Methods for treating or preventing atherosclerosis by administering inhibitors of ANGPTL3

Abstract

The present invention provides a method and composition for treating or preventing atherosclerosis in a subject. The method of the present invention comprises administering an inhibitor of angiopoietin-like protein-3 (ANGPTL3) to a subject who has atherosclerosis or is at risk of developing atherosclerosis. In certain embodiments, the ANGPTL3 inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to ANGPTL3.

Description

[Technical field]

[0001] Sequence description This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on February 8, 2017, is named 0431_23-PCT_SL.txt and is 14,141 bytes in size.

[0002] FIELD OF THEINVENTION The present invention relates to the field of therapeutic treatment of atherosclerotic disease and related disorders. More particularly, the present invention relates to the use of ANGPTL3 inhibitors to treat or prevent atherosclerosis, as well as to methods of eliminating or reducing atherosclerotic plaque formation and / or atherosclerotic lesions in a subject. [Background technology]

[0003] background Atherosclerosis is a syndrome that affects arterial blood vessels. It results from a chronic inflammatory response in the vascular wall and is the leading cause of mortality and cardiovascular morbidity. Atherosclerosis involves thickening of the vascular wall and is characterized by the development of arterial plaque and the eventual restriction or blockage of blood flow. Current atherosclerosis treatments include dietary and lifestyle modifications (e.g., smoking cessation), pharmaceutical interventions (e.g., statins, anticoagulants, etc.), and surgery (e.g., angioplasty, stents, bypass surgery, etc.). Direct evaluation of the efficacy of pharmaceutical interventions on atherosclerosis development in human subjects is problematic due to the difficulty of accurately detecting and measuring plaque and arterial lesions in living subjects. Thus, animal models of atherosclerosis are very useful for the development and testing of novel therapeutic treatments for atherosclerosis. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need in the art for new methods for the treatment, prevention, or amelioration of atherosclerosis. [Means for solving the problem]

[0005] Summary of the Invention The present invention provides methods, uses and compositions for treating, preventing or reducing atherosclerosis in a subject. The methods of the present invention include administering one or more doses of an angiopoietin-like protein 3 (ANGPTL3) inhibitor to a subject. The therapeutic methods of the present invention result in a reduction in atherosclerotic plaques and atherosclerotic lesions in a subject in need thereof. The methods of the present invention are also useful for reducing serum total cholesterol, LDL cholesterol, and triglycerides in a subject, and for treating diseases and disorders treatable by reducing one or more of total cholesterol, LDL cholesterol, and / or triglycerides.

[0006] According to certain embodiments of the present invention, before or at the start of treatment with one or more doses of an ANGPTL3 inhibitor, the subject is diagnosed with heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH), and / or elevated lipoprotein(a) (Lp[a]). The present invention also includes a method for treating, preventing, or reducing atherosclerosis in a subject with hypercholesterolemia that is not familial in nature (i.e., non-familial hypercholesterolemia). In certain embodiments, the subject treatable by the method of the present invention includes a subject who has been diagnosed or will be diagnosed with cardiovascular disease (e.g., atherosclerotic cardiovascular disease). In certain embodiments, the subject treatable by the method of the present invention is a subject who has suffered from stroke or myocardial infarction.

[0007] According to certain embodiments of the invention, an ANGPTL3 inhibitor is administered to a patient as add-on therapy to the patient's existing lipid-lowering therapy (eg, in addition to the patient's background statin therapy).

[0008] Exemplary ANGPTL3 inhibitors that can be used in the context of the methods of the invention include, for example, anti-ANGPTL3 antibodies, nucleic acid-based ANGPTL3 inhibitors, small molecule ANGPTL3 inhibitors, and scaffold-based ANGPTL3 binding molecules.

[0009] Other embodiments of the present invention will become apparent from consideration of the following detailed description. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a study flow chart illustrating the procedures, administrations and measurements performed on animals during the course of the study described in Example 2 herein. [Diagram 2] Figures 2A and 2B show cholesterol levels of animals from different treatment groups (control-treated, filled squares [■]; or evinacumab-treated, filled circles [●]) at different time points during the course of the study described in Example 2 herein. Figure 2A shows the mean cholesterol levels (mM) over time (weeks). Figure 2B shows the total cholesterol exposure (mM x week) at week 13. [Diagram 3] FIG. 3 shows triglyceride levels (mM) of animals from different treatment groups (control-treated, filled squares [■]; or evinacumab-treated, filled circles [●]) at different time points during the course of the study described in Example 2 herein. [Figure 4]4A, 4B, and 4C show cholesterol lipoprotein profiles (mM, after lipoprotein separation) of animals from different treatment groups (control-treated, filled squares [■]; or evinacumab-treated, filled circles [●]) at different time points during the course of the study described in Example 2 herein. Lipoproteins were separated on a Superose column, and the values ​​shown are absolute values ​​from cholesterol measurements in pooled plasma per group at the various time points indicated. FIG. 4A shows cholesterol levels from different fractions of plasma collected at time=0 of the study. FIG. 4B shows cholesterol levels from different fractions of plasma collected at time=6 weeks of the study. FIG. 4C shows cholesterol levels from different fractions of plasma collected at time=13 weeks of the study. [Diagram 5] 5A, 5B, and 5C show triglyceride lipoprotein profiles (mM, after lipoprotein separation) of animals from different treatment groups (control-treated, filled squares [■]; or evinacumab-treated, filled circles [●]) at different time points during the course of the study described in Example 2 herein. Lipoproteins were separated on a Superose column, and the values ​​shown are absolute values ​​from triglyceride measurements in pooled plasma per group at the various time points indicated. FIG. 5A shows triglyceride levels from different fractions of plasma collected at time=0 of the study. FIG. 5B shows triglyceride levels from different fractions of plasma collected at time=6 of the study. FIG. 5C shows triglyceride levels from different fractions of plasma collected at time=13 weeks of the study. [Figure 6] Figures 6A and 6B show the atherosclerotic lesion area of ​​animals from different treatment groups (control treatment, open bars or circles; or evinacumab treatment, filled bars or circles) at week 13 during the course of the study described in Example 2 herein. Figure 6A shows the average atherosclerotic lesion area (x1000 μm2) per cross section for the different treatment groups. Figure 6B is a scatter plot of the total lesion area, showing the atherosclerotic lesion area (x1000 μm2) per cross section for individual animals in the different treatment groups. [Figure 7] Figures 7A and 7B show the average necrotic content of cross sections from atherosclerotic lesions classified as type IV or type V observed in animals from different treatment groups (control-treated, open bars; or evinacumab-treated, filled bars) at week 13 of the study described in Example 2 herein. Figure 7A shows the absolute value of necrotic area per cross section (μm2×1000). Figure 7B shows the relative value of necrotic content of lesions (% of lesion area). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description Before describing the present invention, it is to be understood that the present invention is not limited to the specific methods and experimental conditions described, since such methods and experimental conditions may vary. It is also to be understood that the terms used herein are not intended to be limiting, since they are for the purpose of describing specific embodiments only, and the scope of the present invention is limited only by the appended claims.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. The term "about" used herein, when used with reference to a specific recited value, means that the value may vary by 1% or less from the recited value. For example, the expression "about 100" used herein includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0013] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference as if set forth in their entirety.

[0014] Methods for preventing or reducing atherosclerosis The present invention generally relates to methods and compositions for preventing or reducing atherosclerosis in a subject. The methods of the present invention include administering one or more doses of an angiopoietin-like protein-3 (ANGPTL3) inhibitor to a subject in need thereof. In certain embodiments, the present invention results in a reduction in atherosclerotic plaque formation in the subject. For example, the methods of the present invention can result in one or more of a reduction in the area of ​​atherosclerotic lesions and / or a reduction in the necrotic area of ​​atherosclerotic lesions in the subject.

[0015] Patient Selection According to certain embodiments of the present invention, subjects treatable by the method of the present invention include, but are not limited to, subjects who have been diagnosed with or will be diagnosed with atherosclerosis, or subjects at risk of developing atherosclerosis.Accordingly, the method of the present invention includes selecting a subject who has atherosclerosis or is at risk of developing atherosclerosis, and administering one or more doses of angiopoietin-like protein-3 (ANGPTL3) inhibitor to the subject.As used herein, a subject "at risk of developing atherosclerosis" includes a subject who has or exhibits one or more risk factors for atherosclerosis.Risk factors for atherosclerosis are well known in the art, and include, but are not limited to, elevated serum low-density lipoprotein (LDL) cholesterol levels, elevated serum triglyceride (TG) levels, decreased serum high-density lipoprotein (HDL) cholesterol levels, hypertension, diabetes mellitus, family history, and smoking. Methods for assessing atherosclerosis risk factors for a given subject are also well known in the art.

[0016] According to certain embodiments of the present invention, the subject treatable by the method of the present invention is diagnosed with the occurrence of hypercholesterolemia different from heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), elevated lipoprotein(a) (Lp[a]), autosomal dominant hypercholesterolemia (ADH, e.g., ADH associated with one or more gain-of-function mutations in the LDLR gene, ApoB gene, and / or PCSK9 gene), autosomal recessive hypercholesterolemia (ARH, e.g., ARH associated with a mutation in LDLRAP1), or even familial hypercholesterolemia (nonFH) before or at the start of treatment with one or more doses of an ANGPTL3 inhibitor. The diagnosis of familial hypercholesterolemia (e.g., heFH or hoFH) can be made by genotyping and / or clinical criteria. For patients who are not genotyped, the clinical diagnosis can be based on either the criteria for definite FH on the Simon Broome criteria or a score of >8 points on the WHO / Dutch Lipid Network criteria.

[0017] According to certain embodiments of the present invention, the subject treatable by the method of the present invention is a subject diagnosed with cardiovascular disease (CVD) before or at the start of treatment with one or more doses of an ANGPTL3 inhibitor. The subject may also be selected based on having a history of coronary heart disease (CHD). As used herein, "history of CHD" (or "recorded history of CHD") includes one or more of the following: (i) acute myocardial infarction (MI); (ii) asymptomatic MI; (iii) unstable angina; (iv) coronary revascularization (e.g., percutaneous coronary intervention [PCI] or coronary artery bypass graft surgery [CABG]); and / or (v) clinically significant CHD diagnosed by invasive or non-invasive testing (e.g., coronary angiography, treadmill stress testing, stress echocardiography or nuclear imaging).

[0018] According to certain embodiments of the invention, subjects treatable by the methods of the invention may be selected on the basis of having non-coronary heart disease cardiovascular disease ("non-CHD CVD"). As used herein, "non-CHD CVD" includes one or more of: (i) a documented previous ischemic attack with focal ischemic neurological deficit lasting longer than 24 hours believed to be of atherothrombotic origin; (ii) peripheral artery disease; (iii) abdominal aortic aneurysm; (iv) atherosclerotic renal artery stenosis; and / or (v) carotid artery disease (transient ischemic attack or >50% occlusion of the carotid artery).

[0019] According to certain embodiments of the invention, subjects treatable by the methods of the invention may be selected based on having one or more additional risk factors, such as, for example, (i) documented moderate chronic kidney disease (CKD) defined by 30≦eGFR<60 mL / min / 1.73 m2 for 3 months or more; (ii) type 1 or type 2 diabetes with or without target organ damage (e.g., retinopathy, nephropathy, microalbuminuria); and / or (iii) a calculated 10-year fatal CVD risk score ≧5% (ESC / EAS guidelines for the management of dyslipidemia, Conroy et al., 2003, Eur. Heart J. 24:987-1003).

[0020] According to certain embodiments of the invention, subjects treatable by the methods of the invention may be selected based on having one or more additional risk factors selected from the group consisting of age (e.g., greater than 40, 45, 50, 55, 60, 65, 70, 75, or 80 years of age), race, nationality, sex (male or female), exercise habits (e.g., sedentary or active), other pre-existing medical conditions (e.g., type II diabetes, hypertension, etc.), and current medical conditions (e.g., currently receiving beta-blockers, niacin, ezetimibe, fibrates, omega-3 fatty acids, bile acid resins, etc.).

[0021] In certain embodiments of the present invention, the subject treatable by the method of the present invention shows elevated levels of one or more inflammatory markers. Any marker of systemic inflammation can be utilized for the purposes of the present invention. Suitable inflammatory markers include, but are not limited to, C-reactive protein, cytokines (e.g., IL-6, IL-8, and / or IL-17), and cell adhesion molecules (e.g., ICAM-1, ICAM-3, BL-CAM, LFA-2, VCAM-1, NCAM, and PECAM).

[0022] According to an embodiment of the present invention, subjects treatable by the method of the present invention can be selected based on one or more combinations of the above selection criteria or treatment characteristics.For example, according to a particular embodiment, subjects suitable for treatment with the method of the present invention can be further selected based on: (i) having a documented history of CHD, (ii) non-CHD CVD, and / or (iii) having heFH or non-FH in combination with diabetes with target organ damage; such patients can also be selected based on having a serum LDL-C concentration equal to or greater than 70 mg / dL.

[0023] According to certain embodiments of the invention, subjects treatable by the methods of the invention have hypercholesterolemia that is not adequately controlled by a daily moderate therapeutic statin regimen, and may be further selected based on either having heFH or non-FH without CHD, or having non-CHD CVD, but (i) a calculated 10-year fatal CVD risk score of > 5%; or (ii) diabetes without target organ damage; such subjects may also be selected based on having a serum LDL-C concentration equal to or greater than 100 mg / dL.

[0024] According to a particular embodiment of the invention, the subject treatable by the methods of the invention is a subject with Familial Chylomicronemia Syndrome (FCS; also known as Lipoprotein Lipase Deficiency).

[0025] According to certain embodiments of the invention, subjects treatable by the methods of the invention are subjects who have undergone or have recently undergone lipoprotein apheresis (e.g., within the last 6 months, within the last 12 weeks, within the last 8 weeks, within the last 6 weeks, within the last 4 weeks, within the last 2 weeks, etc.).

[0026] Add-on ANGPTL3 inhibitors The present invention includes methods of preventing or reducing atherosclerosis in a subject, where the subject is administered an ANGPTL3 inhibitor according to a particular dosage and frequency, and where the ANGPTL3 inhibitor is administered as an add-on to the patient's existing lipid-lowering therapy (LMT), such as an add-on to the patient's existing daily therapeutic statin regimen (specific exemplary daily therapeutic statin regimens to which an ANGPTL3 inhibitor can be added may be described elsewhere herein).

[0027] For example, the method of the present invention includes an add-on treatment regimen in which the ANGPTL3 inhibitor is administered as an add-on therapy to the same stable daily therapeutic statin regimen (i.e., the same dosage of statin) that the patient was undergoing before receiving the ANGPTL3 inhibitor. In other embodiments, the ANGPTL3 inhibitor is administered as an add-on therapy to a therapeutic statin regimen that includes a statin at a dose that is greater or less than the dose of statin that the patient was undergoing before receiving the ANGPTL3 inhibitor. For example, after starting a treatment regimen that includes an ANGPTL3 inhibitor administered at a certain dosage frequency and amount, the daily dose of statin administered or prescribed to the patient may (a) remain the same, (b) increase, or (c) decrease (e.g., up-titrate or down-titrate) compared to the daily statin dose that the patient was receiving before starting the ANGPTL3 inhibitor treatment regimen, depending on the patient's treatment needs.

[0028] Treatment Efficacy The methods of the invention result in a reduction in atherosclerotic plaque formation in an artery of a subject and / or prevention of the progression of atherosclerosis in a subject. For example, the methods of the invention are useful for limiting or reducing the area of ​​atherosclerotic lesions and / or the area of ​​necrotic cores in atherosclerotic lesions in a subject.

[0029] The methods of the present invention may further result in a reduction in serum levels of one or more lipid components selected from the group consisting of LDL-C, ApoB100, non-HDL-C, total cholesterol, VLDL-C, triglycerides, Lp(a) and remnant cholesterol. For example, according to certain embodiments of the invention, administration of a pharmaceutical composition comprising an ANGPTL3 inhibitor to a suitable subject may result in a mean percent reduction from baseline in serum low density lipoprotein cholesterol (LDL-C) of at least about 25%, 30%, 40%, 50%, 60%, or more; a mean percent reduction from baseline in ApoB100 of at least about 25%, 30%, 40%, 50%, 60%, or more; a mean percent reduction from baseline in non-HDL-C of at least about 25%, 30%, 40%, 50%, 60%, or more; a mean percent reduction from baseline in total cholesterol of at least about 10%, 15%, 20%, 25%, 30%, 35%, or more; a mean percent reduction from baseline in VLDL-C of at least about 5%, 10%, 15%, 20%, 25%, 30%, or more; a mean percent reduction from baseline in triglycerides of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or more; and / or a mean percent reduction from baseline in Lp(a) of at least about 5%, 10%, 15%, 20%, 25% or more.

[0030] ANGPTL3 inhibitors The methods of the invention include administering to a patient a therapeutic composition comprising an ANGPTL3 inhibitor. As used herein, an "ANGPTL3 inhibitor" is any substance that binds to or interacts with human ANGPTL3 in vitro or in vivo and inhibits the normal biological function of ANGPTL3. Non-limiting examples of classes of ANGPTL3 inhibitors include small molecule ANGPTL3 antagonists, nucleic acid-based inhibitors of ANGPTL3 expression or activity (e.g., siRNA or antisense), peptide-based molecules that specifically interact with ANGPTL3 (e.g., peptibodies), receptor molecules that specifically interact with ANGPTL3, ANGPTL3-binding scaffold molecules (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, fibronectin-based scaffold constructs, and other scaffolds based on naturally occurring repeat proteins, etc. [see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]), and anti-ANGPTL3 aptamers or portions thereof. According to certain embodiments, the ANGPTL3 inhibitors that can be used in the context of the present invention are anti-ANGPTL3 antibodies or antigen-binding fragments of antibodies that specifically bind to human ANGPTL3.

[0031] As used herein, the term "human angiopoietin-like protein-3" or "human ANGPTL3" or "hANGPTL3" refers to ANGPTL3 having the amino acid sequence of SEQ ID NO:1 (NCBI Accession No. NP_055310), or a biologically active fragment thereof.

[0032] The term "antibody" as used herein is intended to refer to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or VL). HThe heavy chain constant region is made up of three domains: H 1. C H 2 and C H Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with regions that are more conserved, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-ANGPTL3 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. The amino acid sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0033] The term "antibody" as used herein also includes antigen-binding fragments of a complete antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from a complete antibody molecule using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable domains and optionally constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and engineered chemically or using molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, or to introduce codons, to create cysteine ​​residues, to modify, add or delete amino acids.

[0034] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) a minimal recognition unit consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0035] Antigen-binding fragments of antibodies typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and generally contains at least one CDR that is contiguous or in frame with one or more framework sequences. L Domain and Related V H In an antigen-binding fragment having a domain, H and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer, with the V H -V H , V H -V L Or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a monomeric V H Or V L It may contain domains.

[0036] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to each other or linked by a complete or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that create a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the invention may be linked to each other and / or to one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, with the domains non-covalently associated (eg, by disulfide bonds).

[0037] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, where each variable domain can specifically bind to a separate antigen or a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of antigen-binding fragments of antibodies of the invention using routine techniques available in the art.

[0038] The constant region of an antibody is important in the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, an antibody isotype can be selected based on whether it is desirable for the antibody to mediate cytotoxicity.

[0039] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, the human antibodies of the invention may include amino acid residues (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs and particularly CDR3, that are not encoded by human germline immunoglobulin sequences. However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, e.g., a mouse, have been grafted onto human framework sequences.

[0040] The term "recombinant human antibody" is intended to include all human antibodies that are produced, expressed, created or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies produced, expressed, created or isolated by any other means including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus the V and V regions of the recombinant antibodies are not necessarily modified. H and V L The amino acid sequence of the region is H and V L Derived from the sequence and human germline V H and V L Although related in sequence, they may not naturally occur within the human antibody germline repertoire in vivo.

[0041] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of about 150-160 kDa, where the dimers are linked by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds, and a molecule of about 75-80 kDa is formed, composed of covalently coupled light and heavy chains (half antibodies). These forms have been very difficult to separate, even after affinity purification.

[0042] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the hinge region isotype of the antibody. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed using a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention provides a method for the identification of a second form that is highly selective for the hinge region of an antibody. H 2 or C H Included are antibodies with one or more mutations in three regions, which may be desirable in manufacturing, for example, to improve yield of the desired antibody form.

[0043] An "isolated antibody" refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present invention. An isolated antibody also includes an antibody in situ within a recombinant cell. An isolated antibody is an antibody that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0044] The term "specifically binds" or the like means that an antibody or an antigen-binding fragment thereof forms a relatively stable complex with an antigen under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, an antibody that "specifically binds" to ANGPTL3 as used in the context of the present invention is one that binds to ANGPTL3 or a portion thereof with a K of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by a surface plasmon resonance assay. D However, an isolated antibody that specifically binds human ANGPTL3 may have cross-reactivity to other antigens, such as ANGPTL3 molecules from other (non-human) species.

[0045] Anti-ANGPTL3 antibodies useful for the methods of the invention may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The invention includes methods that include the use of antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or one or more CDR regions are mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce a large number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll framework and / or CDR residues within the domain are backmutated to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are backmutated to the original germline sequence, e.g., only mutated residues found within the first eight amino acids of FR1 or the last eight residues of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the invention may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., where certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Uses of antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.

[0046] The invention also includes methods that include the use of anti-ANGPTL3 antibodies that include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the invention includes the use of anti-ANGPTL3 antibodies that have HCVR, LCVR, and / or CDR amino acid sequences that include, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0047] The term "surface plasmon resonance" is used, for example, in BIAcore TMThis refers to an optical phenomenon that allows the analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix using a 3D RT-PCR system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0048] As used herein, the term "K D " is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0049] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions of an antigen and have different biological effects. Epitopes may be either conformational or linear. Conformational epitopes are those that arise from spatially adjacent amino acids from different segments of a linear polypeptide chain. Linear epitopes are those that arise from adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include sugar, phosphoryl, or sulfonyl moieties on the antigen.

[0050] According to certain embodiments, the anti-ANGPTL3 antibody used in the method of the present invention is an antibody with pH-dependent binding properties. As used herein, the term "pH-dependent binding" means that the antibody or antigen-binding fragment thereof exhibits "reduced binding to ANGPTL3 at acidic pH compared to neutral pH" (for the purposes of this disclosure, both terms may be used interchangeably). For example, antibodies with "pH-dependent binding properties" include antibodies and antigen-binding fragments thereof that bind to ANGPTL3 with higher affinity at neutral pH than at acidic pH. In certain embodiments, the antibodies and antigen-binding fragments of the present invention bind to ANGPTL3 with at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more times higher affinity at neutral pH than at acidic pH.

[0051] According to this aspect of the invention, an anti-ANGPTL3 antibody with pH-dependent binding properties may have one or more amino acid mutations compared to a parent anti-ANGPTL3 antibody. For example, an anti-ANGPTL3 antibody with pH-dependent binding properties may include, for example, one or more histidine substitutions or insertions in one or more CDRs of a parent anti-ANGPTL3 antibody. Thus, according to certain embodiments of the invention, a method is provided that includes administering an anti-ANGPTL3 antibody that includes a CDR amino acid sequence (e.g., heavy and light chain CDRs) that is identical to the CDR amino acid sequence of a parent anti-ANGPTL3 antibody, except that one or more amino acids in one or more CDRs of the parent antibody are substituted with histidine residues. An anti-ANGPTL3 antibody with pH-dependent binding may have, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or more histidine substitutions within a single CDR of the parent antibody or distributed throughout multiple (e.g., 2, 3, 4, 5, or 6) CDRs of the parent anti-ANGPTL3 antibody. For example, the invention includes the use of anti-ANGPTL3 antibodies that have pH-dependent binding and that comprise one or more histidine substitutions in HCDR1, one or more histidine substitutions in HCDR2, one or more histidine substitutions in HCDR3, one or more histidine substitutions in LCDR1, one or more histidine substitutions in LCDR2, and / or one or more histidine substitutions in LCDR3 of a parent anti-ANGPTL3 antibody.

[0052] The term "acidic pH" as used herein means a pH of 6.0 or less (e.g., less than about 6.0, less than about 5.5, less than about 5.0, etc.). The term "acidic pH" includes pH values ​​of about 6.0, 5.95, 5.90, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0 or less. The term "neutral pH" as used herein means a pH of about 7.0 to about 7.4. The term "neutral pH" includes pH values ​​of about 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0053] Non-limiting examples of anti-ANGPTL3 antibodies that may be used in the context of the present invention include, for example, evinacumab, as well as any of the exemplary anti-ANGPTL3 antibodies set forth in U.S. Pat. No. 9,018,356 (Regeneron Pharmaceuticals, Inc.), the disclosure of which is incorporated herein by reference in its entirety, or any of the exemplary anti-ANGPTL3 antibodies set forth in U.S. Pat. No. 8,742,075 (Lexicon Pharmaceuticals, Inc.), the disclosure of which is incorporated herein by reference in its entirety.

[0054] Human antibody production Anti-ANGPTL3 antibodies can be produced according to any antibody production / isolation method known in the art.For example, antibodies for use in the methods of the present invention can be produced by hybridoma technology, phage display, yeast display, etc.Antibodies for use in the methods of the present invention can be, for example, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0055] Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present invention to generate human antibodies that specifically bind to ANGPTL3.

[0056] For example, VELOCIMMUNE TM Using techniques (see, e.g., US 6,596,541, Regeneron Pharmaceuticals) or any other known method for generating monoclonal antibodies, a high affinity chimeric antibody against ANGPTL3 having a human variable region and a mouse constant region is first isolated. (R)The technique involves the generation of a transgenic mouse having a genome that includes human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces antibodies that include human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

[0057] In general, VELOCIMMUNE (R) Mice are challenged with an antigen of interest, and lymphocytes (e.g., B cells) are harvested from the mice that express antibodies. The lymphocytes may be fused with a myeloma cell line to produce a hybridoma cell line, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains may be isolated and linked to constant regions of the desired isotypes of heavy and light chains. Such antibody proteins may be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains may be isolated directly from antigen-specific lymphocytes.

[0058] First, a high affinity chimeric antibody having a human variable region and a mouse constant region is isolated. The antibody is characterized and selected for desired characteristics including affinity, selectivity, epitope, etc. using standard procedures known in the art. The mouse constant region is replaced with the desired human constant region to generate a fully human antibody of the invention, e.g., wild-type or modified IgG1 or IgG4. The constant region selected can vary depending on the particular application, the high affinity antigen binding properties and the target specificity properties that belong to the variable region.

[0059] In general, the antibodies that can be used in the methods of the present invention have high affinity, as described above, as measured by binding to antigen either immobilized on a solid phase or in solution phase. The mouse constant region is replaced with the desired human constant region to generate the fully human antibody of the present invention. The constant region selected can vary depending on the particular application, the high affinity antigen binding properties and the target specificity properties that belong to the variable region.

[0060] Specific examples of human antibodies or antigen-binding fragments of antibodies that specifically bind to ANGPTL3 that can be used in the context of the methods of the invention include antibodies or antigen-binding proteins that comprise six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) from the heavy chain and light chain variable region (HCVR / LCVR) amino acid sequence pair comprising SEQ ID NO:2 / 3.

[0061] In certain embodiments of the invention, the anti-ANGPTL3 antibody or antigen-binding fragment thereof that can be used in the methods of the invention comprises heavy and light chain complementarity determining regions (HCDR1-HCDR2-HCDR3 / LCDR1-LCDR2-LCDR3) comprising the amino acid sequences of SEQ ID NOs: 4, 5, 6, 7, 8 and 9.

[0062] In certain embodiments of the invention, an anti-ANGPTL3 antibody or antigen-binding fragment thereof that can be used in the methods of the invention comprises a HCVR having the amino acid sequence of SEQ ID NO:2 and a LCVR having the amino acid sequence of SEQ ID NO:3.

[0063] Pharmaceutical Compositions and Methods of Administration The present invention includes methods comprising administering an ANGPTL3 inhibitor to a patient, wherein the ANGPTL3 inhibitor is contained within a pharmaceutical composition. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide for suitable transport, delivery, tolerability, and the like. A large number of suitable formulations can be found in formularies known to every pharmacist: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN, acetaminophen ... TM ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowax emulsions (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0064] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents.

[0065] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. Moreover, for subcutaneous delivery, a pen delivery device has easy applicability in delivering the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. A disposable pen delivery device does not have a replaceable cartridge. Rather, the disposable pen delivery device is sold pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0066] A number of reusable pen and auto-injector delivery devices have applicability in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, the AUTOPEN, to name just a few. TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM Penn (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM , OPTIPEN PRO TM, OPTIPEN STARLET TM , and OPTICLIK TM (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen-type delivery devices that have applicability in the subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR, to name just a few examples. TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk), and KWIKPEN TM (Eli Lilly), SURECLICK TM Auto-injector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM Penn (Abbott Labs, Abbott Park IL) is one example.

[0067] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, pumps can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, controlled release systems can be placed in the vicinity of the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0068] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations may be manufactured by known methods. For example, the injectable preparations may be manufactured by dissolving, suspending or injecting the above-mentioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other auxiliary agents, and the like, which may be used in combination with suitable solubilizing agents such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. As oily media, for example, sesame oil, soybean oil, and the like may be used in combination with solubilizing agents such as benzyl benzoate, benzyl alcohol, and the like. The injectable preparations thus manufactured are preferably filled into suitable ampoules.

[0069] Advantageously, the pharmaceutical compositions for oral or parenteral use are prepared in a dosage form with a unit dose suitable for the dose of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0070] Dosage The amount of ANGPTL3 inhibitor (e.g., anti-ANGPTL3 antibody) administered to a subject according to the method of the present invention is generally a therapeutically effective amount.The phrase "therapeutically effective amount" as used herein refers to the dose of ANGPTL3 inhibitor that causes a detectable decrease (at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more from baseline) in one or more parameters selected from the group consisting of LDL-C, ApoB100, non-HDL-C, total cholesterol, VLDL-C, triglycerides, Lp(a) and remnant cholesterol, or an amount that prevents or reduces atherosclerosis in a subject (as described elsewhere herein).

[0071] In the case of an anti-ANGPTL3 antibody, a therapeutically effective amount is about 0.05 mg to about 600 mg of an anti-ANGPTL3 antibody, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 64 The dosage of the ANGPTL3 inhibitor may be about 300 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg. Other dosage amounts of the ANGPTL3 inhibitor will be apparent to one of skill in the art and are contemplated within the scope of the present invention.

[0072] The amount of anti-ANGPTL3 antibody contained within an individual dose is expressed in milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). For example, an anti-ANGPTL3 antibody may be administered to a patient at a dose of about 0.0001 to about 10 mg / kg of the patient's body weight.

[0073] Combination therapy As described elsewhere herein, the methods of the invention can include administering an ANGPTL3 inhibitor to a patient in combination with ("in addition to") a lipid-lowering therapy for which the patient has previously been prescribed. For example, in the context of preventing or reducing atherosclerosis, an ANGPTL3 inhibitor can be administered to a patient in combination with a stable therapeutic daily statin regimen. Exemplary daily therapeutic statin regimens with which an ANGPTL3 inhibitor may be administered in the context of the present invention include, for example, atorvastatin (10, 20, 40 or 80 mg daily), (atorvastatin / ezetimibe 10 / 10 or 40 / 10 mg daily), rosuvastatin (5, 10 or 20 mg daily), cerivastatin (0.4 or 0.8 mg daily), pitavastatin (1, 2 or 4 mg daily), fluvastatin (20, 40 or 80 mg daily), simvastatin (5, 10, 20, 40 or 80 mg daily), simvastatin / ezetimibe (10 / 10, 20 / 10, 40 / 10 or 80 / 10 mg daily), lovastatin (10, 20, 40 or 80 mg daily), pravastatin (10, 20, 40 or 80 mg daily), and combinations thereof. Other lipid-lowering therapies in combination with which an ANGPTL3 inhibitor may be administered in the context of the present invention include, for example, (1) drugs that inhibit cholesterol uptake and / or bile acid reabsorption (e.g., ezetimibe); (2) agents that increase lipoprotein catabolism (e.g., niacin); and / or (3) activators of LXR transcription factors that play a role in cholesterol efflux, such as 22-hydroxycholesterol.

[0074] In certain embodiments, the present invention includes a method for preventing or reducing atherosclerosis in a subject by administering to the subject an ANGPTL3 inhibitor in combination with an inhibitor of ANGPTL4 (e.g., an anti-ANGPTL4 antibody or an antigen-binding fragment thereof), an inhibitor of ANGPTL8 (e.g., an anti-ANGPTL8 antibody or an antigen-binding fragment thereof), and / or an inhibitor of PCSK9 (e.g., an anti-PCSK9 antibody or an antigen-binding fragment thereof). Non-limiting examples of anti-PCSK9 antibodies that may be used in the context of the present invention include, for example, alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, or an antigen-binding portion of any of the aforementioned antibodies.

[0075] Dosing regimen According to certain embodiments of the present invention, multiple doses of an ANGPTL3 inhibitor (i.e., a pharmaceutical composition comprising an ANGPTL3 inhibitor) can be administered to a subject over a defined time course (e.g., in addition to a daily therapeutic statin regimen or other background lipid-lowering therapy). The method according to this aspect of the present invention comprises sequentially administering multiple doses of an ANGPTL3 inhibitor to a subject. As used herein, "sequentially administering" means that each dose of an ANGPTL3 inhibitor is administered to a subject at different times, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes a method comprising sequentially administering to a patient a single initial dose of an ANGPTL3 inhibitor, followed by one or more secondary doses of an ANGPTL3 inhibitor, and optionally followed by one or more tertiary doses of an ANGPTL3 inhibitor.

[0076] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the time sequence of individual doses of pharmaceutical compositions containing an ANGPTL3 inhibitor. Thus, an "initial dose" is the dose administered at the beginning of a treatment regimen (also called "baseline dose"); a "secondary dose" is the dose administered after the initial dose; and a "tertiary dose" is the dose administered after the secondary dose. The initial, secondary and tertiary doses may all contain the same amount of ANGPTL3 inhibitor, but generally may differ from each other in terms of administration frequency. However, in certain embodiments, the amount of ANGPTL3 inhibitor contained in the initial, secondary and / or tertiary doses differs from each other during treatment (e.g., adjusted up or down as necessary). In certain embodiments, two or more (e.g., 2, 3, 4 or 5) doses are administered at the beginning of a treatment regimen as a "loading dose", followed by subsequent doses administered less frequently (e.g., "maintenance dose").

[0077] According to an exemplary embodiment of the invention, each secondary and / or tertiary dose is administered within 1 to 26 (e.g., 1, 1 1 / 2, 2, 2 1 / 2, 3, 3 1 / 2, 4, 4 1 / 2, 5, 5 1 / 2, 6, 6 1 / 2, 7, 7 1 / 2, 8, 8 1 / 2, 9, 9 1 / 2, 10, 10 1 / 2, 11, 11 1 / 2, 12, 12 1 / 2, 13, 13 1 / 2, 14, 14 1 / 2, 15, 15 1 / 2, 16, 16 1 / 2, 17, 17 1 / 2, 18, 18 1 / 2, 19, 19 1 / 2, 20, 20 1 / 2, 21, 21 1 / 2, 22, 22 1 / 2, 23, 23 1 / 2, 24, 24 1 / 2, 25, 25 1 / 2, 26, 26 1 As used herein, the phrase "immediately preceding dose" refers to a dose of an antigen-binding molecule that is administered to a patient in a multiple dose sequence prior to administration of the immediately succeeding dose in the sequence, with no intervening administrations.

[0078] The method according to this aspect of the present invention can include administering any number of secondary and / or tertiary doses of ANGPTL3 inhibitor to a patient.For example, in certain embodiments, only a single secondary dose is administered to a patient.In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to a patient.Similarly, in certain embodiments, only a single tertiary dose is administered to a patient.In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to a patient.

[0079] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2, 4, 6, 8 or more weeks after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 1-2, 4, 6, 8 or more weeks after the immediately preceding dose. Alternatively, the frequency at which the secondary and / or tertiary doses are administered to the patient may vary during the treatment regimen. The frequency of administration may also be adjusted during treatment by the physician according to the needs of the individual patient after clinical examination. EXAMPLES

[0080] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0081] Example 1. Generation of human antibodies against human ANGPTL3 Human anti-ANGPTL3 antibodies were generated as described in US Patent No. 9,018,356. The exemplary ANGPTL3 inhibitor used in the following examples is a human anti-ANGPTL3 antibody designated "H4H1276S", also known as "evinacumab". Evinacumab has the following amino acid sequence characteristics: heavy chain comprising SEQ ID NO: 10 and light chain comprising SEQ ID NO: 11; heavy chain variable region (HCVR) comprising SEQ ID NO: 2 and light chain variable domain (LCVR) comprising SEQ ID NO: 3; heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 4, HCDR2 comprising SEQ ID NO: 5, HCDR3 comprising SEQ ID NO: 6, light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 7, LCDR2 comprising SEQ ID NO: 8 and LCDR3 comprising SEQ ID NO: 9.

[0082] Example 2. Anti-ANGPTL3 antibody prevents atherosclerosis development in a mouse model introduction The objective of this study was to evaluate the effect of ANGPTL3 inhibition on the development of atherosclerosis in APOE*3Leiden.CETP mice, a well-established model of hyperlipidemia with all the features of mixed or familial dysbetalipoproteinemia and atherosclerosis (e.g., see Kuehnast et al., 2014, J Lipid Res 55: 2103-2112 for a general description of the APOE*3Leiden.CETP mouse model (alternatively referred to herein as "E3L.CETP mouse") and its use in evaluating the effects of agents on atherosclerosis development).

[0083] ANGPTL3 inhibition was achieved using an anti-ANGPTL3 antibody. The particular anti-ANGPTL3 antibody used in these studies was the antibody called evinacumab described above. The control antibody used in these studies was an isotype-matched antibody against an unrelated target.

[0084] research design A description of the study design is shown in Figure 1. Evinacumab is a fully human IgG4 antibody. Therefore, when evaluated in different mouse strains, some mice will develop anti-drug antibodies that will eliminate the efficacy of the therapeutic agent. The percentage of mice that develop this anti-drug response may differ for each mouse model / genetic background. For this reason, more mice per group were included in this initial study in APOE*3Leiden.CETP mice to determine how many mice develop an anti-human immune response in this particular model. After removing the mice that developed an anti-human immune response, the remaining mice could be used to evaluate the effect of evinacumab on atherosclerosis.

[0085] Sixty-three female APOE*3Leiden.CETP mice were fed diet T (a semisynthetic modified diet described by Nishina et al. 1990, J Lipid Res 31:859) supplemented with 0.15% cholesterol and 15% cocoa butter. After a 4-week run-in period, 13 low-responder mice were removed from the study and the remaining 50 mice were subdivided into a control group of 20 mice and an evinacumab-treated group of 30 mice, with body weight, plasma cholesterol, and triglycerides matched after a 4-hour fast (t=0).

[0086] Treatment with evinacumab or control antibody was performed by subcutaneous injection once weekly at a dose of 25 mg / kg / week. The injection volume during the treatment period was 10 mL / kg using 0.9% NaCl as vehicle and adjusted to the last measured body weight value. Body weight and food intake (per cage) were measured at weeks 0, 3, 6, 8, 11 and 13.

[0087] After 0, 2, 4, 6, 8, 11 and 13 weeks of treatment, blood samples were taken after 4 hours of fasting. Plasma cholesterol and triglycerides were measured individually, and EDTA plasma was also collected for hFc and mouse anti-human antibody evaluation. After 6 weeks of treatment, the primary anti-drug response was evaluated, and 3 mice in the control group and 5 mice in the treatment group with high levels of anti-drug antibodies were removed from the study.

[0088] Lipoprotein profiles were measured in pooled plasma samples per group at weeks 0, 6 and 13. Feces were collected per cage for 12 weeks (twice every 48 hours). Mice were sacrificed non-fasting at t=13. EDTA-plasma was obtained by cardiac puncture and several tissues were collected. Atherosclerosis development in the aortic root was measured by measuring lesion number, lesion area and lesion severity in 15 mice per group (after exclusion of mice that developed an anti-drug response). In addition, lesion composition was determined.

[0089] (1) The parameter measurement schedule is as follows: (2) body weight (0, 3, 6, 8, 11, and 13 weeks); (3) dietary intake (per cage, 0, 3, 6, 8, 11, and 13 weeks); (4) plasma total cholesterol and triglycerides (after a 4-h fast at weeks 0, 2, 4, 6, 8, 11, and 13); (5) collection of 20 μl EDTA plasma for hFc and mouse anti-human antibody assessment (after a 4-h fast at weeks 0, 2, 4, 6, 8, 11, and 13); (6) lipoprotein profile (cholesterol and triglycerides, pooled per group; 0, 6, and 13 weeks); (7) Atherosclerosis development in the aortic root (15 mice per group): (a) lesion number, (b) total lesion area, (c) lesion severity, (d) lesion composition (e.g., macrophages, smooth muscle cells in the cap, necrosis and collagen content), and (e) monocyte adhesion (at sacrifice [13 weeks], aortas were collected from all mice, but atherosclerosis analysis was performed in 15 mice per group; mice that did not develop anti-drug antibodies were selected).

[0090] method Body weight and food intake measurements were performed according to standard procedures.

[0091] Plasma blood samples were obtained according to standard procedures. Total plasma cholesterol and triglycerides were determined using commercially available kits.

[0092] Lipoprotein profile determination by FPLC analysis using an AKTA instrument from Amersham Biosciences. Analysis was performed on pooled samples per group. Cholesterol and triglycerides were measured in fractions using commercially available kits.

[0093] Atherosclerotic lesion area and severity were assessed in the aortic root region as previously reported (see, e.g., Kuehnast et al., 2012, J Hypertens 30:107-116). The aortic root was identified by the appearance of the aortic valve cusps, and serial sections (5 μm thick with 50 μm intervals) throughout the aortic root region were mounted on 3-aminopropyltriethoxysilane-coated slides and stained with hematoxylin-phloxine-saffron (HPS). For each mouse, lesion area was measured in four serial sections. Each section consisted of three parts (separated by valves). For the determination of atherosclerotic lesion size and severity, lesions were classified into five categories according to the American Heart Association (AHA) (see, Stary et al., 1995, Arterioscler Thromb Vasc Biol 15:1512-1531): Type 1 : early fatty streak (up to 10 foam cells in the intima without other changes); Type 2 : Usually fatty streaks (10 or more foam cells in the intima without other changes); Type 3 : Mild plaque (foam cells with a fibrous cap); Type 4 : intermediate plaque (more advanced lesions with affected media but no loss of media structure); Type 5 : Severe plaque (the media is severely affected; broken elastic fibers, cholesteric clefts, calcification and necrosis are frequently observed).

[0094] Images were taken with an Olympus BX51 microscope and areas were measured with image processing software. Total lesion area and lesion number were calculated per section. Unaffected sections were calculated as a percentage of the total section. Lesion severity as a percentage of total lesion was also determined. Type I-III lesions were classified as mild lesions and type IV-V lesions were classified as severe lesions.

[0095] The number of monocytes adherent to the activated endothelium was counted in each segment used for lesion quantification after immunostaining with sAIA 31240 antiserum (1:1000; Accurate Chemical and Scientific, New York, NY, USA) and calculated per cross section.

[0096] Lesion macrophage area was measured after immunostaining with anti-mouse Mac-3 (1:50; BD Pharmingen, the Netherlands). Sirius red staining was performed to stain collagen. Smooth muscle cell area of ​​the fibrous cap was measured after area staining with anti-alpha smooth muscle actin (1:400; PROGEN Biotechnik GmbH, Heidelberg, Germany), which cross-reacts with mouse alpha actin. Lesion photographs / images were taken with an Olympus BX51 microscope. Lesion macrophage, collagen, necrotic core and smooth muscle cell areas were quantified using imaging software. Lesion macrophage, smooth muscle cell (SMC), necrotic core and collagen content were calculated as a percentage of lesion area. Plaque vulnerability / stability index was calculated by dividing the sum of stabilizing factors SMC and collagen by the sum of destabilizing factors macrophages and necrotic core.

[0097] At week 13, mice were sacrificed by CO2 without fasting. No final antibody injection was administered prior to sacrifice during week 13. After sacrifice, as much EDTA-plasma as possible was collected (by cardiac puncture) and stored at -70°C until further use. Hearts including aortic roots were fixed in 10% formalin. Thoracic aortas were collected and fixed in 10% formalin.

[0098] statistics The significance of differences between groups was calculated non-parametrically for independent samples using the computer program SPSS and the Mann-Whitney U test for normality. A P value <0.05 was considered statistically significant. Statistical differences of treated groups compared to the control group are indicated in the figures with asterisks at the measurement points.

[0099] result body weight Body weight results are summarized in Table 1. Values ​​are absolute (grams) and mean ± SD from 14-15 mice per group.

[0100] [Table 1]

[0101] There were no differences in body weight between the evinacumab-treated groups compared to the control group.

[0102] Food intake Food intake results are summarized in Table 2. Values ​​are absolute (grams / mouse / day) and mean ± SD of 17 cages (all mice; before matching) or 4–6 cages (after matching and treatment initiation).

[0103] [Table 2]

[0104] There were no differences in food intake between the evinacumab-treated groups compared to the control group.

[0105] Plasma cholesterol Plasma cholesterol results are summarized in Tables 3 and 4 and Figures 2A and 2B. Values ​​are absolute (mM) and mean ± SD from 14-15 mice per group.

[0106] [Table 3]

[0107] [Table 4]

[0108] Treatment with evinacumab resulted in a significant reduction in plasma cholesterol compared to the control group at all time points (mean reduction of 52%). At the end of the study, cholesterol exposure during the study (weeks 0-13 + run-in phase) was significantly reduced with treatment with evinacumab compared to the control group (41%, p<0.001).

[0109] Plasma triglycerides Plasma triglyceride results are summarized in Table 5 and Figure 3. Values ​​are absolute (mM) and mean ± SD of 14-15 mice per group.

[0110] [Table 5]

[0111] Treatment with evinacumab resulted in significant reductions in plasma glycerides compared to the control group at all time points (mean reduction of 84%).

[0112] Lipoprotein Profile Lipoproteins were separated on a Superose column. Cholesterol and triglyceride fractions at t = 0, t = 6 weeks, and t = 13 weeks are shown in Figures 4A, 4B, and 4C [cholesterol] and 5A, 5B, and 5C [triglycerides]. Values ​​are absolute values ​​from cholesterol and triglyceride measurements in pooled plasma by group at t = 0, 6, and 13 weeks. For the purposes of this analysis, fractions 3-7 were considered VLDL; 8-16 IDL / LDL, and 17-24 HDL.

[0113] At t=0, before treatment began, no differences in lipoprotein profiles were observed, but after 6 and 13 weeks of treatment with evinacumab, plasma cholesterol levels measured in pooled samples by group appeared to decrease the VLDL-LDL peak and increase the HDL peak compared to the control group (see Figures 4B and 4C). Triglyceride profiles showed a similar pattern (Figures 5A, 5B, and 5C).

[0114] These data are consistent with the reduction in total cholesterol and triglyceride levels in individual animals observed during the study.

[0115] Atherosclerotic Lesion Area Atherosclerotic lesion area is summarized in Table 6 and Figures 6A and 6B. Values ​​are absolute lesion area in the aortic root per cross section and are expressed as the mean ± SD of 14-15 mice per group.

[0116] [Table 6]

[0117] The total lesion area in the aortic root of the control group was 216082 μm per cross section at sacrifice at t = 13 weeks. 2 Treatment with evinacumab significantly reduced total lesion area by 39% compared to controls at the time of sacrifice (p<0.001).

[0118] Atherosclerosis: Number of Lesions The number of atherosclerotic lesions per cross section is summarized in Table 7. Values ​​are absolute (number of lesions) and expressed as the mean ± SD of 14-15 mice per group.

[0119] [Table 7]

[0120] There was no difference in the number of lesions between the evinacumab-treated groups compared with the control group.

[0121] Atherosclerosis: Lesion Severity Atherosclerotic lesion severity results are summarized in Table 8. Values ​​are percentages of total lesions and are expressed as the mean ± SD of 14-15 mice per group.

[0122] [Table 8]

[0123] There were no differences in lesion severity between the evinacumab-treated groups compared with the control group.

[0124] Atherosclerosis: Unaffected Segments The number of unaffected atherosclerotic areas is summarized in Table 9. Values ​​represent the percentage of nonatherosclerotic ("unaffected" or healthy) areas. Values ​​are the mean ± SD of 14-15 mice per group.

[0125] [Table 9]

[0126] There was no difference in the amount of unaffected tissue between the evinacumab-treated groups compared to the control group.

[0127] Atherosclerosis: Macrophage Content The macrophage content of the lesions is summarized in Table 10 and Figure 7A. Values ​​are absolute (μm 2 *1000) or relative values ​​(% of lesions) and expressed as the mean ± SD of 14–15 mice per group.

[0128] [Table 10]

[0129] Treatment with evinacumab tended to reduce macrophage content per cross section in type IV-V atherosclerotic lesions compared with controls, but this difference was not observed after correcting for total lesion area and expressing it as a percentage of macrophages in type IV-V lesions.

[0130] Atherosclerosis: Necrotic Content The necrotic content of the lesions is summarized in Table 11 and Figure 7A. Values ​​are absolute (μm 2 *1000) or relative values ​​(% of lesions) and expressed as the mean ± SD of 14–15 mice per group.

[0131] [Table 11]

[0132] Treatment with evinacumab significantly reduced the necrotic content per cross section in type IV-V atherosclerotic lesions compared to the control group (69%, p<0.001). After correction for total lesion area, the percentage of necrotic area in type IV-V lesions was also significantly reduced compared to the control group (45%, p=0.001) (Figure 7A).

[0133] Atherosclerosis: smooth muscle cells in the fibrous cap Quantification of smooth muscle cells (SMCs) in the fibrous cap of the lesions is summarized in Table 12 and Figure 7B. Values ​​are absolute (μm 2 *1000) or relative values ​​(% of lesions) and expressed as the mean ± SD of 14–15 mice per group.

[0134] [Table 12]

[0135] Treatment with evinacumab significantly reduced smooth muscle cell (SMC) area per cross-sectional area in type IV-V atherosclerotic lesions compared to controls (39%, p=0.009). However, after correction for total lesions, the percentage of SMCs in the fibrous cap in type IV-V lesions was not significantly reduced compared to controls (Figure 7B).

[0136] Atherosclerosis: Collagen Content The collagen content of the lesions is summarized in Table 13 and Figure 7B. Values ​​are absolute (μm 2 *1000) or relative values ​​(% of lesions) and expressed as the mean ± SD of 14–15 mice per group.

[0137] [Table 13]

[0138] Treatment with evinacumab significantly reduced collagen content per cross section in type IV-V atherosclerotic lesions compared to the control group (40%, p=0.009). However, after correction for total lesion area, the percentage of collagen in type IV-V lesions was not significantly reduced compared to the control group (Figure 7B).

[0139] Atherosclerosis: Monocyte adhesion The percentage of monocytes per cross section is summarized in Table 14. Values ​​are absolute (number of adherent monocytes per cross section) and are expressed as the mean ± SD of 14-15 mice per group.

[0140] [Table 14]

[0141] Monocyte adhesion to activated endothelium in the vessel wall of the aortic root was not affected by treatment with evinacumab compared to the control group.

[0142] Atherosclerosis: Stability Index Lesion plaque stability (represented by the stability index) is summarized in Table 15. The stability index is defined as the sum of the stable markers SMC and collagen content divided by the sum of the unstable markers macrophage and necrosis content. This value provides a measure of plaque stability. Values ​​are expressed as the mean ± SD of 14-15 mice per group.

[0143] [Table 15]

[0144] Treatment with evinacumab had no effect on stability indicators compared to the control group.

[0145] Abstract Safety aspect No effects on body weight (gain) and food intake were observed with evinacumab treatment compared to control.

[0146] Reduction of VLDL and LDL levels APOE*3Leiden.CETP mice are a well-established model for familial dysbetalipoproteinemia (FD) in humans, characterized by accumulation of remnant lipoproteins and an increased VLDL-C to LDL-C ratio. The lipoprotein profile in APOE*3Leiden.CETP mice reflects that of FD patients with a similar response to lipid-lowering therapy. This is explained by the comparable reduction of cholesterol in all apoB-containing lipoprotein subfractions with statin treatment25 and with fenofibrate and niacin.

[0147] In this study, treatment with evinacumab significantly reduced mean total cholesterol (TC) (-52%, p<0.001) and triglycerides (TG) (-84%, p<0.001) compared to controls, which was restricted to the non-HDL fraction.

[0148] Reduction in lesion size and necrotic area In the current study, evinacumab significantly reduced lesion size compared to the control group (-39%, p<0.001). No significant differences were seen in lesion count, unaffected area, or lesion severity.

[0149] Treatment with evinacumab significantly reduced necrotic content as a percentage of total lesion size in severe type IV-V lesions (with 45%, p=0.001), but did not affect macrophage content, collagen content, or SMC area, resulting in lesion stability indices similar to controls. Evinacumab did not affect monocyte recruitment to the activated endothelium.

[0150] Necrotic content was the only marker of altered plaque composition, both in terms of absolute area and as a percentage of total lesion area. A possible explanation may be a shift in the macrophage population in plaques from more proinflammatory M1 "killer" macrophages to more healing "repair" M2 macrophages that may remove cholesterol from the plaque.

[0151] conclusion Collectively, these results presented in this example show that the anti-ANGPTL3 monoclonal antibody evinacumab reduces plasma lipids and prevents the progression of atherosclerosis in APOE*3Leiden.CETP mice. Evinacumab had no effect on lesion number, lesion severity, or plaque stability, but significantly reduced lesion area and necrotic core area.

[0152] The present invention should not be limited to the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A medicament comprising an angiopoietin-like protein 3 (ANGPTL3) inhibitor for use in a method for preventing or reducing atherosclerosis in a subject, the method comprising selecting a subject having atherosclerosis or at risk of developing atherosclerosis, and administering to the subject one or more doses of an ANGPTL3 inhibitor, wherein the ANGPTL3 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to ANGPTL3; wherein the antibody or antigen-binding fragment thereof that specifically binds to ANGPTL3 comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs: 4, 5, 6, 7, 8, and 9, and the subject is on stable background lipid-lowering therapy (LMT) prior to or at the initiation of treatment with one or more doses of an ANGPTL3 inhibitor, or concurrently with administration of one or more doses of an ANGPTL3 inhibitor, where the stable background LMT is low-dose, moderate-dose, or high-dose statin therapy.

2. The pharmaceutical composition of claim 1, wherein the subject has been diagnosed with heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH), and / or elevated lipoprotein(a) (Lp[a]), prior to or at the start of treatment with one or more doses of an ANGPTL3 inhibitor.

3. The pharmaceutical agent of claim 1, wherein the subject has been diagnosed with cardiovascular disease (CVD) or has suffered a stroke or myocardial infarction prior to or at the time of initiation of treatment with one or more doses of an ANGPTL3 inhibitor.

4. Administering one or more doses of an ANGPTL3 inhibitor to a subject comprises: (a) a reduction in serum total cholesterol (TC) levels; (b) a reduction in serum triglyceride (TG) levels; (c) a reduction in serum low density lipoprotein (LDL) levels, and (d) Reduction of Very Low Density Lipoprotein (VLDL) Levels producing one or more therapeutic outcomes selected from the group consisting of: The pharmaceutical agent according to any one of claims 1 to 3, wherein the reduction in (a), (b), (c) and / or (d) is determined relative to the subject's serum TC level, serum TG level, serum LDL level, and / or serum VLDL level before or at the start of treatment with one or more doses of an ANGPTL3 inhibitor.

5. The pharmaceutical agent according to any one of claims 1 to 4, wherein administration of one or more doses of an ANGPTL3 inhibitor to a subject results in a reduction in atherosclerotic plaque formation.

6. The pharmaceutical composition of claim 1 , wherein the antibody or antigen-binding fragment thereof that specifically binds to ANGPTL3 comprises an HCVR having the amino acid sequence of SEQ ID NO:2 and an LCVR having the amino acid sequence of SEQ ID NO:

3.

7. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof that specifically binds to ANGPTL3 is evinacumab.

8. The pharmaceutical composition of any one of claims 1 to 7, further comprising administering to the subject one or more doses of a proprotein convertase subtilisin / kexin-9 (PCSK9) inhibitor, wherein the PCSK9 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to PCSK9.

9. The pharmaceutical agent of claim 8, wherein the antibody or antigen-binding fragment thereof that specifically binds to PCSK9 is selected from the group consisting of alirocumab, evolocumab, bococizumab, roderucizumab, and ralpancizumab.

10. The pharmaceutical composition of any one of claims 1 to 9, further comprising administering to the subject one or more doses of an ANGPTL4 inhibitor and / or an ANGPTL8 inhibitor, wherein the ANGPTL4 inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to ANGPTL4, and the ANGPTL8 inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to ANGPTL8.

11. A pharmaceutical composition for use in preventing or ameliorating atherosclerosis in a subject, wherein the subject has atherosclerosis or is at risk of developing atherosclerosis, the composition comprises an angiopoietin-like protein 3 (ANGPTL3) inhibitor, and one or more doses of the angiopoietin-like protein 3 (ANGPTL3) inhibitor are administered to the subject, wherein the ANGPTL3 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to ANGPTL3. wherein the antibody or antigen-binding fragment thereof that specifically binds to ANGPTL3 comprises heavy and light chain CDR amino acid sequences having SEQ ID NOs: 4, 5, 6, 7, 8, and 9, and the subject is on stable background lipid-lowering therapy (LMT) prior to or at the initiation of treatment with one or more doses of an ANGPTL3 inhibitor, or concurrently with administration of one or more doses of an ANGPTL3 inhibitor, wherein the stable background LMT is low-dose, moderate-dose, or high-dose statin therapy.

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