of claim 20, wherein the linker comprises 2 to 20 amino acids and primarily comprises serine, glycine, threonine and / or alanine.

22. The composition of claim 21, wherein the linker comprises 4 to 10 amino acids and consists essentially of serine and / or glycine.

23. The composition of any one of claims 1 to 22, wherein the HSA amino acid sequence comprises at least 500 amino acids.

24. The composition of claim 23, wherein the HSA amino acid sequence comprises SEQ ID NO: 2, optionally with 1 to 10 amino acid substitutions, deletions or insertions.

25. The composition of claim 23 or 24, wherein the HSA amino acid sequence comprises an alanine residue at position 34 relative to SEQ ID NO:

2.

26. The composition of any one of claims 1 to 25, wherein the PCSK-9 binding fusion protein has the amino acid sequence of SEQ ID NO: 3, optionally with 1 to 10 amino acid substitutions, insertions and / or deletions.

27. The composition of any one of claims 1 to 26, wherein the composition is contained in an injection pen.

28. The composition of claim 27, wherein the injection pen contains or delivers a unit dose of about 30 to about 450 mg of the PCSK9-binding fusion protein.

29. The composition of claim 28, wherein the injection pen contains and delivers a unit dose of about 275 mg to about 300 mg of the PCSK9-binding fusion protein.

30. The composition of claim 29, wherein the injection pen contains and delivers a unit dose of about 300 mg of the PCSK9-binding fusion protein.

31. The composition of any one of claims 28 to 30, wherein the volume of the unit dose is no more than about 1.5 mL, or no more than about 1 mL.

32. The composition of claim 31 , wherein the volume of the unit dose does not exceed 0.8 mL.

33. The composition of claim 31, wherein the volume of the unit dose does not exceed about 0.25 mL.

34. A method for treating a PCSK9-related disorder comprising administering to a subject in need thereof the composition of any one of claims 1 to 33.

35. The method of claim 34, wherein the subject is a human suffering from a condition selected from the group consisting of lipid disorders, hypercholesterolemia, dyslipidemia, coronary heart disease, atherosclerosis, hypertension, type 2 diabetes, and cholestatic liver disease.

36. The method of claim 35, wherein the subject has hypercholesterolemia.

37. The method of claim 36, wherein the subject has familial hypercholesterolemia.

38. The method of claim 35, wherein the subject has or is at high risk for atherosclerotic cardiovascular disease (ASCVD).

39. The method of any one of claims 34 to 38, wherein the subject is not undergoing statin therapy or is not undergoing treatment with lipid-lowering oral therapy.

40. The method of any one of claims 34 to 38, wherein the subject is not undergoing statin therapy or another lipid-lowering oral therapy.

41. The method of claim 39, wherein the subject has statin intolerance.

42. The method of any one of claims 34 to 41, wherein the composition is administered subcutaneously, intramuscularly, intradermally, or intravenously.

43. The method of any one of claims 34 to 42, wherein the subject receives a unit dose of the composition from once weekly to once every two months.

44. The method of claim 43, wherein the subject receives a unit dose of the composition about once a month or about once every four weeks.

45. The method of claim 44, wherein the unit dose has a volume of 0.7 to 1.5 mL.

46. ​​The method of claim 43, wherein the subject receives a unit dose of the composition approximately once a week.

47. The method of claim 46, wherein the unit dose has a volume of about 0.05 to about 0.25 mL.

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