EA040975B1 - METHODS FOR TREATMENT OR PREVENTION OF ATHEROSCLEROSIS BY ADMINISTRATION OF ANGPTL3 INHIBITOR - Google Patents

Description

Commentary on Sequences

The application for this invention contains a sequence listing, which is presented in electronic form in ASCII format and is incorporated into the present description by reference in its entirety. The specified ASCII copy, created on February 8, 2017, is named 0431_23-PCT_SL.txt and is 14141 bytes in size.

Technical field

The present invention relates to the field of therapeutic treatment of atherosclerotic diseases and related disorders. More specifically, the invention relates to the use of ANGPTL3 inhibitors for the treatment or prevention of atherosclerosis, as well as to methods for eliminating or reducing the formation of atherosclerotic plaques and/or atherosclerotic lesions in a patient.

State of the art

Atherosclerosis is a syndrome characterized by damage to the blood vessels. Atherosclerosis is the result of a chronic inflammatory response in the arterial walls and is a major cause of death and morbidity from cardiovascular disease. Atherosclerosis involves the thickening of the walls of the arteries and is characterized by the development of arterial plaque and possible restriction or blockage of blood flow. Current treatments for atherosclerosis include diet and lifestyle modification (eg, smoking cessation), pharmaceutical intervention (eg, statins, anticoagulants, etc.), and surgery (eg, angioplasty, stents, bypass, etc.). Direct assessment of the effectiveness of pharmaceutical interventions in the development of atherosclerosis in humans is problematic because of the difficulty in accurately detecting and measuring plaque and arterial lesions in living patients. Therefore, animal models of atherosclerosis are very useful for the development and testing of new therapeutic methods for the treatment of atherosclerosis.

New methods of treating, preventing or improving atherosclerosis are needed in the art.

Brief summary of the invention

The present invention relates to methods, uses, and compositions for the treatment, prevention, or amelioration of atherosclerosis in a patient. The method of the present invention comprises administering one or more doses of an angiopoietin-like protein 3 (ANGPTL3) inhibitor to a patient. The therapeutic methods of the present invention result in a reduction in the formation of atherosclerotic plaques and atherosclerotic lesions in patients in need thereof. The methods of the present invention are also useful for lowering total serum cholesterol, LDL cholesterol and triglycerides in a patient and for treating diseases and disorders that can be treated by lowering one or more of total cholesterol, LDL cholesterol and/or triglycerides.

In some embodiments, the patient is diagnosed with heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH) and/or elevated lipoprotein (a) (Lp[a]) before or at the time of initiation of treatment with one or more doses of an ANGPTL3 inhibitor, in accordance with some embodiments of the present invention. . The present invention also includes methods for treating, preventing, or attenuating atherosclerosis in a patient who has hypercholesterolemia that is not familial (ie, non-familial hypercholesterolemia). Patients that are amenable to treatment with the methods of the present invention, in certain embodiments, include those patients who have or have been diagnosed with cardiovascular disease (eg, atherosclerotic cardiovascular disease). In some embodiments, the patient being treated with the methods of the present invention is a patient who has had a stroke or myocardial infarction.

According to some embodiments of the present invention, the ANGPTL3 inhibitor is administered to the patient as an add-on therapy to the patient's existing lipid-lowering therapy (eg, while the patient is on a statin therapy).

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

Other embodiments of the present invention will become apparent from a review of the following detailed description.

Brief description of the drawings

In FIG. 1 is a flowchart illustrating the procedures, administrations, and measurements performed on animals throughout the study described in Example 2 of this disclosure.

In FIG. 2A and 2B show the cholesterol levels of animals from different treatment groups (control treatments, solid squares, or evinacumab treatments, solid circles [•]) at different time points throughout the study described in Example 2 herein.

In FIG. 2A shows mean cholesterol levels (mM) over time (in weeks).

- 1 040975

In FIG. 2B shows total cholesterol exposure (mM x wk) at 13 weeks.

In FIG. 3 shows triglyceride levels (mM) of animals from different treatment groups (control treatments, solid squares [], or evinacumab treatments, solid circles [•]) at different time points throughout the study described in example 2 of this description.

In FIG. 4A-4C are lipoprotein cholesterol profiles (mM after lipoprotein separation) of animals from different treatment groups (control treatments, solid squares [], or evinacumab treatments, solid circles [•]) at various times throughout the study described in Example 2 of this descriptions. Lipoproteins were separated on a superose column and the values shown are absolute values based on measurements of total plasma cholesterol per group at the various time points indicated.

In FIG. 4A shows cholesterol levels from different plasma fractions corresponding to study time = 0.

In FIG. 4B shows cholesterol levels from different plasma fractions corresponding to time = 6 weeks of study.

In FIG. 4C shows cholesterol levels from different plasma fractions corresponding to time = 13 weeks of study.

In FIG. 5A-5C are lipoprotein profiles of triglycerides (mM after lipoprotein separation) of animals from different treatment groups (control treatments, solid squares [], or evinacumab treatments, solid circles [•]) at various times throughout the study described in Example 2 of the present. descriptions. Lipoproteins were separated on a superose column and the values reported are absolute values based on total plasma triglyceride measurements per group at the various time points indicated.

In FIG. 5A shows triglyceride levels from different plasma fractions corresponding to study time = 0.

In FIG. 5B shows triglyceride levels from different plasma fractions corresponding to study time = 6 weeks.

In FIG. 5C shows triglyceride levels from different plasma fractions corresponding to time = 13 weeks of study.

In FIG. 6A and 6B show the area of atherosclerosis in animals from different treatment groups (control treatment group, open bar or circles; or evinacumab treatment group, solid bar or circles) at week 13 of the study described in Example 2 herein.

In FIG. 6A shows the average atherosclerotic lesion area per transverse section (x 1000 μm ² ) for various treatment groups.

In FIG. 6B is a scatterplot of total lesion area showing atherosclerotic lesion area per cross section (x1000 μm ² ) for individual animals in different treatment groups.

In FIG. 7A and 7B show the mean necrotic content of transverse sections from atherosclerotic lesions classified as type IV or type V observed in animals from different treatment groups (control treatment group, open bar, or evinacumab treatment group, solid bar) at week 13 of the study described in example 2 of the present description.

In FIG. 7A shows the absolute value (µm ² x 1000) of necrotic area per cross section.

In FIG. 7B shows the relative values (as a percentage of the area of the lesion) of the necrotic content of the lesions.

Detailed description

Before the present invention is described, it should be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is only intended to describe specific embodiments and is not intended to limit them, as the scope of the present invention will only be limited by the appended claims.

Unless otherwise indicated, all technical and scientific terms used in the present description have the same meaning as generally understood by a person skilled in the art to which the present invention pertains. Used in the present description, the term approximately when used in relation to a specific specified numerical value means that the value may differ from the specified value by no more than 1%. For example, as used herein, the expression about 100 includes 99 and 101 and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

While any materials and methods similar or equivalent to those described in this document

- 2 040975 mente, can be used in the practice of the present invention, the preferred materials and methods will be described here. All publications mentioned in the present description are incorporated into the description by reference in their entirety.

Ways to prevent or reduce atherosclerosis

The present invention relates generally to methods and compositions for preventing or attenuating atherosclerosis in a patient. The methods of the present invention include administering to a patient in need thereof one or more doses of an angiopoietin-like protein 3 (ANGPTL3) inhibitor. In some embodiments, the methods of the present invention result in a reduction in atherosclerotic plaque formation in a patient. For example, the methods of the present invention may result in one or more of a reduction in the area of an atherosclerotic lesion and/or a reduction in the necrotic area of an atherosclerotic lesion in a patient.

Selection of patients.

In accordance with some embodiments of the present invention, patients who are amenable to treatment with the methods of the invention include, but are not limited to, patients who have or have been diagnosed with atherosclerosis, or who are at risk of developing atherosclerosis. Accordingly, the methods of the present invention include selecting a patient having atherosclerosis or at risk of developing atherosclerosis and administering to the patient one or more doses of an angiopoietin-like protein 3 (ANGPTL3) inhibitor. As used herein, patients who are at risk for developing atherosclerosis include patients who have or exhibit one or more risk factors for atherosclerosis. Risk factors for atherosclerosis are well known in the art and include, without limitation, elevated serum low-density lipoprotein (LDL) cholesterol, elevated serum triglycerides (TG), decreased serum high-density lipoprotein (HDL) cholesterol, hypertension, diabetes mellitus, family history and cigarette smoking. Methods for assessing atherosclerosis risk factors for a given patient are also well known in the art.

According to some embodiments of the present invention, a patient that is treatable with the methods of the invention is a patient who is diagnosed with heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), elevated lipoprotein (a) (Lp[a]), autosomal dominant hypercholesterolemia (ADH, e.g. ARH associated with mutations in LDLRAP1), as well as cases of hypercholesterolemia that differ from familial hypercholesterolemia (nonFH). The diagnosis of familial hypercholesterolemia (eg, heFH or hoFH) can be obtained by genotyping and/or clinical criteria. For patients who are not genotyped, the clinical diagnosis can be based on either the Simon Broom criteria, the criteria for specific FHs, or the WHO/Dutch Lipid Network criteria with a score of >8 points.

According to some embodiments of the present invention, a patient being treated with the methods of the invention is a patient who has been diagnosed with cardiovascular disease (CVD) prior to or at the time of initiation of treatment with one or more doses of an ANGPTL3 inhibitor. The patient may also be selected based on a history of coronary heart disease (CHD). As used herein, the term history of CHD (or documented history of CHD) includes one or more of (i) acute myocardial infarction (MI); (ii) covert MI; (iii) unstable angina; (iv) coronary artery revascularization procedures (eg, percutaneous coronary intervention [PCI] or coronary artery bypass surgery [CABG]) and/or (v) clinically significant CHD diagnosed by invasive or non-invasive testing (eg, coronary angiography, stress testing with using a treadmill, stress echocardiography, or nuclear imaging).

In accordance with some embodiments of the present invention, a patient to be treated with the methods of the invention may be selected based on the presence of cardiovascular disease other than coronary artery disease (non-CHD cardiovascular disease). As used herein, the term non-CHD-CVD includes one or more of (i) documented prior ischemic stroke with focal ischemic neurological deficit that persists for more than 24 hours and is believed to be atherothrombotic in origin; (ii) peripheral arterial disease; (iii) abdominal aortic aneurysms; (iv) atherosclerotic renal artery stenosis; and/or (v) carotid disease (transient ischemic attacks or >50% carotid obstruction).

According to some embodiments of the present invention, a patient who is amenable to treatment with the methods of the invention may be selected based on the presence of one or more additional risk factors, such as, for example, (i) documented moderate chronic kidney disease (CKD), as determined by 30 <eGFR<60 ml/min/1.73 m ² for 3 months or more; (ii) type 1 or type 2 diabetes mellitus with or without target organ damage

- 3 040975 (e.g. retinopathy, nephropathy, microalbuminuria) and/or (iii) calculated 10-year fatal cardiovascular risk >5% (ESC/EAS guidelines for the treatment of dyslipidaemia, Conroy et al., 2003, Eur. Heart J. 24:987-1003).

According to some embodiments of the present invention, a patient who is amenable to treatment with the methods of the invention may be selected based on the presence of one or more additional risk factors selected from the group consisting of age (e.g., older than 40, 45, 50, 55, 60, 65 , 70, 75, or 80 years old), race, national origin, gender (male or female), physical habits (eg, regular exercise, no exercise), other pre-existing medical conditions (eg, type II diabetes, high blood pressure etc.) and current drug status (e.g. currently taking beta-blockers, niacin, ezetimibe, fibrates, omega-3 fatty acids, bile acid sequestrants, etc.).

According to some embodiments of the present invention, a patient who is being treated with the methods of the invention exhibits elevated levels of one or more inflammatory markers. Any marker of systemic inflammation can be used 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 RESAM).

In accordance with some embodiments of the present invention, a patient who is amenable to treatment with the methods of the invention may be selected based on a combination of one or more of the above selection criteria or therapeutic characteristics. For example, in accordance with some embodiments, a patient suitable for treatment with the methods of the present invention may be further selected based on the presence of heFH or non-FH in combination with (i) a documented history of CHD, (ii) non-CHD-CVD, and/ or (iii) diabetes mellitus with end organ damage; such patients may also be selected based on a serum LDL-C concentration greater than or equal to 70 mg/dl.

In accordance with some embodiments of the present invention, the patient being treated with the methods of the invention has hypercholesterolemia that is not adequately controlled by a moderate daily statin therapeutic regimen and may be further selected based on the presence of heFH, or non-FH without CHD, or non-CHD CVD, but having either (i) a calculated 10-year risk estimate for fatal CVD >5%; or (ii) diabetes mellitus without target organ damage; such patients may also be selected based on a serum LDL-C concentration greater than or equal to 100 mg/dl.

According to some embodiments of the present invention, the patient who is treatable with the methods of the invention is a patient who has familial chylomicronemia syndrome (FCS, also known as lipoprotein lipase deficiency).

According to some embodiments of the present invention, the patient being treated with the methods of the invention is a patient who is undergoing or has recently undergone lipoprotein apheresis (e.g., within the past 6 months, within the past 12 weeks, within the past 8 weeks, within the past 6 weeks, within the last 4 weeks, within the last 2 weeks, etc.).

Administration of an ANGPTL3 inhibitor as add-on therapy.

The present invention includes methods for preventing or attenuating atherosclerosis in a patient, wherein an ANGPTL3 inhibitor is administered to the patient according to a specific amount and dosing frequency, and wherein the ANGPTL3 inhibitor is administered as an adjunct to a patient's pre-existing lipid-normalizing therapy (LMT), such as in addition to a pre-existing daily therapeutic regimen. statin regimen. (Specific exemplary daily statin therapeutic regimens to which an ANGPTL3 inhibitor may be added are described elsewhere herein).

For example, the methods of the present invention include additional therapeutic regimens in which the ANGPTL3 inhibitor is administered as add-on therapy to the same stable daily statin regimen (i.e., the same statin dosage amount) that the patient was taking prior to receiving the ANGPTL3 inhibitor. . In other embodiments, the ANGPTL3 inhibitor is administered as add-on therapy to a statin therapeutic regimen containing a statin in an amount greater than or less than the statin dose the patient was on prior to taking the ANGPTL3 inhibitor. For example, after starting a therapeutic regimen that includes an ANGPTL3 inhibitor administered at a certain frequency and dose amount, the daily dose of a statin administered or administered to a patient may (a) remain unchanged, (b) increase, or (c) decrease (e.g., with increasing dose or dose reduction) compared to the daily dose of statins that the patient was taking before starting the ANGPTL3 inhibitor therapeutic regimen, depending on the patient's therapeutic needs.

Therapeutic efficacy.

The methods of the present invention result in a reduction in the formation of atherosclerotic plaque in a patient's arteries and/or in the prevention of progression of atherosclerosis in a patient. On

- 4 040975 example, the methods of the present invention are useful for limiting or reducing the area of atherosclerotic lesions and/or the area of the necrotic nucleus in atherosclerotic lesions in a patient.

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 residual cholesterol. For example, in some embodiments of the present invention, administering to a suitable patient a pharmaceutical composition containing an ANGPTL3 inhibitor will result in an average percentage reduction in baseline serum low-density lipoprotein (LDL-C) cholesterol levels of at least about 25%, 30, 40, 50, 60% or more; an average percentage reduction in ApoB100 from baseline of at least about 25%, 30%, 40%, 50%, 60% or more; an average percentage reduction in non-HDL-C from baseline of at least about 25%, 30%, 40%, 50%, 60% or more; an average percentage reduction from baseline in total cholesterol of at least about 10%, 15%, 20%, 25%, 30%, 35% or more; an average percentage reduction in VLDL-C from baseline of at least about 5%, 10%, 15%, 20%, 25%, 30% or more; an average percentage reduction in triglycerides from baseline of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or more, and/or an average percentage reduction in Lp(a) from baseline of at least about by 5, 10, 15, 20, 25% or more.

ANGPTL3 inhibitors.

The methods of the present invention include administering to a patient a therapeutic composition containing an ANGPTL3 inhibitor. As used herein, the term ANGPTL3 inhibitor is any agent that binds to or interacts with human ANGPTL3 and inhibits the normal biological function of ANGPTL3 in vitro or in vivo. Non-limiting examples of categories of ANGPTL3 inhibitors include small molecule antagonists of ANGPTL3, nucleic acid-based inhibitors of ANGPTL3 expression or activity (e.g., siRNAs or antisense RNAs), peptide-based molecules that specifically interact with ANGPTL3 (e.g., peptibody), receptor molecules that specifically interact with ANGPTL3, ANGPTL3-binding structural molecules (e.g. DARPin, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, structural constructs based on fibronectin and other structures based on naturally occurring repeat proteins, etc. [see, for example, Boersma and Pluckthun, 2011, Curr Opin Biotechnol 22:849-857 and references cited there]) and aptamers or anti-ANGPTL3 thereof. In some embodiments, ANGPTL3 inhibitors that can be used in the context of the present invention are anti-ANGPTL3 antibodies or antigen-binding antibody fragments that specifically bind to human ANGPTL3.

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 (see also NCBI accession number NP_055310) or a biologically active fragment thereof.

As used herein, the term antibody is intended to refer to immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains, and two light (L) chains linked by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated here as HCVR or VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) that alternate with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, N-terminal to C-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, the FR of an anti-ANGPTL3 antibody (or an antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. The consensus amino acid sequence can be determined based on a parallel analysis of two or more CDRs.

As used herein, the term antibody also includes antigen-binding fragments of full-length antibody molecules. The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like as used herein include any naturally occurring, enzymatically available, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of an antibody can be obtained, for example, from full-length antibody molecules using any suitable standard methods, such as proteolytic cleavage,

- 5 040975 or using recombinant genetic engineering methods, including the manipulation and expression of DNA encoding the antibody and, optionally, constant domains. Such DNA is known and/or readily available, for example from commercial sources, DNA libraries (including, for example, antibody phage libraries), or can be synthesized. The DNA can be sequenced and processed chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, insert or delete amino acids, etc.

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 molecules (scFv); (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic an antibody hypervariable region (e.g., a complementarity-determining region (CDR) such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies, tetrabodies, mini-antibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.) .), small modular protein (SMIP) immunopharmaceuticals, and shark IgNAR variable domains are also included in the expression antigen binding fragment as used herein.

An antigen binding fragment of an antibody typically contains at least one variable domain. The variable domain may be of any size or amino acid composition, and typically contains at least one CDR that is adjacent to or in-frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be arranged relative to each other in any suitable order. For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may comprise a VH or VL monomeric domain.

In some embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary variable and constant domain configurations that may be present in an antigen-binding fragment of an antibody of the present invention include (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the illustrative configurations above, the variable and constant domains may either be directly linked to each other or may be linked through a complete or partial hinge or linker region. The hinge region may be at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible bond between adjacent variable and/or constant domains in a single polypeptide molecule. In addition, an antigen-binding fragment of an antibody of the present invention may comprise a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more VH or VL monomeric domains (e.g. , via disulfide bond(s)).

As with full-length antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding antibody fragment will typically contain at least two distinct variable domains, where each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats described herein, can be adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using conventional techniques available in the art.

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

As used herein, the term human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may, however, include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutation), for example, in CDRs and in particular CDR3 . However, the term human antibody as used herein is not intended to include antibodies in which CDR sequences derived from the germline of other mammalian species

- 6040975 such as mouse were grafted onto human framework sequences.

As used herein, the term recombinant human antibody is intended to include all human antibodies that have been generated, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described below), antibodies, isolated from a recombinant combinatorial library of human antibodies (described further below), antibodies isolated from an animal (e.g., mouse) that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992), Nucl. Acids Res. 20:6287-6295), or antibodies produced, expressed, generated, or isolated by any other method that involves splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when a transgenic animal is used for human Ig sequences, somatic in vivo mutagenesis), and therefore the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, being derived from and associated with human germline VH and VL sequences, may not naturally exist in the human germline repertoire in vivo.

Human antibodies can exist in two forms that are associated with hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-strand construct of approximately 150-160 kDa, in which the dimers are held together by an interstrand disulfide bond. In the second form, the dimers are not linked via interchain disulfide bonds, and a molecule of about 75-80 kDa is formed, consisting of covalently linked light and heavy chains (half-antibody). These forms are extremely difficult to separate even after affine purification.

The frequency of appearance of the second form in various intact IgG isotypes is due to structural differences associated with the isotype of the hinge region of the antibody, but is not limited to this. A single amino acid substitution at the human IgG4 hinge region can significantly reduce the appearance of the second form (Angal et al. (1993), Molecular Immunology, 30:105) to levels normally seen with the human IgG1 hinge. The present invention encompasses antibodies having one or more mutations in the hinge region, CH2 or CH3, which may be desirable, for example, when obtained to improve the yield of the target form of the antibody.

As used herein, the term isolated antibody means an antibody that has been identified and isolated and/or isolated from at least one component of its natural environment. For the purposes of the present invention, an isolated antibody is an antibody that has been separated or removed from at least one component of an organism, or tissue, or cell in which the antibody naturally exists or from which it is naturally produced. An isolated antibody also includes an antibody in situ in a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In accordance with some embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

The term specifically binds or the like means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds 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, includes antibodies that bind to ANGPTL3, or a portion thereof, with a KD of less than about 1000 nM, less than about 500 nM, less than about 300 pM, 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 in surface plasmon resonance analysis. However, an isolated antibody that specifically binds to human ANGPTL3 is cross-reactive with other antigens, such as ANGPTL3 molecules from other (non-human) species.

Anti-ANGPTL3 antibodies useful in the methods of the present 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 they were derived. antibodies. Such mutations can be easily detected by comparing the amino acid sequences presented herein with germline sequences available, for example, from public antibody databases.

- 7 040975

The present invention includes methods comprising the use of antibodies and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework and/or CDR regions have been mutated to the corresponding residue(s) the germline sequence from which the antibody was derived, or up to the corresponding residue(s) of another human germline sequence, or up to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as germline mutations). One skilled in the art, starting from the heavy and light chain variable region sequences described herein, can easily produce numerous antibodies and antigen-binding fragments that contain one or more individual germline mutations, or combinations thereof. In some embodiments, all framework and/or CDR residues in the VH and/or VL domains are mutated back to residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR regions, for example, where certain individual residues are mutated to the corresponding residue of a particular germline sequence, while some other residues that differ from the original germline sequence are retained or mutated to the corresponding residue of a different germline sequence. Once generated, antibodies and antigen-binding fragments that contain one or more germline mutations can be readily tested for one or more desirable properties such as improved binding specificity, improved binding affinity, improved or enhanced antagonistic or agonistic biological properties (if any) , reduced immunogenicity, etc. The use of antibodies and antigen-binding fragments obtained by this general method is included in the present invention.

The present invention also includes methods comprising the use of anti-ANGPTL3 antibodies containing variants of any of the HCVR, LCVR and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes the use of anti-ANGPTL3 antibodies having the amino acid sequences HCVR, LCVR, and/or CDR, e.g., containing 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions with respect to any from the HCVR, LCVR and/or CDR amino acid sequences described herein.

As used herein, the term surface plasmon resonance refers to an optical phenomenon that allows analysis of interactions in real time by detecting changes in protein concentration in a biosensor array, for example, using the BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ) .

As used herein, the term KD is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

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 a paratope. One antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and may have different biological effects. Epitopes can be either conformational or linear. A conformational epitope is formed by spatially closely spaced amino acids from different segments of a linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include fragments of saccharides, phosphoryl groups, or sulfonyl groups on an antigen.

In some embodiments, the anti-ANGPTL3 antibody used in the methods of the present invention is an antibody with pH dependent binding characteristics. As used herein, the expression pH-dependent binding means that the antibody or antigen-binding fragment thereof exhibits reduced binding to ANGPTL3 at acidic pH compared to neutral pH (both expressions may be used interchangeably for the purposes of this disclosure). For example, antibodies with pH-dependent binding characteristics include antibodies and antigen-binding fragments thereof that bind to ANGPTL3 with higher affinity at neutral pH than at acidic pH. In some embodiments,

The antibodies and antigen-binding fragments of the present invention bind to ANGPTL3 with an affinity that is 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.

According to this aspect of the invention, anti-ANGPTL3 antibodies with pH dependent binding characteristics may have one or more amino acid variations relative to the parent anti-ANGPTL3 antibody. For example, an anti-ANGPTL3 antibody with pH dependent binding characteristics may contain one or more histidine substitutions or insertions, for example, in one or more CDRs of the parent anti-ANGPTL3 antibody. Thus, in accordance with some embodiments of the present invention, methods are provided comprising administering an anti-ANGPTL3 antibody that contains amino acid sequences of CDRs (e.g., heavy and light chain CDRs) that are identical to the amino acid sequences of the CDRs of the parent anti-ANGPTL3 antibody, except for replacement with a histidine residue. one or more amino acids of one or more CDRs of the parent antibody. Anti-ANGPTL3 antibodies with pH dependent binding may have, for example, 1, 2, 3, 4, 5, b, 7, 8, 9 or more histidine substitutions, either within a single CDR of the parent antibody or distributed over a plurality (e.g., 2, 3, 4, 5 or 6) CDR of parental anti-ANGPTL3 antibody. For example, the present invention includes the use of pH-dependent binding anti-ANGPTL3 antibodies comprising one or more HCDR1 histidine substitutions, one or more HCDR2 histidine substitutions, one or more HCDR3 histidine substitutions, one or more LCDR1 histidine substitutions, one or more histidine substitutions in LCDR2; and/or one or more histidine substitutions in LCDR3 of the parent anti-ANGPTL3 antibody.

As used herein, the term acidic pH means pH 6 or less (eg, less than about 6, less than about 5.5, less than about 5, etc.). The expression acidic pH includes pH values of about 6, 5.95, 5.9, 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 or less. Used in the present description, the term neutral pH means a pH from about 7 to about 7.4. The expression neutral pH includes pH values of about 7, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

Non-limiting examples of anti-ANGPTL3 antibodies that can be used in the context of the present invention include, for example, evinacumab, as well as any of the exemplary anti-ANGPTL3 antibodies as set forth in US Pat. description by reference in its entirety, or any of the exemplary anti-ANGPTL3 antibodies as set forth in US Pat. No. 8,742,075 (Lexicon Pharmaceuticals, Inc.), the disclosure of which is incorporated by reference in its entirety.

Obtaining human antibodies.

Anti-ANGPTL3 antibodies may be prepared 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 generated using hybridoma technologies, phage display, yeast display, and the like. Antibodies for use in the methods of the present invention may be, for example, chimeric antibodies, humanized antibodies, or fully human antibodies.

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

For example, using VELOCIMMUNE™ technology (see, for example, US 6594541, Regeneron Pharmaceuticals) or any other known method for producing monoclonal antibodies, high affinity chimeric antibodies to ANGPTL3 are initially isolated with a human variable region and a mouse constant region. VELOCIMMUNE® technology involves generating a transgenic mouse having a genome containing human heavy and light chain variable regions operably linked to endogenous constant region loci such that the mouse produces an antibody comprising a human variable region and a mouse constant region in response to antigen challenge. The DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to the DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

Typically, VELOCIMMUNE® mice are challenged with an antigen of interest and lymphatic cells (such as B cells) that express antibodies are isolated from the mice's blood. The lymphatic cells can be fused to a myeloma cell line to obtain immortalized hybridoma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the heavy chain and light chain variable regions can be isolated and linked to the desired isotypic heavy chain and light chain constant regions. Such an antibody protein can be produced in a cell, such as a CHO cell. Alternatively, DNA

- 9 040975 encoding antigen-specific chimeric antibodies or variable domains of light and heavy chains, can be isolated directly from antigen-specific lymphocytes.

Initially, high-affinity chimeric antibodies are isolated having a human variable region and a mouse constant region. Antibodies are characterized and selected for the presence of desirable characteristics, including affinity, selectivity, epitope, etc., using standard procedures known to those skilled in the art. The mouse constant regions are replaced with a human target constant region to produce a fully human antibody of the invention, eg wild-type or modified IgG1 or IgG4. Although the constant region chosen may vary depending on the particular use, the variable region contains high affinity antigen binding and target specificity characteristics.

In general, antibodies that can be used in the methods of the present invention have high affinity, as described above, as measured by antigen binding, either immobilized on a solid phase or in solution. Mouse constant regions are replaced with the required human constant regions to generate fully human antibodies of the invention. Although the constant region chosen may vary depending on the particular use, the variable region contains high affinity antigen-binding and targeted targeting characteristics.

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

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

In some embodiments, an anti-ANGPTL3 antibody or antigen-binding fragment thereof that can be used in the methods of the present invention 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.

Pharmaceutical compositions and methods of administration.

The present invention includes methods that include administering an ANGPTL3 inhibitor to a patient, wherein the ANGPTL3 inhibitor is contained in a pharmaceutical composition. Pharmaceutical compositions of the invention are formulated with suitable carriers, excipients and other agents that provide suitable transmission, delivery, tolerability and the like. Many suitable formulations can be found in the reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water- in oil, carbovax emulsions (polyethylene glycols of various molecular weights), semi-solid gels and semi-solid mixtures containing carbovax. See also Powell et al. Compendium of excipients for parenteral formulations, PDA (1998), J. Pharm. sci. Technol. 52:238-311.

Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, eg liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J Biol Chem 262:4429-4432). Routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes of administration. The composition can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucosal surfaces (eg, oral mucosa, rectal and intestinal mucosa, etc.) and together with other biologically active agents.

The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen-shaped delivery device can easily have applications for delivering the pharmaceutical composition of the present invention. Such a pen-shaped delivery device may be reusable or disposable. A pen-shaped reusable delivery device typically uses a replaceable cartridge that contains a pharmaceutical composition. After introducing all the pharmaceutical composition that was inside the cartridge, and after the cartridge becomes empty, the empty cartridge can be easily discarded and replaced with a new one that will contain the pharmaceutical composition. The pen-shaped delivery device can then be reused. There is no replaceable cartridge in the disposable pen-shaped delivery device. On the contrary, one

- 10 040975 a pen-shaped disposable delivery device is filled with a pharmaceutical composition held in a reservoir within the device. After the reservoir is empty due to the use of the entire pharmaceutical composition, the entire device is discarded.

Numerous reusable pen-shaped delivery devices and auto-injectors have applications for subcutaneous delivery of the pharmaceutical composition of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ handle (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™ handle, HUMALOG™ handle, HUMALIN 70/ 30™ (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lake, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany) to name but a few. Examples of disposable pen-shaped delivery devices having applications for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk) and KWIKPEN™ (Eli Lilly), SURECLICK™ auto-injector (Amgen, Thousand Oaks, CA), PENLETTM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and the HUMIRATM pen (Abbott Labs, Abbott Park Illinois) to name but a few.

In certain situations, the pharmaceutical composition may be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled release system may be located near the target of the composition so that only a fraction of the systemic dose is required (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, p. 115-138) . Other controlled release systems are reviewed by Langer, 1990, Science, 249:1527-1533.

Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, etc. These injectable preparations can be obtained by known methods. For example, injectable preparations may be prepared, for example, by dissolving, suspending or emulsifying an antibody or salt thereof, as described above, in a sterile aqueous or oily medium commonly used for injection. As the aqueous injection medium, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc. are used, which can be used in combination with a suitable solubilizing agent such as an alcohol (for example, ethanol) (for example, propylene glycol, polyethylene glycol), non-ionic surfactant (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil), etc.) As the oil medium, for example, sesame oil, soybean oil, etc. .d., which can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus obtained is preferably filled into an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteral administration described above are formulated in unit dosage forms appropriate to match the dosages of the active ingredients. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, and the like.

Dosage.

The amount of an ANGPTL3 inhibitor (eg, an anti-ANGPTL3 antibody) administered to a patient in accordance with the methods of the present invention is typically a therapeutically effective amount. As used herein, the term therapeutically effective amount means the dose of an ANGPTL3 inhibitor that results in a detectable decrease (at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75% or more of baseline) of one or more parameters selected from the group consisting of LDL-C, ApoB100, non-HDL-C, total cholesterol, VLDL-C, triglycerides, Lp(a) and residual cholesterol, or an amount that prevents or attenuates atherosclerosis in a patient (as described hereinafter).

In the case of an anti-ANGPTL3 antibody, a therapeutically effective amount may be from about 0.05 to about 600 mg, for example, about 0.05 mg, about 0.1 mg, about 1 mg, about 1.5 mg, about 2 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,

- 11 040975 about about about about about about about

260 mg, approx.

310 mg, approx.

360 mg, approx.

410 mg, approx.

460 mg, approx.

510 mg, approximately

270 mg, approximately 280

320 mg, approximately 330

370 mg, approximately 380

420 mg, approximately 430

470 mg, approximately 480

520 mg, about 530 mg, about 290 mg, about 340 mg, about 390 mg, about 440 mg, about 490 mg, about 540 mg, about 300 mg mg, about 350 mg mg, about 400 mg mg, about 450 mg mg , approximately 500 mg mg, approximately 550 mg

560 mg, about 570 mg, about 580 mg, about 590 mg or about 600 mg anti-ANGPTL3 antibody. Other dosage amounts of ANGPTL3 inhibitors will be apparent to those skilled in the art and are contemplated within the scope of the present invention.

The amount of anti-ANGPTL3 antibody contained in individual doses may be expressed as milligrams of antibody per kg of patient body weight (ie, mg/kg). For example, the 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.

Combined therapies.

As described hereinafter, the methods of the present invention may include administering an ANGPTL3 inhibitor to a patient in combination (in the background) with a lipid-lowering therapy previously administered to the patient. For example, in the context of preventing or attenuating atherosclerosis in a patient, an ANGPTL3 inhibitor may be administered in combination with a stable daily statin therapy regimen. Exemplary daily therapeutic regimens for statins with which an ANGPTL3 inhibitor may be administered in combination 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) an agent that inhibits cholesterol uptake and/or bile acid reuptake (eg, ezetimibe); (2) an agent that increases lipoprotein catabolism (such as niacin); and/or (3) activators of the transcription factor LXR, which plays a role in the elimination of cholesterol, such as 22-hydroxycholesterol.

In some embodiments, the present invention includes methods of preventing or attenuating atherosclerosis in a patient by administering to the patient an ANGPTL3 inhibitor in combination with an ANGPTL4 inhibitor (e.g., an anti-ANGPTL4 antibody or antigen-binding fragment thereof), an ANGPTL8 inhibitor (e.g., an anti-ANGPTL8 antibody or antigen-binding fragment thereof). ) and/or a PCSK9 inhibitor (eg, an anti-PCSK9 antibody or antigen-binding fragment thereof). Non-limiting examples of PCSK9 antibodies that can be used in the context of the present invention include, for example, alirocumab, evolocumab, bococizumab, lodelcizumab, ralpancizumab, or antigen binding portions of any of the above antibodies.

Introduction schemes.

In accordance with some embodiments of the present invention, multiple doses of an ANGPTL3 inhibitor (i.e., a pharmaceutical composition containing an ANGPTL3 inhibitor) may be administered to a patient over a period of time (e.g., during a daily statin regimen or other lipid-lowering background therapy) . Methods in accordance with this aspect of the invention include sequential administration of several doses of an ANGPTL3 inhibitor to a patient. As used herein, the term sequential administration means that each dose of an ANGPTL3 inhibitor is administered to a patient at different time points, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that include 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.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of individual doses of a pharmaceutical composition containing an ANGPTL3 inhibitor. Thus, the initial dose is the dose that is administered at the beginning of the treatment regimen (also called the base dose); secondary doses - doses administered after the initial dose; and tertiary doses are doses administered after secondary doses. Initial, secondary, and tertiary doses may contain the same amount of ANGPTL3 inhibitor, but may generally differ from each other in terms of frequency of administration. However, in some embodiments, the amount of ANGPTL3 inhibitor contained in the initial, secondary, and/or tertiary doses varies from one another (eg, adjusted up or down as needed) during treatment. In some embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the start of a treatment regimen as bolus doses, followed by doses that are administered less frequently (by

- 12 040975 example, maintenance doses).

According to exemplary embodiments of the present invention, each secondary and / or tertiary dose is administered through 1-26 (for example, through 1, _11/2 , 2, 21/2, 3, 3, 4, 41/2, 5, 51/2, 6, _61/2 , 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2, 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22, 221/ 2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2 or more) weeks immediately after the previous dose. The term "immediately preceding dose" as used herein means, in a sequence of multiple administrations, the dose of an antigen-binding molecule that is administered to a patient prior to the next dose in the sequence without intermediate doses.

Methods in accordance with this aspect of the invention may include administering to a patient any number of secondary and/or tertiary doses of an ANGPTL3 inhibitor. For example, in some embodiments, only one secondary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Similarly, in some embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient.

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 a 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 a patient 1-2, 4, 6, 8 or more weeks after the immediately preceding dose. Alternatively, the frequency with which secondary and/or tertiary doses are administered to a patient may vary over the course of treatment. The frequency of administration may also be adjusted during the course of treatment by the physician, depending on the needs of the individual patient after clinical examination.

Examples

The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the authors consider to be their invention. Efforts have been made to ensure accuracy with respect to the numerical values used (eg amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or about atmospheric.

Example 1 Preparation of human antibodies to human ANGPTL3.

Anti-human ANGPTL3 antibodies were prepared as described in US Pat. No. 9,018,356. An exemplary ANGPTL3 inhibitor used in the following example is a human anti-ANGPTL3 antibody designated H4H1276S, also known as evinacumab. Evinacumab has the following amino acid sequence characteristics:

a heavy chain containing SEQ ID NO: 10 and a light chain containing SEQ ID NO: 11;

a heavy chain variable region (HCVR) containing SEQ ID NO: 2 and a light chain variable domain (LCVR) containing SEQ ID NO: 3;

HCDR1, heavy chain complementarity-determining region 1, containing SEQ ID NO: 4, HCDR2, containing SEQ ID NO: 5, HCDR3, containing SEQ ID NO: 6;

LCDR1, light chain complementarity determining region 1, containing SEQ ID NO: 7, LCDR2, containing SEQ ID NO: 8, and LCDR3, containing SEQ ID NO: 9.

Example 2 An anti-ANGPTL3 antibody prevents the development of atherosclerosis in a mouse model.

Introduction.

The aim 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 features of mixed or familial dys-beta-lipoproteinemia and atherosclerosis. (See, for example, Kuhnast 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 the E3L.CETP mouse) and its use to evaluate influence of a pharmacological agent on the development of atherosclerosis).

Inhibition of ANGPTL3 was achieved using an anti-ANGPTL3 antibody. The specific anti-ANGPTL3 antibody used in these studies is an antibody called evinacumab, as described above. The control antibody used in these studies is an antibody of the same isotype against an irrelevant target.

Experiment design.

An illustration of the study design is shown in FIG. 1. Evinacumab is a fully human IgG4 antibody. Thus, when evaluated in different mouse strains, some mice develop anti-drug antibodies that preclude the effectiveness of the therapeutic agent. The percentage of mice developing this response against drugs may be different for each mouse model/genetic background. For this reason, more mice in

- 13 040975 group was included in this first study in APOE*3Leiden.CETP mice to determine how many mice develop an immune response against human proteins in this particular model. After exclusion of mice that develop an immune response against human proteins, the remaining mice can be used to evaluate the effect of evinacumab on atherosclerosis.

female APOE*3Leiden.CETP mice were placed on a T diet (semi-synthetic 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 administration period 13 low response mice were removed from the study, and the remaining 50 mice were subdivided into one control group of 20 mice and one evinacumab treatment group of 30 mice matched for body weight, plasma cholesterol and triglyceride levels after 4 hours of fasting (t=0 ).

Treatment with evinacumab or control antibody was administered weekly via subcutaneous injections 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. Body weight and food intake (per cell) were measured at weeks 0, 3, 6, 8, 11 and 13.

At 0, 2, 4, 6, 8, 11 and 13 weeks of treatment, blood samples were collected after 4 hours of fasting. Plasma cholesterol and triglyceride levels were measured individually and additional EDTA plasma was collected to assess hFc and mouse antibodies against human proteins. After 6 weeks of treatment, the first anti-drug response was assessed and 3 control group mice and 5 treatment group mice with high levels of anti-drug antibodies were removed from the study.

Lipoprotein profiles were measured at weeks 0, 6 and 13 in plasma samples pooled per group. Feces were collected in each cage during week 12 (twice within 48 h). At time t=13 mice were sacrificed without starvation. EDTA plasma was obtained by cardiac puncture and several tissues were collected. The development of atherosclerosis in the aortic root was measured in 15 mice per group (after excluding mice that developed an anti-drug response) by measuring the number of lesions, the area of the lesion and the severity of the lesion. In addition, the composition of the lesion was determined.

(1) The parameter measurement graph is as follows:

(2) body weight (weeks 0, 3, 6, 8, 11 and 13);

(3) food intake (per cell, weeks 0, 3, 6, 8, 11, and 13);

(4) total cholesterol and plasma triglycerides (following 4 hours of fasting at weeks 0, 2, 4, 6, 8, 11 and 13);

(5) collection of 20 µl of EDTA plasma for hFc and mouse anti-human protein assessment (after 4 hours of fasting at weeks 0, 2, 4, 6, 8, 11 and 13);

(6) lipoprotein profiles (cholesterol and triglycerides totaled per group, at weeks 0, 6, and 13);

(7) development of atherosclerosis in the aortic root (15 mice per group): (a) number of lesion, (b) total area of lesion, (c) severity of lesion, (d) composition of lesion (e.g. macrophage, capped smooth muscle cell, necrosis and collagen content) and (e) aggregation of monocytes. (At sacrifice [week 13], aortas were collected from all mice, however atherosclerosis analysis was performed with 15 mice per group, mice were selected that did not develop anti-drug antibodies).

Methods.

Body weight and food intake were measured according to standard procedures.

Blood plasma samples were obtained according to standard procedures. Total plasma cholesterol and triglyceride levels were determined using commercially available kits.

Lipoprotein profiles were measured by FPLC analysis using the AKTA apparatus from Amersham Biosciences. The analysis was performed on total samples for the group. Cholesterol and triglycerides were measured in fractions using commercially available kits.

Atherosclerotic lesion area and severity was assessed in the aortic root region as previously reported (see, for example, Kuhnast et al., 2012, J. Hypertens, 30:107-116). The aortic root was identified by the appearance of aortic valve leaflets, and successive transverse sections of the entire aortic root region (5 µm thick at 50 µm intervals) were mounted on 3-aminopropyltriethoxysilane-coated slides and stained with hematoxylin-phloxin-saffron (HPS). For each mouse, the lesion area was measured in 4 consecutive sections. Each slice consists of three segments (separated by flaps). To determine the size and severity of atherosclerotic lesions, they 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 fat streaks (up to 10 foam cells in the intima without any changes);

type 2: regular fat streaks (ten or more foam cells in the intima with no other changes);

type 3: soft plaque (foamy cells with fibrous cap);

type 4: moderate plaque (more progressive lesions with affected media but no loss of media architecture);

type 5: severe plaque (medium is severely damaged, broken elastic fibers, cholesterol fissures, calcification and necrosis are often observed).

Images were taken with an Olympus BX51 microscope and areas were measured with image processing software. The total lesion area and number of lesions were calculated for each transverse section. Healthy segments were calculated as a percentage of the total number of segments. The severity of the lesion was also determined as a percentage of the total number of lesions. Type I-III lesions were classified as moderate lesions and type IV-V lesions were classified as severe lesions.

The number of monocytes adhering to the activated endothelium was counted in each segment used for quantification of the lesion after immunostaining with antiserum AIA 31240 (1:1000, Accurate Chemical and Scientific, New York, New York, USA), and the calculation was performed on a cross-sectional cut.

Macrophage area of lesions was measured after immunostaining with anti-mouse Mac-3 antibody (1:50, BD Pharmingen, The Netherlands). Sirius Red staining was performed to stain collagen. The area of fibrous cap smooth muscle cells was measured after immunostaining with anti-smooth muscle alpha actin antibody (1:400, PROGEN Biotechnik GmbH, Heidelberg, Germany), which is cross-reactive with mouse alpha actin. Photographs/images were taken with an Olympus BX51 microscope. Macrophage, collagen, necrotic nucleus area and smooth muscle cell area were quantified using imaging software. The content of macrophages, smooth muscle cells (SMC), necrotic nucleus and collagen in lesions was calculated as a percentage of the area of the lesion. The plaque sensitivity/stability index was calculated by dividing the sum of SMC and collagen stabilization factors by the sum of macrophage and necrotic nucleus destabilization factors.

At week 13, mice were sacrificed without starvation with CO2. No final antibody injection was given for 13 weeks prior to sacrifice. After sacrifice, as much EDTA plasma as possible was collected (via cardiac puncture) and stored at -70° C. until further use. Hearts, including the aortic root, were fixed in 10% formalin. The thoracic aorta was collected and fixed in 10% formalin.

Statistics.

Depending on normality, the significance of differences between groups was calculated non-parametrically using the SPSS computer program and the Mann-Whitney U-test for independent samples. A P value <0.05 was considered statistically significant.

Statistical differences in the treatment group compared to the control group are indicated in the drawings with an asterisk at the time of measurement.

Results.

Body mass.

The results by body weight are summarized in Table. 1. Values are absolute values (grams) and are mean values ± SD for 14-15 mice per group.

Non-parametric analysis: Mann-Whitney U-test.

There was no difference in body weight between the evinacumab group and the control group.

Eating.

The results of food intake are summarized in table. 2. Values are absolute values (g/mouse/day) and are mean ± SD for 17 cells (all mice, before matching) or for 4-6 cells (after matching and start of treatment).

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table 2

food intake (g/m/d) Time (weeks) t=0 t=3 t=6 T=8 t=ll t=13 Control AV AVG 2.9 2, 6 2.4 2.3 2, 6 2.5 SD 0.2 0, 1 0.2 0.2 0.4 0.4 Evinacumab AVG 2.9 2.7 2.4 2.3 2.5 2.5 SD 0.2 0, 1 0.2 0.2 0.3 0.4

Meal: Rvalue t=0 t=3 t=6 T-8 t=ll t=13 Control vs. n/a 0.47 6 0.91 4 0.91 4 0.91 4 1.00 0 evinacumab

Non-parametric analysis: Mann-Whitney U-test.

There were no differences in food intake between the evinacumab group and the control group.

Plasma cholesterol.

The results for plasma cholesterol levels are summarized in Table 1. 3 and 4 and in FIG. 2A and 2B. Values are absolute values (mM) and are mean ± SD for 14-15 mice per group.

Table 3

Cholesterol (mm) Time (weeks) t=0 t=2 t=4 t=6 t=8 t=ll t=13 Control AVG 24.1 29, 9 28.0 26, 7 27.6 22.1 20.9 AB SD 3.1 50 4.5 3.3 4.5 5.4 4.6 Evinacumab AVG 23.7 12.6 14.0 14.1 13.6 10, 8 9, 6 SD 2.4 2.4 19 2.2 2.5 1.7 2.1

Cholesterol : P-value t=0 t=2 t=4 t=6 t=8 t=ll t=13 Control versus evinacumab 0.9, 1 4 <0.00 1 <0.00 1 <0.00 1 <0.00 1 <0.00 1 <0.00 1

Non-parametric analysis: Mann-Whitney U-test.

Table 4

Cholesterol (mm) Impact on general Cholesterol (mM*week) Time (weeks) t=13 Control AB AVG SD 393.5 49.7 Evinacumab AVG SD 230.2 23, 8

Effect on Cholesterol: P-value t=13 Control versus evinacumab <0.001

Non-parametric analysis: Mann-Whitney U-test.

Evinacumab treatment 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 experiment, cholesterol exposure during the experiment (0-13 weeks + run-in period) was significantly reduced with evinacumab treatment (41%, p < 0.001) compared with the control group.

Plasma triglycerides.

The results on the level of plasma triglycerides are summarized in table. 5 and in FIG. 3. Values are absolute values (mM) and are mean values ± SD for 14-15 mice per group.

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Table 5

Triglycerides (mm) t=2 t=4 t=6 t=8 t=ll t=13 Time (weeks t=0 Control ) AVG 7.5 5.5 4.9 4.8 3, 6 4.0 3.4 AB SD 2.6 1.3 1.6 1.5 1.3 1,o 1.2 Evinacumab AVG 7.4 0.7 0.7 0.7 0.7 0.7 0.6 SD 2.0 0.3 0.2 0.3 0.2 0.2 0.2

Triglycerides : P-value t=0 t=2 t=4 t=6 t=8 t=ll t=13 Control versus evinacumab 0.74 7 <0.00 1 <0.00 1 <0.00 1 <0.00 1 <0.001 <0.00 1

Non-parametric analysis: Mann-Whitney U-test.

Treatment with evinacumab resulted in a significant reduction in plasma triglycerides compared to the control group at all time points (mean reduction of 84%).

lipoprotein profiles.

Lipoproteins were separated on a superose column. Fractions of cholesterol and triglycerides at t=0, 6 and 13 weeks are shown in FIG. 4A-4C [cholesterol] and 5A-5C [triglycerides]. Values are absolute values from total plasma cholesterol and triglyceride measurements per group at t=0, 6 and 13 weeks. For the purposes of this analysis, fractions 3-7 were considered VLDLs; 8-16 as IDL/LDL and 17-24 as HDL.

At t=0, no differences in lipoprotein profiles were observed prior to treatment, and after 6 and 13 weeks of evinacumab treatment, plasma cholesterol levels measured in per-group summed samples decreased at the VLDL-LDL peak compared to the control group and, apparently increased at the HDL peak (see FIGS. 4B and 4C). The triglyceride profiles showed a similar pattern (see FIGS. 5A-5C).

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

Area of atherosclerotic lesion.

Data on the area of atherosclerotic lesions are summarized in Table. 6 and in FIG. 6A and 6B. Values are absolute values of aortic root lesion area per transverse section and are expressed as mean ± SD for 14-15 mice per group.

Table 6

Lesion area per cross section (*1000 µm ² ) Time (weeks) t=13 Control AV AVG SD 216.1 49, 3 Evinacumab AVG SD 133.0 52.8 Damage area: Rvalue t=13 Control versus evinacumab

Non-parametric analysis: Mann-Whitney U-test.

The total area of the lesion in the aortic root of the control group was 216082 μm ² per transverse section when sacrificed at time t=13 weeks. Evinacumab treatment significantly reduced the total lesion area by 39% (p<0.001) compared to the control group, as found at sacrifice.

Atherosclerosis: number of lesions.

The number of atherosclerotic lesions per transverse section is summarized in Table 1. 7. Values are absolute values (number of lesions) and are expressed as means ± SD for 14-15 mice per group.

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Table 7

Number of lesions per cross section (*1000 µm ² ) Time (weeks) t=13 Control AV AVG SD 50 0.8 Evinacumab AVG SD 50 eleven Number of defeats: Rvalue t=13 Control versus evinacumab 0.880

Non-parametric analysis: Mann-Whitney U-test.

There was no difference in the number of lesions between the evinacumab group and the control group.

Atherosclerosis: severity of lesions.

The results regarding the severity of atherosclerotic lesions are summarized in table. 8. Values are percentages of total lesions and are expressed as means ± SD for 14-15 mice per group.

Table 8

Injury severity (%) Time (weeks) Moderate Lesions (Type IIII) Severe lesions (Type IVV) Control AB AVG SD 27.7 10.6 72.3 10.6 Evinacumab AVG SD 36, 8 26, 8 63.2 26, 8

Severity value defeats: R- Moderate (ι-sh) Heavy (IV-V) Control compared evinacumab from 0.451 0.451

Non-parametric analysis: Mann-Whitney U-test.

There was no difference in the severity of lesions between the evinacumab group and the control group.

Atherosclerosis: healthy segments.

The number of healthy atherosclerotic segments is summarized in Table. 9. Values represent the percentage of non-atherosclerotic (healthy) segments. Values are means ± SD for 14-15 mice per group.

Table 9

Healthy segments (%) Time (weeks) t=13 Control AV AVG SD 2.4 8.9 Evinacumab AVG SD 2.8 8.7

Healthy segments: P-value t=13 Control vs. 0.813 evinacumab

Non-parametric analysis: Mann-Whitney U-test.

There was no difference in the number of healthy segments between the evinacumab group and the control group.

Atherosclerosis: the content of macrophages.

The content of macrophages in lesions is summarized in Table. 10 and in FIG. 7A. Values are absolute values (μm ² x 1000) or relative values (% lesion) and are expressed as means±SD for 14-15 mice per group.

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Table 10

Macrophage area/cross section (µm ² *1000) The content of macrophages (% damage) Type III-V Type IV-V Type IIIV Type IV-V Control AB AVG SD 25, 9 8.3 22.7 8.3 12.9 4.7 12.1 4, 9 Evinacumab AVG SD 19, 3 9, 7 15, 9 10.6 17.2 7.7 14.6 5, 6

Area with macrophages: P-value Area with macrophages/cross section (µm ² *1000) The content of macrophages (% damage) Type III-V Type IV-V Type III- V Type IV-V Control vs. 0.070 0.070 0.102 0.227

Non-parametric analysis: Mann-Whitney U-test.

Evinacumab treatment tended to decrease macrophage per cross section in type IV-V atherosclerotic lesions compared to controls. However, this difference was not observed after adjusting for the total area of the lesion, which was expressed as a percentage of macrophages in type IV-V lesions (FIG. 7A).

Atherosclerosis: necrotic content.

The necrotic content of the lesions is summarized in Table 1. 11 and in FIG. 7A. Values are absolute values (μm ² x 1000) or relative values (% lesion) and are expressed as means±SD for 14-15 mice per group.

Table 11

Necrotic area/cross section (µm ² *1000) Necrotic content (% damage) Type IIIV Type IV-V Type III-V Type IV-V Control AB AVG SD 19, 4 10.2 18, 8 10.2 8, 8 3.4 9.0 3, 6 Evinacumab AVG SD 6.0 3, 8 5, 8 3, 9 4.5 2.0 4, 9 18

Necrotic Area: Rvalue Necrotic area/cross section (µm ² *1000) Necrotic content (% damage) Type III- V Type IV-V Type III-V Type IV-V Control versus evinacumab <0.001 <0.001 <0.001 0.001

Non-parametric analysis: Mann-Whitney U-test.

Evinacumab treatment significantly reduced the necrotic content in cross section in type IV-V atherosclerotic lesions compared with the control group (by 69%, p<0.001). After correction of the total area of the lesion, the percentage of necrotic tissue area in type IV-V lesions also significantly decreased compared with the control group (by 45%, p=0.001) (Fig. 7A).

Atherosclerosis: smooth muscle cells in a fibrous cap.

Data on the quantification of smooth muscle cells (SMC) in the fibrous cap of the lesions are summarized in Table. 12 and in FIG. 7B. Values are absolute values (μm ² x 1000) or relative values (% damage) and are expressed as means ± SD for 14-15 mice per group.

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Table 12

SMC area/cross section (µm ² * 10 00) SMC (% defeat) Type IIIV Type IV-V Type III-V Type IV-V Control AB AVG SD 24.9 7.7 23, 4 7.7 12.1 3.2 12.1 3, 3 Evinacumab AVG SD 15, 8 7.5 14.4 8.0 13.5 4.2 13, 7 4, 6

SMC area: R value SMC area/cross section (μm ² * 1000) SMC content(% hit) Type IIIV Type IV-V Type III-V Type IV-V Control versus evinacumab 0.005 0.009 0.217 0.210

Non-parametric analysis: Mann-Whitney U-test.

Evinacumab treatment significantly reduced smooth muscle cell (SMC) area per transverse section in type IV-V atherosclerotic lesions compared with controls (by 39%, p=0.009). However, after correction of the total lesion, the percentage of SMC in the fibrous cap in type IV-V lesions did not decrease significantly compared to the control group (Fig. 7B).

Atherosclerosis: collagen content

The content of collagen in lesions is summarized in table. 13 and in FIG. 7B. Values are absolute values (μm ² x 1000) or relative values (% damage) and are expressed as means ± SD for 14-15 mice per group.

Table 13

Collagen area/cross section (µm ² *1000) Collagen content (% damage) Type III-V Type IV-V Type III-V Type IV-V Control AB AVG SD 102.6 34.0 96.9 34.7 48.7 8.7 48.5 9, 2 Evinacumab AVG SD 62.0 29, 2 58.1 30.5 48.7 10.3 52.3 8.2

Collagen area: R value Collagen area/cross section (µm ² * 1000) Collagen content (% lesion) Type III-V Type IV-V Type III-V Type IV-V Control versus evinacumab 0.005 0.009 0.813 0.227

Non-parametric analysis: Mann-Whitney U-test.

Treatment with evinacumab significantly reduced the collagen content in the cross section of atherosclerotic lesions of type IV-V compared with the control group (by 40%, p=0.009). However, after correction of the total area of the lesion, the percentage of collagen in type IV-V lesions did not significantly decrease compared to the control group (Fig. 7B).

Atherosclerosis: adhesion of monocytes.

The percentage of monocytes per cross section is summarized in Table 1. 14. Values are absolute values (number of attached monocytes per cross section) and are expressed as means ± SD for 14-15 mice per group.

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Table 14

Number of monocytes per cross section Control _AVG AB SD 3, 4 1.2 Evinacumab AVG SD 3, 5 1.2

Monocytes: P - value The number of monocytes per cross section Control versus evinacumab 0.983

Non-parametric analysis: Mann-Whitney U-test.

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

Atherosclerosis: stability index.

Data on the stability of plaque lesions (expressed in units of the stability index) are summarized in table. 15. The stability index is defined as the sum of SMC stabilization markers and collagen content divided by the sum of macrophage destabilizing markers and necrotic content. This value provides a measure of plaque stability. Values are means ± SD for 14-15 mice per group.

Table 15

Stability index in Types IV-V (% SMC+% collagen)/(% macrophages+% necrosis) Reference AV AVG SD 3.2 1.3 Evinacumab AVG SD 3.7 1.7

Stability index: P-value Stability index in Types IV-V (% SMC+% collagen)/(% macrophages+% necrosis) Control versus evinacumab 0.329

Non-parametric analysis: Mann-Whitney U-test.

Evinacumab treatment did not affect the stability index compared to the control group.

Conclusion.

Aspects of security.

There was no effect on body weight (gain) and food intake when treated with evinacumab compared to controls.

Decreased VLDL and LDL levels.

The APOE*3Leiden.CETP mice are a well-established model for familial dys-beta lipoproteinemia (FD) in humans, which is characterized by accumulation of residual lipoproteins and an increased ratio of VLDL-C to LDL-C. The lipoprotein profile in APOE*3Leiden.CETP mice reflects the corresponding level of FD patients with a similar response to lipid-normalizing therapies. This is illustrated by comparable cholesterol reduction across all apoB-containing lipoprotein subfractions with statin and fenofibrate and niacin treatment.

In the present study, evinacumab treatment significantly reduced mean total cholesterol (TC) (-52%, p<0.001) and triglycerides (TG) (-84%, p<0.001) compared to controls that were restricted to non-HDL fractions.

Reducing the size of the lesion and necrotic area.

In the current study, evinacumab significantly reduced lesion size (-39%, p<0.001) compared to controls. No significant differences were found in the number of lesions,

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Claims (7)

healthy segments or severity of lesions. Evinacumab treatment significantly reduced necrotic content as a percentage of total lesion size in severe type IV-V lesions (from 45%, p=0.001), but did not affect macrophage content, collagen content, or SMC area, resulting in a similar lesion stability index as in the control group. Evinacumab had no effect on monocyte recruitment to activated endothelium. The necrotic content was the only marker of plaque composition that varied both in absolute area and in percentage of the total area of the lesion. A potential explanation could be a shift in the composition of the macrophage population in plaque from more pro-inflammatory M1 killer macrophages to more healing M2 repair macrophages that can remove cholesterol from plaque. Conclusion. Taken together, the results presented in this example demonstrate that the anti-ANGPTL3 monoclonal antibody, evinacumab, reduces plasma lipids and prevents the progression of atherosclerosis in APOE*3Leiden.CETP mice. Evinacumab does not affect the number of lesions, the severity of the lesion, or the stability of the plaques, but significantly reduces the area of the lesion and the area of the necrotic core. The scope of the present invention should not be limited to the specific embodiments presented in the present description. Indeed, various modifications of the invention in addition to those presented in the present description will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to be covered by the appended claims. CLAIM 1. A method of reducing the size of a lesion and/or the necrotic content of atherosclerotic lesions in a subject in a patient, the method comprising selecting a patient who has atherosclerosis or is at risk of developing atherosclerosis and administering to the patient one or more doses of an angiopoietin-like protein 3 (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 contains the heavy and light chain CDRs of the HCVR/LCVR amino acid sequence pair containing SEQ ID NO: 2/ 3, where the patient is receiving stable background lipid-lowering therapy (LMT) before or at the time of initiation of treatment with one or more doses of an ANGPTL3 inhibitor, where the stable background LMT is low, moderate, or high-dose statin therapy, and where the administration of one or more doses inhibitor of ANGPTL3 to a patient leads to reduced lesion size and/or necrotic content of atherosclerotic lesions in the subject. 2. The method according to claim 1, characterized in that the patient was diagnosed with heterozygous familial hypercholesterolemia (HeFH) or homozygous familial hypercholesterolemia (HoFH) and/or elevated lipoprotein (a) (Lp[a]) before or at the time of initiation of treatment using one or more doses of an ANGPTL3 inhibitor. 3. The method of claim 1, wherein the patient has been diagnosed with cardiovascular disease (CVD) prior to or at the time of initiation of treatment with one or more doses of an ANGPTL3 inhibitor. 4. The method according to claim 1, characterized in that the patient has suffered a stroke or myocardial infarction before or at the time of initiation of treatment using one or more doses of an ANGPTL3 inhibitor. 5. The method according to any one of claims 1 to 4, wherein the subject received a stable background lipid-lowering therapy (LMT) concomitantly with the administration of one or more doses of an ANGPTL3 inhibitor, where the stable background LMT is low, moderate or high dose statin therapy . 6. A method according to any one of claims 1 to 5, wherein administration of one or more doses of an ANGPTL3 inhibitor to the patient results in one or more therapeutic outcomes selected from the group consisting of: (a) lowering serum total cholesterol (TC), (b) lowering serum triglycerides (TG), (c) lowering serum low-density lipoprotein (LDL) levels, and (d) lowering very-low-density lipoprotein (VLDL) levels ) in serum, where the reduction in (a), (b), (c) and/or (d) is measured relative to the patient's serum TC level, the patient's serum TG level, the patient's serum LDL level, and/or the patient's serum VLDL level before or at the time of initiation of treatment with one or more doses of an ANGPTL3 inhibitor. 7. A method according to any one of claims 1 to 6 wherein administration of one or more doses of an ANGPTL3 inhibitor to a patient results in a reduction in atherosclerotic plaque formation. -